Journal of Distilling Science, Winter 2021

JOURNAL OF DISTILLING SCIENCE VOLUME 1 NUMBER 1

Winter 2021

SOCIETY OF DISTILLING SCIENTISTS AND TECHNOLOGISTS

AN ARTISAN SPIRIT MEDIA PUBLICATION

SPECIAL THANKS TO OUR SPONSORS Lallemand Biofuels & Distilled Spirits is the industry leader in supplying fermentation products and value-added services to the distilled spirits industry.

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For details, visit mgpingredients.com/distilled-spirits.

Rudolph Research Analytical is pleased to be a sponsor of the Journal of Distilling Science and this represents part of our commitment to the Alcohol Production Industry. Rudolph is a 60-year-old manufacturer of high accuracy, high quality laboratory instruments. Rudolph serves the Alcohol production industry with its TTB Approved DDM 2911 PLUS Density Meter and the innovative AlcoTest-RI® system which measures the alcohol content of obscured beverages, without distillation. As an industry partner Rudolph seeks to assist you in the areas of beverage quality, consistency, and compliance so you can provide your customers the highest quality products. Rudolph is always available to help you with quality instruments backed by a team of qualified scientists, engineers, technicians, and experienced staff.

JOURNAL OF DISTILLING SCIENCE

THE OFFICIAL PUBLICATION OF THE SOCIETY OF DISTILLING SCIENTISTS AND TECHNOLOGISTS

TABLE OF CONTENTS

JOURNAL OF DISTILLING SCIENCE

VOLUME 1 NUMBER 1

Winter 2021

THE OFFICIAL PUBLICATION OF THE SOCIETY OF DISTILLING SCIENTISTS AND TECHNOLOGISTS

EDITORIAL

ORIGINAL PAPERS

EDITORIAL

5

A Hearty Welcome to the Journal of Distilling Science

Gary Spedding

7

Overview and Scope

Edition 2021

8

Applications of the Partial Molar Volume Concept in Whisky and Gin Dilutions with Water

Franklin M. Chen, Nada Abdi, and Nolan Torres

17

Lignin-derived phenolic compounds in cachaça aged in barrels from tropical wood species

Mariana C. Castro, Giovanni C. Silvello, and André R. Alcarde

26

Investigation of Appropriate Cleaning Solutions for Removal of Denatonium Benzoate from Distillery Equipment

Lauren E. Mehanna, Kara A. Davis, Shankar C. Miller-Murthy, Tracy A. Gastineau-Stevens, Bert C. Lynn, and Brad J. Berron

37

Chinese Baijiu - Finding a channel to design a defined starter culture

Bowen Wang, Huiyi Hao, Hehe Li, Jinyuan Sun, and Baoguo Sun

50

General Guide for Contributors/Authors

Edition 2021

53

Guidelines and Notes for Reviewers

Edition 2021

57

Manuscript Formatting & Preparation

THE JOURNAL OF DISTILLING SCIENCE

Edition 2021 VOLUME 1 NUMBER 1

Winter 2021

3

JOURNAL OF DISTILLING SCIENCE

THE OFFICIAL PUBLICATION OF THE SOCIETY OF DISTILLING SCIENTISTS AND TECHNOLOGISTS

VOLUME 1

NUMBER 1

Winter 2021

EDITOR Gary Spedding, Ph.D.

EDITORIAL BOARD Andre R. Alcarde

Seth DeBolt

Jacob Lahne

Darrin L. Smith

Denise R. Anderson

Jeannine Delwiche

Ray Marsili

Nermina Spaho

Robert J. Arnold

Roy D. Desroches

Rob McCaughey

Alex Speers

Luis Ayala

John Edwards

Dean McDonald

Molly Troupe

Jamie Baxter

Christopher J. Findlay

Aaron McLeod

Toshio Ueno

Bradley Berron

Christopher Gerling

Gregory H. Miller

Richard Curtis Bird

Patrick M. Hayes

Conor O’Driscoll

Ana Guadalupe Valenzuela-Zapata

Gordon Burns

Patrick Heist

Matthew Pauley

Derek D. Bussan

Annie Hill

Chris Paumi

Keith R. Cadwallader

Reade Huddleston

Guillaume de Pracomtal

Adam Carmer

Paul Hughes

Andrei Prida

Miguel Cedeno

Frances Jack

Michael Qian

Franklin Chen

John D.E. Jeffery

Elizabeth Liz Rhodes

Thomas S. Collins

Nathan Kreel

Kurt A. Rosentrater

Bowen Wang Akira Wanikawa Stuart Joseph Williams N.A. “Nik” Willoughby Alan Wolstenholme Steve Wright

EDITORIAL POLICIES The Journal of Distilling Science (JDS) is both an online and in-print peer-reviewed journal with an overarching reach for reviews and original research papers dealing with all the science and technology disciplines involved in the production of potable distilled spirits and related alcoholic beverages. The journal will become an integral part of the Society of Distilling Scientists and Technologists (SDST) to be formed in 2022.

Papers will be accepted for review from universities and colleges, research institutes and industrial laboratories, distilleries, raw materials producers, and allied industries supporting the testing and quality control functions of distilling operations. All submissions are sent to three reviewers for assessment with due consideration to confidentiality.

To submit your article please email GSpedding@jdsed.com

AN ARTISAN SPIRIT MEDIA PUBLICATION The Journal of Distilling Science (©2021 Artisan Spirit Media) with all rights reserved has been accepted as the official publication of the Society of Distilling Scientists and Technologists (SDST) and is under the editorship of an independent review body appointed and staffed by the founding members of the SDST. Upon formation of the SDST and the full incorporation of the journal into its jurisdiction the editorial committee to be formally approved will assume the greater role of governance over the editorial process. ©2021 ARTISAN SPIRIT MEDIA

PO BOX 31494, SPOKANE, WA 99223

WWW. ARTISANSPIRITMAG.COM/JOURNALDS

EDITORIAL

A Hearty Welcome to the Journal of Distilling Science Intended to be a worldwide accessible and respected journal of excellence in the distilling sciences and technologies arena

A year in the making, and a covidtime undertaking. Now entering fall and nearing the end of the second year of a virus epidemic that hit the world hard in ways we could not have fully imagined. The world of conferences went virtual, and most scientists and technologists were confined to home or office. Many editors believed that articles and chapters and books would fly off the desk without office distractions.

Well, this editor can tell you otherwise. Not just from our new journal perspective – which I should welcome you to by the way – but from other writing commitments and discussions with peers, we all ended up getting beyond deadlines in a more extended way than usual.

Those deadlines just whooshed by.

But enough discussion of procrastinating by me – we did put the cart before the horse after all. What you ask? The journal you now hold in your hands, or view on your computer screen or smartphone, is the companion organ to the new Society of Distilling Scientists and Technologists (SDST). Four years in the making, the SDST itself has been delayed in launch and, to my chagrin, has only been so delayed based in part on the covid situation.

That said, it will be forthcoming.

THE JOURNAL OF DISTILLING SCIENCE

Many of us within the distilling industry have felt the need for regularly convening a coherent scientific body to serve our specific needs and wants. We do lag far behind the enological and brewing sciences, especially in lacking a specific society and a defined methods manual for testing of our beverages, as well as in the realm of sensory evaluation studies and programs. We also addressed the need for a defined organ for the presentation of scientific and technological findings and developments.

Many relevant scientific papers appear in approximately 250-300 of the more than 30,000 journals the world currently offers us. However, most distillers are not taught how to access the literature, nor would expect to find relevant material in journals with very obscure sounding titles. I am not putting down such journals and will not mention any by name – they have solidly reliable, peer-reviewed, information – with the occasional gem of an article highlighting distilling somewhere in its title or context. It is also sometimes expensive to find and obtain such rare articles within the body of many other worldly unrelated topics of coverage. As best we can discern there is no journal dedicated solely to distilled spirits and the world of distilling. A central home where one can find many articles of interest in one place.

Well, we hope you will need to look no further to find the base and advanced information. At the same time, you will be able to discover the names of those other relevant 250+ journals. You can then dig deeper via their citation by the authors of the papers presented within the pages of the Journal of Distilling Science.

VOLUME 1 NUMBER 1

Winter 2021

This inaugural edition of our new journal began to germinate in January of 2021. A very talented group of scientists from across the globe joined as reviewers, and the work creating instructions for authors and reviewers began to unfold. For this first issue manuscript submissions were invited, however, no bias was entertained, nor special privilege granted. A strict but fair review of several manuscripts led to some revisions to the papers you see before you and to some that will appear in 2022. While authors were delayed in research, teaching, and writing due to covid-related events, we are not naïve to the fact that an inaugural issue needs to be seen to be believed – a term we in the US refer to as a Catch-22. The publisher and I are proud to present to you this issue replete with a handful of novel and interesting papers, the instructions to authors and to the reviewers for future reference, and a hearty invitation to help us make this publication a world-class organ to best serve all distillers and our allied industries.

I thank all authors for their contributions and revisions – a lot of work went into them – especially working to the confines of a new journal and its emerging policies and instructions. Thanks also to the set of reviewers involved in this round of peer review, and to those others on our deep roster interested in reviewing future submissions. As I look forward to working with you all in this new venture, I now eagerly await your manuscripts ready for a full publication schedule.

Gary Spedding (Appointed as the initial lead science editor by the organizing body of the forthcoming Society of Distilling Scientists and Technologists) 10/23/2021

5

EDITORIAL

JOURNAL OF DISTILLING SCIENCE

OVERVIEW AND SCOPE EDITION 2021

The Journal of Distilling Science (JDS) is both an online and in-print journal with an overarching reach for reviews and original research papers dealing with all the science and technology disciplines involved in the production of potable distilled spirits and related alcoholic beverages. The journal will become an integral part of the Society of Distilling Scientists and Technologists (SDST) to be formed in 2021.

The JDS is thus open to an international audience and involved in the publishing of high-quality and original scientific papers. Such materials communicating significant research, technical reports, detailing applicable analytical techniques and methods, and providing reviews, that deal with the scientific and technical disciplines applied primarily to the distilling of high quality potable alcoholic beverages. Biology/microbiology, chemistry/biochemistry, molecular biology, physics and engineering.

Acceptable research and review areas include those related to the brewing, fermentation distilling, maturation and distribution of quality distilled spirits and to their associated raw materials and by-products. Fermentation coverage to include multiple parallel and solid-state fermentation and koji production. Non-distilled higher alcoholic strength foods and beverages also would fit within the purview of the journal’s mission directives, including saké for example.

As a technical discipline, theoretical and mathematical modeling papers would also apply in relation to distillation.

Papers dealing with the sensory aspects of such beverages also belong here. The editor may be contacted prior to the submission of any manuscript that might fall outside the above listed topic descriptions.

Papers will be accepted for review from universities and colleges, research institutes and industrial laboratories, distilleries, raw materials producers, and allied industries supporting the testing and quality control functions of distilling operations.

REACH As a result of its mission, executive, supervisory, and active distillation personnel involved in formulation, production, engineering, quality control, research, water quality, sanitation, raw materials handling, co-products, spent materials operations, stability/packaging of potable alcoholic beverages, and associated industries, including educational establishments, will utilize this scientific resource. Members of the forthcoming Society of Distilling Scientists and Technologists (SDST) will have full access to the journal, though membership in the SDST is not a requirement for either publication subscription or individual article purchase.

CALL FOR PAPERS The call for papers is now on open invite with the lead science editor appointed. Authors should contact Gary Spedding, Ph.D. (gspedding@jdsed.com) in all matters concerning manuscript submission and addressing suitable areas of coverage.

The Journal of Distilling Science (© Artisan Spirit Media) with all rights reserved has been accepted as the official publication of the Society of Distilling Scientists and Technologists (SDST) and is under the editorship of an independent review body appointed and staffed by the founding members of the SDST. It is devoted to the advancement and publication of basic and applied knowledge related primarily to the production of potable alcoholic beverages.

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7

ORIGINAL PAPER

Applications of the Partial Molar Volume Concept in Whisky and Gin Dilutions with Water Franklin M. Chen1*, Nada Abdi1, and Nolan Torres1 1 University of Wisconsin-Green Bay, 2420 Nicolet Drive, Green Bay, WI 54311

KEYWORDS alcohol by volume (A v/v) alcohol by weight (A w/w) partial molar volume Additivity Theorem alcohol strength dilution single shot or multi-shot gin flavor dilution Grain Neutral Spirit (GNS)

The meaning of the partial molar volume, the analytical methods for finding the partial molar volumes and the additivity theorem of the partial molar volumes are explained. The additivity theorem of the partial molar volume is used for whisky alcohol strength dilutions and for single shot or multi-shots for gin in both flavor dilution with GNS and alcohol strength dilution.

INTRODUCTION

Whisky, rum, gin, and other spirits are projected to contribute up to $507,465 million in global revenue in RECEIVED: April 29, 2021 2021, and the market ACCEPTED: June 6, 2021 is expected to grow * CORRESPONDING AUTHOR: annually by 5.49%2 Franklin M. Chen [1]. Bourbon and othE-MAIL: chenf@uwgb.edu er whiskies are made © 2021 BY THE SOCIETY OF from the aging of barDISTILLING SCIENTISTS AND TECHNOLOGISTS rel-filled grain alcohol which is often 100-150 proof3 (50-75% ABV (Alcohol by Volume)).

The final bottled spirit products are often 76-90 US degrees of proof (38-45% ABV). For a spirit sensory test, the spirit is then served at bottle strength or often diluted to around 20% ABV. In making gins [2], whether it is made via a single-shot, or multi-shot process, the botanical compounds, extracted via maceration and distillation or via vapor infusion through a gin basket, may first be mixed with a large amount of neutral spirit approximately at 96% with water then added to attain a final alcohol content of 40% ABV (gins maybe bottled at between 37.5 and up to 55% ABV). In all three examples, barrel to whisky,

whisky diluted to samples for sensory tests, or in the process of making gins, ethanol is mixed with water to obtain a desired final ABV. In all those processes, the questions pertaining to the distillery are: (1) How much water should be added for dilution to obtain a desired ABV? (2) What is the final product volume?

(3) If a fixed amount of water and neutral spirit of 96% ABV are mixed, what is the final ABV, and how much water should the distillery add to obtain the final and accurate 40% ABV? Both the U.S. Gauging Manual Method [3] and Travagli’s method [4-5] are the available math-oriented methods used in the distilling industry. In this paper, we offer another approach based on the partial molar volume concept to solve those problems.

Partial Molar Volume is a thermodynamic definition [6]. In a two-component system (e.g., ethanol and water) at constant temperature T and pressure P, the partial molar volume of water (component 1) is usually denoted as4

[Equation 1]

Partial molar volume is additive. In a two-component system (e.g., water and ethanol) at constant temperature T and pressure p, we have

2 According to the Distilled Spirit Council of the United States, in 2020 spirits gained the largest share of the US beverage alcohol market. The 1.3% increase was more than double the average share gain over the past four years. https://www.just-drinks.com/analysis/has-the-spirits-category-emergedstronger-from-covid-19-fight-analysis_id132749.aspx 3 Proof here refers to US proof, which is two times alcohol by volume. A vodka that is 40 percent ABV is 80 proof and one that is 45 percent ABV is 90 proof 4 Throughout the paper, the subscript “1” implies the water phase, while superscript “2” implies the ethanol phase. Thus n1 means number of moles of means the partial molar volume of ethanol. In addition, when mole fraction “x” is referred to, it means the mole fraction of ethanol water, 8

THE JOURNAL OF DISTILLING SCIENCE

VOLUME 1 NUMBER 1

Winter 2021

Applications of the Partial Molar Volume Concept in Whisky and Gin Dilutions with Water

Chen et al.

[Equation 2]

Likewise, in a three-component system (e.g. water, ethanol, and sugar) at constant temperature T and pressure p, we have

[Equation 3]

Equation [3] is exact and its proof can be seen in many physical chemistry textbooks [7]. In the context of this paper we focus only on the water-ethanol system. The partial molar volume is different from the molar volume because of the formation of the hydrogen bonds between the solute and the solvent [8-10].

MATERIALS AND METHODS Ethyl alcohol is 200 U.S. Proof from Pharmco (Cottrellville, MI). Water is a distilled water prepared from the UW-Green Bay double distilled faFIGURE 1 Finding the Partial Molar Volume of the Solute by the Graphical cility. The density meter used is a DMA 4500 DensiMethod [11]. ty Meter, Anton Paar. (Ashland, Virginia) To produce a spreadsheet for data analysis, it is usual to prepare approximately 10 binary mixtures calculation of the partial molar volumes are made using the of ethanol and water gravimetrically, with ethanol weight following procedures: percentage ranging from 0% to 100% with a 5% interval.

(1) GRAPHICAL APPROACH The density for each mixture is then determined by a density meter at 20 °C. The data tables published by the legal We will start with a binary solution of n1 moles of the metrology organization known as the OIML tables [10] are solvent (water), and n2 moles of the solute (ethanol), and consulted for data analysis. Once spreadsheets are prepared, the volume of the solution V. If we are interested in find-

FIGURE 2 Density data from the OIML table (blue) and from UW-Green Bay (red).

THE JOURNAL OF DISTILLING SCIENCE

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ing the partial molar volume of ethanol, the first step is to plot V/n1 against n2 /n1 . An example of the plot is shown in Figure 1. The partial molar volume of ethanol at a specific n2 , say point A in the graph, is determined by the slope of the curve at point A. Alternately, we can construct an Excel spreadsheet starting with a set of density and weight percentages. Such a data table exists in the Table Vb of the legal metrology tables known as the OIML tables [11].

Figure 2 shows the plot of density of ethanol-water mixtures against ABW (alcohol by weight) from two different data sets: one from the OIML table and the other from the UW-Green Bay. Both data sets agree within 0.5% of error tolerance.

9

Applications of the Partial Molar Volume Concept in Whisky and Gin Dilutions with Water

Chen et al.

TABLE 1 An example set of data illustrating how the partial molar volumes of ethanol are calculated.

Table 1 presents an example set of data values illustrating how the partial molar volumes of water are calculated.

For an example, the cell A3 has ABW=92%, which stands for 92g of ethanol and 8g of water in a 100g of ethanol-water mixture. The solution volume in column E is weight/density, or 100g over the density value in column B. The cell E3 value is obtained as Cell values in column C are obtained by dividing the water mass by the molar mass of water, which is 18.015g/mol.

Cell values in column D are obtained by dividing the ethanol mass by the molar mass of ethanol, which is 46.068g/ mol. The mole fraction of ethanol is then defined as

[Equation 4]

Mole fraction values of ethanol for each ABW are shown in column F of Table 1.

If we are interested in calculating the partial molar volume of ethanol (the second component in our illustration), everything has to be normalized by the number of moles of water (the first component in our illustration). This includes V (total)/n1, and n2/n1 shown in columns G and H of Table 1.

The partial molar volume of ethanol can then be calculated as:

[Equation 5]

Considering the first two data rows (Rows 2 and 3 in Table 1):

in Table 1. The resulting value is shown in cell I2. The cell values in I3 to I8 are calculated using a similar equation.

This is the numerical value shown on cell I3 of Table 1.

Using the same procedures, we can also calculate the partial molar volumes of water which are shown as cell values between J2 and J8.

The values shown in column K in Table 1 are the additivity tests for the partial molar volumes of water and ethanol using the procedures seen through the application of equations 5 and 6. For the additivity test, it implies equation 2 must be strictly followed. The values in column K should exactly match the values in column E in Table 1. This is indeed the case, except we have an outlier, K8 with a value of 132.5779 mL while the value of E8 is 121.1094 mL. This is because the spreadsheet is truncated on row 8. In this limited set of data, we are not able to carry out ΔVtotal and Δn2 calculations for Equations 5 and 6 on Row 8 and beyond.

This problem can, however, be overcome by plotting partial molar volumes of either ethanol (column I) or water (column J) against the mole fraction of ethanol (column D). This is shown in Figure 3, plotting partial molar volume of ethanol and water against the mole fraction of ethanol; and Table 2 which expresses the regression equations of the partial molar volumes of both ethanol and water as a function of ethanol mole fraction.

Regression equations are obtained from the two plots of partial molar volumes shown in Figure 3 and Table 2: TABLE 2 Regression Equation Expressions of the Partial Molar Volumes of Ethanol and Water as a Function of Mole Fraction of Ethanol from Table 1.

PARTIAL MOLAR VOLUME AS A FUNCTION OF ETHANOL MOLE FRACTION (x)

Water + 11.076 x + 13.414 1 = − 10.57 x

0.9971

Ethanol 2 = − 2.1002 x + 4.8896 x +55.611 2

0.9976

2

[Equation 6]

Where ΔVtotal is the difference of two cell values G3 and G2, and Δn2 is the difference of two cell values H3 and H2 10

R2

THE JOURNAL OF DISTILLING SCIENCE

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Applications of the Partial Molar Volume Concept in Whisky and Gin Dilutions with Water

Chen et al.

In row 8 of Table 1, the mole fraction of ethanol is 0.7239. Using the two regression equations from the plots, we have the new partial molar volumes of ethanol and water: 58.05 and 15.89 mL/mol. The additivity test would be (0.7205) (15.89) +(1.889) (58.05) =121.105 mL/mol, which differs from 121.109 mL/mol (cell E8) within 0.003%.

Note that the example we provide in Table 1 has a very limited data set with mole fractions of ethanol ranging only from 0.7239 to 0.8390. The OIML table provides us alcohol density data with ethanol ranging from FIGURE 3 Plotting of Partial Molar Volumes of Ethanol and Water as a Function of Mole 0% to 100% with 0.1 ABW incremenFraction of Ethanol from Table 1. tal intervals. Regression formulas for finding partial molar volumes of ethanol and water are useful. The regression from Equation 1, we have equations5 for ethanol and water from the OIML table are:

[Equation 7]

In solving all the distillery and the brewery problems, we will all use Equations 2, 4, and 7. Equation 2 is simply the additivity property of the partial molar volume. Equation 4 allows us to find the mole fraction of the ethanol so that we can use Equation 7 to calculate the partial molar volumes of both ethanol and water.

All data presented in the OIML tables were obtained at 20 °C. For temperatures other than 20 °C, such as 60 °F (15.56 °C) which is used in the U.S, then the investigators must generate zdata tables similar to the OIML table which can be a formidable task. The U.S. Gauging Manual published in 1918 [3] presents an extensive data set on specific gravity of ethanol-water mixtures at 60 °F. Since specific gravity and density values are related, in the absence of any new data source, this latter reference can be used for spirit dilution practices in both the brewing and distilling industries [5, 13].

[Equation 8]

In our studies, when δn1= 0.06 mol, and δn2= 0.02 mol, the partial molar volumes obtained from Equation 8 agree with data calculated from Equation 7 within 4%. The agreements can be improved if we tweak both the δn1 and the δn2 values.

RESULTS AND DISCUSSION (1) PARTIAL MOLAR VOLUMES ARE DIFFERENT FROM THE MOLAR VOLUMES

There are a few comments to be made regarding partial molar volumes. First, the partial molar volumes are functions of compositions. As the composition changes, so do partial molar volumes. Second, the partial molar volumes of ethanol are close to, but not the same as, the actual molar

(3) SPOT-CHECKING APPROACH

volume of ethanol which is

Our lab has also adopted a technique used by Trandum et al. [14] which can be used to find partial molar volumes at a specific molecular composition without establishing an extensive data table. This is illustrated in the following: According to the definition of the partial molar volume

In fact, all the partial molar volumes calculated for ethanol range from 52.59 mL/mol to 58.37 mL/mol and are lower than the molar volume of the ethanol. Similarly, the partial molar volumes of water ranging from 13.82 to

5 Supplemental Excel Spreadsheet -1 THE JOURNAL OF DISTILLING SCIENCE

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Applications of the Partial Molar Volume Concept in Whisky and Gin Dilutions with Water

18.05 mL/mol are different from the molar volume of water which is 18.05 mL/mol. The differences between the partial molar volumes and the molar volumes of both water and ethanol are the origins of the volume contractions that are also composition dependent. The origin of the difference between the partial molar volume and the molar volume is the hydrogen bond formation between the ethanol and water [8-10].

The difference between the molar volume and partial molar volume is relevant for the distillery industry and is a well known volume-contraction phenomena where ethanol and water are mixed. We shall explore this volume contraction phenomenon in the following application examples.

(2) APPLICATIONS OF THE PARTIAL MOLAR VOLUMES ON ALCOHOL DILUTIONS IN WHISKY

Bourbon and other whiskies are made by aging grain alcohol at strengths ranging from 100-125 proof (50-62.5% ABV) in charred or toasted oak barrels [15]. The Alcohol and Tobacco Tax and Trade Bureau (TTB) specifies that the alcohol strength in the barrel is 125 proof (62.5% ABV) or less [16]. The spirits undergo reduction in strength with water prior to bottling. The final strength of the bottled products is often found in the 76-90 proof (38-45% ABV) range, in many cases 40% ABV [15, 16]. For a spirit sensory test, the spirit is then often served undiluted (bottle strength) or often diluted to around 20% ABV (20-23%) depending on the investigators [17-20].

For this presentation, we will work on three dilution problems: 65% ABV to 40% ABV, 40% ABV to 23% ABV, and lastly 65% ABV to 23% ABV. We are interested in answering the following questions: (1) How much water (mass and volume) are required for each stage of the dilution? (2) What are the final solution volumes for each stage of the dilution? (3) What is the volume contraction for each stage of the dilution?

It is noted that the mathematical procedures for diluting 40% ABV to 23% ABV and for 65% ABV to 23% ABV are the same as those for 65% ABV to 40%. Therefore, we will just simply explain the dilution for 65% ABV to 40% ABV in the details here. We then set up an Excel spreadsheet and use the algorithm in the sheet to find the answers for the other two dilutions.

Mathematical Procedures for 65% ABV to 40% ABV Dilution

Chen et al.

of ethanol in 65% ABV is calculated as follows:

[Equation 9]

Note that the ethanol mass is conserved which implies this mass does not change after the dilution.

For a 40% ABV, the density is 0.94805 g/mL (OIML, Table IVa) and is equivalent to 33.3 % ABW (OIML, Table IVb).

The total mass of the final diluted solution is calculated as:

[Equation 10]

The water mass of the final solution is

[Equation 11]

The number of moles of water (n1 ) and ethanol (n2 ) are then calculated as follows:

[Equation 12]

Note that the molar masses of water and ethanol are 18.015 g/mol and 46.07 g/mol respectively.

The mole fraction (x) of ethanol is calculated as follows:

[Equation 13]

Using Equation 7, the partial molar volumes of ethanol and water are then calculated. The answers are:

[Equation 14]

Using the data generated from Eqs. 12-14, the final volume as calculated from the additivity theorem of partial molar volumes in Equation 2 is then

[Equation 15]

To calculate the mass of water needed to be added to achieve a final 162.1886 mL 40% ABV, we need to find the difference of the water masses before and after the dilution.

This is calculated as follows:

The procedures require the use of Tables IVa and IVb of the OIML data [10]. Let us start with 100 mL of 65% ABV which is equivalent to 57.15% ABW (OIML, Table IVb) and has a density of 0.89765 g/mL (OIML, Table IVa). The mass

12

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[Equation 16]

The density of water at 20 °C is 0.99820 g/mL. The volume VOLUME 1 NUMBER 1

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Applications of the Partial Molar Volume Concept in Whisky and Gin Dilutions with Water

Chen et al.

TABLE 3 Summary of Whisky Dilution—Comparison of the Results between This Work and Travagli’s Method.

FINAL VOLUME (mL)

VOLUME (mL) OF WATER ADDED FOR DILUTION

VOLUME CONTRACTION

INITIAL VOLUME (mL)

THIS WORK

TRAVAGLI METHOD [4]

THIS WORK

TRAVAGLI METHOD [4]

THIS WORK

TRAVAGLI METHOD [4]

65%ABV to 40% ABV

100

162.19

162.5

64.21

64.29

2.02

1.79

40%ABV to 23% ABV

100

173.01

173.90

73.36

73.93

0.35

0.03

65%ABV to 23% ABV

100

281.14

282.60

183.62

184.44

2.48

1.84

DILUTION

of water to be added for this dilution is then [Equation 17]

The volume contraction in this dilution is

[Equation 18]

This lengthy mathematical manipulation can be streamlined with an Excel spreadsheet. Such a spreadsheet is provided as a supplemental material6 in this paper.

Table 3 provides the summary of the work that includes 65% ABV to 40% ABV, 40% ABV to 23% ABV and 65% ABV to 23 % ABV. Included in the table are the mass and volume of the water to be added, the final solution volume and the extent of volume contraction. Also included are the results obtained from Travagli’s method of solving ethanol solution dilutions [4].

The agreement between this partial molar volume-based dilution method and the Travagli method [4] are within 0.5% in final volume, and 0.7% in the volume to be added.

The trend of volume contraction of various dilution processes between the two algorithms is also in agreement with each other. It is therefore concluded that both methods are valid for the applications for distillery dilution applications.

Also, it is noted that volume contraction depends both on the starting ABV and the difference between starting ABV and final ABV. The contraction is the largest for 65% ABV to 23% ABV (2.48 mL) and the smallest for 40% ABV to 23% ABV (0.35 mL). This is explored further below.

(3) APPLICATIONS OF THE PARTIAL MOLAR VOLUMES ON GIN MULTI-SHOT FLAVOR DILUTION AND ALCOHOL DILUTION

Distilled gin production [2], does not require aging in charred or toasted barrels, though some innovative

producers do this. A gin product must contain juniper. A distiller will also select other materials (botanicals) to give the final product additional flavor and character. These botanicals are used in carefully determined quantities, so that they complement the juniper. Organic compounds in juniper and the supporting botanicals (terpenes) provide the key flavor components through distillation, generally either via maceration or vapor infusion. Distillation tools and techniques include pot stills, continuous stills, low-pressure vacuum stills, and rotary evaporation [21-24] The botanical loads (in weight per unit volume) influence how a distiller may choose to reduce the concentration of the terpenes in the gin by mixing in “shots” of neutral spirits at ~ 192-194 proof (96.0%-96.2% ABV), in known quantities. These quantities are called liquid alcohol liters (LALs) or proof gallons. If the distiller does not do this, then the product is a single shot gin. If the distiller chooses to do this, then the product is called a multi-shot gin. Increasing the amounts of botanicals in the distillation operation, and then diluting the terpenes and other flavor volatiles extracted via alcohol addition, and then diluting to bottle strength, increases the amount of final gin to be bottled. The number of shots the distiller chooses is often a risk management response to ‘louching’.7 The final bottle strength of gin products, taken globally, is often found in the 75-110 proof (37.5% - 55% ABV) range. That said, there are some products in the 115 proof (57.7% ABV) and above range which are called Navy Strength gins. These too, may be single shot or multi-shot [25].

For the purpose of the mathematical procedure in this problem, we will initially set up blending 100 mL of gin (hypothetically dealing with an extreme 25 times multishot botanical load) with 2500 mL of GNS with 96% ABV. We would like to ask the following questions: (1) What is the final volume of the mixture and the volume contraction due to such a mixing? (2) What is the resulting ABV after

6 Supplemental Excel spreadsheet -2 7 Louching means the forming of colloidal particles in the ethanol-water mixture due to the presence of botanical compounds.

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the mixing? (3) How much water to be added to the mixture to reach a 40% ABV, and finally what are the volume contractions on this step?

In order to find the final volume using the additivity theorem (Eq.2] of the partial molar volumes, we need to find the number of moles of water and ethanol. 96% ABV is equivalent to 93.84% ABW (OIML, Table IVb ) and has a density of 0.80742 g/mL (OIML, Table Iva). The ethanol mass of the mixture can be calculated as:

[Equation 19]

Chen et al.

Now, we invoke Eq.7 to calculate partial molar volume of both water and ethanol, [Equation 23]

The final volume is then calculated as:

The volume contraction of adding 2500 mL of 96% ABV to 100 mL of the original volume of the distilled botanical compounds is then,

The water mass is calculated as follows:

[Equation 20]

Moles of ethanol (n2 ) and water (n1 ) are then calculated as,

[Equation 24]

[Equation 25]

Percentage of volume contraction for stage-1 (flavor strength dilution) is calculated as follows:

[Equation 26]

The ABW of the final mixture is calculated as ethanol mass divided by the total mass, which is

[Equation 21]

Mole fraction of ethanol, x, is calculated as,

[Equation 22].

[Equation 27]

This is equivalent to 89.42% ABW. The OIML Table IIIb indicates that 89.42% ABW is equivalent to 92.84% ABV. This is the answer to the part 1 question: For 1:25 addition of botanical dilution ethanol with a concentration of 96% ABV, the final volume is 2573.9 mL with 92.84% ABV.

TABLE 48 Summary of Single Shot and Multi-Shot Gin Processes Which Include both Flavor- dilution with GNS (96% ABV) and Alcohol Strength Dilution with Water. Volume unit is in mL. The volume of the starting flavor stock is 100 mL. The final alcohol strength is 40% ABV. VOLUME (mL) OF WATER to be added for alcohol strength dilution

FINAL 40% ABV VOLUME (mL) before bottling

VOLUME CONTRACTION in final stage alcohol strength dilution (mL, %)

VOLUME RATIO Flavor stock to GNS

ABV after flavor neutralization

VOLUME (mL) after initial flavor dilution

VOLUME CONTRACTION (mL, %) in Stage-1 (flavor dilution)

1:25

92.84

2573.9

26.1 (1.0%)

3560.9

5974

160.8 (2.6%)

1:20

92.06

2076.8

23.2 (1.1%)

2828.5

4779

126.7 (2.6%)

1:15

90.79

1580.3

19.7 (1.2%)

2098.1

3587

91.5 (2.5%)

1:10

88.33

1084.4

15.6 (1.4%)

1367.3

2394

57.4 (2.3%)

1:5

82.19

590.7

9.3 (1.6%)

647.9

1214

24.9 (2.0%)

1:2

66.46

292.9

7.1 (2.4%)

199.6

487

5.9 (1.2%)

1:1

49.50

193.9

6.1 (3.1%)

47.0

240

1.0 (0.4%)

8 Supplemental Excel spreadsheet-3 14

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In the second part, the questions to be addressed are: (1) How much volume of pure water is needed to be added to dilute 2573.9 mL with 92.84% ABV to 40% ABV? (2) What is the final volume of the finished ready to bottle gin? (3) What is the volume contraction on the post-stage dilution?

These questions are the same as those discussed in the whisky dilution problems. The calculations are provided in the Excel spreadsheet as a supplemental material. The answers are: (1) 3560.9 mL of water to be added, and (2) The final volume is 5962.6 mL. The extent of volume contraction is 160.8 mL. Table 4 summarizes the results of both single shot and multi-shot processes which include (1) What are the ABV values after GNS initial dilution of the flavor? (2) What are the volumes after GNS initial dilution of the flavor? (3) How much water is needed to bring the alcohol ABV to 40% ABV for each process? (4) How much volume of 40% ABV is required for a subsequent bottling? (5) What are the volume contractions for each scenario? For the examples shown volumes are in mL but may be scaled up to production volumes in liters, hectoliters etc.

The results indicate that multi-shot gin processes can yield more bottles of gin than the single-shot gin process.

Also, in the multi-shot process, the operators may encounter more volume contraction issues in multi-shot versus the single-shot process9, especially at stage two with the alcohol strength dilution with water. Interestingly, the multishot process sees less percentage of volume contraction than the single-shot situation at stage one when the flavor is diluted with 96% ABV of alcohol.

Chen et al.

Thompson of Still Magic Pty Limited, (South Wales, Australia) ; and Mr. Tony Aiken, Brewing and Distilling Analytical Services (Lexington, KY) in providing information regarding the single shot versus the emerging multi-shot gin manufacturing processes; to Professor Valter Travagli of University of Siena, ITA for useful discussions of his method for alcohol dilution.

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

ORCID

Franklin M. Chen https://orcid.org/0000-0003-4750-9472 REFERENCES [1] Spirit North America. Statisa: The Statistics Portal, http://

www.statista.com/outlook/10020000/104/spirits/northamerica (accessed, April 22, 2021)

[2] Buglass, A.J.; McKay, M.; Lee, C.G. Distilled Spirits. In

Handbook of Alcoholic Beverages, Technical, Analytical and Nutritional Aspects, edited by Alan J. Buglass, John Wiley and Sons, Chapter 3, 2011, 457-533.

[3] Bulletins of Bureau of Standards Respective Volumes of

Alcohol and Water and The Specific Gravity in Both Air and Vacuum of Spiritus Liquors. 1918, 9, 327-474.

[4] Travagli, V. The Alcohol Dilution https://www.scribd.com/

doc/70865498/Alcohol-Dilution (accessed, April 16, 2021)

[5] Spedding, G.; Weygandt, A.; Linske, M. Alcohol Dilution

Practices for Distillers, Artisan Spirit, Spring 2016; 65-70.

[6] Partial Molar Property https://en.wikipedia.org/wiki/

Partial_molar_property (accessed May 26, 2021)

[7] Kloz, I.M.; Rosenberg, R.M. Chemical Thermodynamics,

CONCLUSION In all three examples discussed in this paper — barrel-to-whisky, whisky diluted to prepare samples for sensory testing, or in the process of making gins — ethanol is mixed with water to obtain a desired final ABV. Thus, the additivity theorem of the partial molar volumes can be successfully applied to both whisky dilution, gin multi-shot flavor programs, and alcohol strength dilution processes. It also demonstrates that in alcohol dilution, volume contraction occurs. Because of volume contraction, one cannot rely on the conventional additive algebra process, but such distillation issues need to call upon more sophisticated math equations such as the use of the partial molar volume concept.

The Benjamin/Cumming Company, Inc., Menlo Park, CA, 1986, 262-263.

[8] Teresawa, S.; Itsuki, H.; Arkawa, B. Contribution of

Hydrogen Bonds to the Partial Molar Volumes of Nonionic Solutes in Water, The Journal of Phys. Chem. 1975, 79, 2345-2351.

[9] Fishman, E.; Drickmar, H.G. Effect of Pressure on the

Frequency of O-H Band in Butanol Solutions J Chem Phys.

1956, 24, 548-553.

[10] Coccia, A.; Indovina, P.L.; Poto, F.; Viti, V. PMR Studies on

the Structure of Water-Ethanol Mixtures, Chem Phy. 1975, 7, 30-40.

[11] International Alcoholometric Tables, published by

International Organization of Legal Metrology, Paris, France. www.oiml.org/en/files/pdf_r/r022-e75.pdf/view (Accessed, April 16, 2021)

[12] Kloz, I.M.; Rosenberg, R.M. Chemical Thermodynamics,

ACKNOWLEDGMENTS

We express gratitude to Mr. Jamie Baxter of Craft Distilling Services, LTD ( Blaby, Leicester UK); Mr. Marcel

The Benjamin/Cumming Company, Inc., Menlo Park, CA, 1986, 368-373.

9 The percentages of volume shrinkage in Stage-1 are in the 1-3%; the higher percentage for the single-shot process.

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[13] Spedding, G. Measuring Alcohol-Three Ways to Proof

the Product, in Worldwide Distilled Spirit Conference Proceeding, edited by F. Jack, D. Dabrowska, S. Davies, M. Garden D. Maskell, D. Murray, Context Product Ltd., Leicestershire, UK. 2017.

[14] Trandum, C.; Westh, P.; Haynes, C.A.; Koga, Y.

Intermolecular Interactions in tert-Butyl Alcohol-Dimethyl Sulfoxide-H2O: Chemical Potentials, Partial Molar Entropies and Volumes, 1998, J. Phys. Chem. B., 102, 51825195.

[15] Baldwin, S.; Andreasen, A.A. Congener Development in

Bourbon Whisky Matured at Various Proofs for Twelve Years, Journal of the AOAC, 1974, 57, 940-950.

[16] TTB Alcohol and Tobacco Tax and Trade Bureau Class and

Type Designation. https://www.ttb.gov/images/pdfs/spirits_ bam/chapter4.pdf. (accessed, October 25, 2021)

[17] Ickes, C., Cadwallader, K. Effect of Ethanol on Flavor

of Rum, Food Science & Nutrition, 2017, 6, 912-924, doi:10.1002/fsn3.62.

[18] Piggott, J.R.; Jardine, S.P. Descriptive Sensory Analysis of

Whisky Flavour, J. Inst. Brew. 1979, 85, 82-85.

[19] Frances, J. Development of Guidelines for the Preparation

and Handling of the Sensory Samples in the Scotch Whisky Industry, J. Inst. Brew. 2003, 109, 114-119.

[20] Wang, Z.; Ickes, C.M.; Cadwallader, K.R. Influence of

Ethanol on Flavor Perception in Distilled Spirit, in ACS Symposium Series 1321, Sex, Smoke, and Spirits: The Role of Chemistry, 2019, edited by Guthrie, B.; Beauchamp, J.D.; Buettner, A.; Toth, S.; and Qian, M.C.; American Chemical Society.

16

Chen et al.

[21] Aumatell, M.R. Gin: production and sensory properties.;

in Alcoholic Beverages, Sensory evaluation and consumer research, edited by J. Piggott., 2012, Woodhead Publishing, 267-279.

[22] REGULATION (EC) No 110/2008 OF THE EUROPEAN

PARLIAMENT AND OF THE COUNCIL of 15 January 2008 on the definition, description, presentation, labelling and the protection of geographical indications of spirit drinks and repealing Council Regulation (EEC) No 1576/89, https://eur-lex.europa.eu/legal-content/EN/TXT/ PDF/?uri=CELEX:32008R0110&from=EN

[23] Single-shot vs. Multi-shot, Gin Magazine, August 16, 2019,

https://gin-mag.com/2019/08/16/single-shot-vs-multi-shot/

[24] Making Gin in a Still with a Thumper Keg | In The Welsh

Wind Distillery Making Gin in a Still with a Thumper Keg | In The Welsh Wind Distillery - YouTube. https://www. youtube.com/watch?v=FOZEHpukXqA&t=16s

[25] Thompson, M. personal conversation, April 27, 2021.

SUPPLEMENTAL MATERIALS

Three Excel spreadsheets are provided to allow interested readers to explore partial molar volumes calculations, whisky alcohol strength dilution and single shot versus multi-shot gin process calculations: http://artisanspiritmag.com/journalds/jds_v1n1/

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ORIGINAL PAPER

Lignin-derived phenolic compounds in cachaça aged in barrels from tropical wood species

Mariana C. Castro1, Giovanni C. Silvello1, and André R. Alcarde1* 1 Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, Av. Pádua Dias 11, CP 9, 13418-900, Piracicaba, SP, Brazil

This study investigated the lignin-derived phenolic compounds in cachaça aged in barrels made from tropical woods. Cachaça was aged for 36 months in toasted new wooden barrels made from amburana (Amburana cearensis), cabreúva (Myrocarpus frondosus) and castanheira (Bertholletia excelsa). New barrels made from European oak (Quercus petraea) and American oak (Quercus alba) were also employed.

Cinnamic aldehydes, benzoic aldehydes and benzoic acids were analysed at the end of the ageing time. A significant effect of wood species was observed on all the studied phenolic compounds. Syringaldehyde and the benzoic acids were the main low-molecular-weight compounds in aged cachaça. All the phenolic families under study were at higher concentrations in cachaça aged in amburana barrels. Cachaça aged in castanheira barrels displayed the highest ratio of gallic acid to vanillin, whereas that aged in cabreúva barrels exhibited the highest ratio of syringaldehyde to vanillin. Cachaça aged in barrels made from amburana had the highest sum of lignin-derived phenolic compounds, followed by cachaça matured in American oak and cabreúva barrels. Amburana showed a great potential to provide lignin-derived phenolic compounds to cachaça during ageing. Cachaça aged in oak barrels exhibited the highest contents of ethyl acetate and acetic acid, whereas the samples aged in European and American oak and amburana barrels reached the highest total score in sensory evaluation. The ageing process in new tropical wood barrels, singly or complementarily to oak, enhanced the flavour complexity of aged cachaça and broadened and diversified its taste and aroma profiles.

INTRODUCTION Cachaça is a typical and exclusive sugar cane spirit of Brazil, containing 38–48% ethanol by volume, obtained by the distillation of fermented sugarcane juice [1]. Although ageing is not mandatory for this distilled spirit, the ageing practices conducted on sugar cane spirit in Brazil are of fundamental importance, inasmuch as ageing in wooden barrels (usually from one to three years) improves cachaça sensory profile.

The effects of the ageing process are primarily influenced by the wood species used for making the barrel [2]. American oak (Quercus alba) and European oak (Quercus petraea) are the most commonly used species to make barrels for ageing distillates [3]. Quercus robur and Quercus petraea are the European oak species most commonly used in tight cooperage [4].

The availability and extractability of ellagitannins, phenolic and volatile compounds, as well as the water THE JOURNAL OF DISTILLING SCIENCE

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KEYWORDS sugarcane spirit ageing tropical woods lignin phenolic compounds

RECEIVED: June 15, 2021 ACCEPTED: August 12, 2021 * CORRESPONDING AUTHOR: André R. Alcarde E-MAIL: andre.alcarde@usp.br © 2021 BY THE SOCIETY OF DISTILLING SCIENTISTS AND TECHNOLOGISTS

tightness of oak tyloses are the main features that make certain oak species the favourite wood type to produce barrels for wine and distilled beverages [5]. However, tropical woods can be a viable option for ageing cachaça in Brazil, where a vast, diverse flora grows.

Some species that have been reported as showing potential for this purpose such as amendoim (Pterogyne nitens), araruva (Centrolobium tomentosum), bálsamo (Myroxylon peruiferum), cabreúva (Myrocarpus frondosus), amburana (Amburana cearensis), grápia (Apuleia leiocarpa), jatobá (Hymenaeae carbouril), ipê roxo (Tabebuia heptaphylla), jequitibá (Cariniana estrellensis), jequitibá rosa (Cariniana legalis), pereira (Platycyamus regnellii) and peroba (Paratecoma peroba) [1,6–8].

Low molecular weight phenolic compounds extracted from wood are incorporated to distillates during ageing [9,10]. Lignin transformations that occur 17

Lignin-derived phenolic compounds in cachaça aged in barrels from tropical wood species

Castro et al.

during the ageing process are among the most important factors that influence the quality of distilled beverages. The level of ageing of Scotch whisky, brandies, grape marc distillate and wine distillates can be designated based on the content of phenolic compounds derived from lignin as well as on their profile [11–13].

Lignin macromolecules have branches of coniferyl (guaiacyl-like compounds) and sinapyl (syringyl-like compounds) alcohols. The former gives rise to coniferaldehyde, which is converted to vanillin and, in turn, oxidized to vanillic acid.

The latter generates sinapaldehyde, which is transformed into syringaldehyde and later oxidized to syringic acid [14] (Figure 1).

This study aimed to evaluate the phenolic compounds derived from the degradation of lignin in cachaça aged for 36 months in new barrels made from three tropical wood species (Amburana cearensis – amburana, Myrocarpus frondosus – cabreúva and Bertholletia excelsa – castanheira) and two oak species (Quercus petraea – European oak and Quercus alba – American oak).

Although some tropical species have already been studied FIGURE 1 Transformations of lignin-derived aromatic compounds during the ageing process of distilled spirits in new wooden barrels. for cachaça ageing, including cabreúva and amburana, the novelty of the present work described [15]. After extraction in a Maqtron M-730 stainlies in the fact that the ageing process was carried out in new barrels from tropical wood less steel presser (Joaçaba, SC, Brazil) the sugarcane juice from variety SP 81-3250 was diluted to 18 °Bx using potaspecies compared to oak species. ble water. Fermentation was carried out in 1,500-L stainless steel tanks (Alambiques Santa Efigênia, Itaverava, MG, Brazil) for 24 h at 30 °C using Saccharomyces cerevisiae MATERIALS AND METHODS The sugarcane spirit used in this study was produced in strain CA-11 (LNF Latino Americana, Bento Gonçalves, 2017 in the distillery of the Department of Agri-Food In- RS, Brazil). Fermentation was considered finished when dustry, Foods and Nutrition, Escola Superior de Agricultura all fermentable sugars were consumed by yeast (Brix sta“Luiz de Queiroz”, Universidade de São Paulo, as previously ble). The fermented must contain 10.1% ethanol by volume.

18

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Castro et al.

Double distillation was carried out in a 1,000L copper pot still (Alambiques Santa Efigênia, Itaverava, MG, Brazil) [16]. The first distillation resulted in a distillate known as phlegma, containing 29.9% ethanol by volume. The phlegma was subjected to the second distillation, in which the “heads” and “tails” fractions were withdrawn. The “heart” fraction characterises the double-distilled cachaça, containing 63.5% ethanol by volume.

The wooden barrels (225L) used for cachaça ageing were made from tropical species: amburana (Amburana cearensis), cabreúva (Myrocarpus frondosus) and castanheira (Bertholletia excelsa), medium toasted (240 °C for 12 min) (Tanoaria Mesacaza, Monte Belo do Sul, RS, Brazil). The oak barrels (225 L) were made from European oak (Quercus petraea) and American oak (Quercus alba), medium toasted (240 °C for 12 min) (Tonelleries de Bourgogne, Meursault, France). These tropical species were chosen because they are the most used wood species in Brazil for cachaça ageing and medium toasting is usually applied to the internal charring of the barrels.

Double-distilled sugarcane spirit was aged for 36 months at room temperature (22 ± 3 °C), 55 ± 10% relative humidity and protected from vibrations. Samples of 25 mL were collected at the same level from the centre of the barrels at the end of the ageing period (36 months) for analyses of maturation-related congeners. Non-aged double-distilled cachaça was used as control in this study.

Ageing congeners were analysed using high-performance liquid chromatography (HPLC) in a Shimadzu equipment, model LC-10 AD (Tokyo, Japan), with two Shimadzu LC-20 AD pumps, an ultraviolet (UV)–visible detector Shimadzu SPD-20A, a system controller CBM20A and an automated injection system (20 μL) with gradient elution [1]. The standards employed in this research were vanillin, vanillic acid, syringaldehyde, sinapaldehyde,

syringic acid and coniferaldehyde, all purchased from Sigma-Aldrich (St. Louis, MO, USA), purity > 99 %. To obtain the calibration curves for standard mixtures (Table 1), their concentrations ranged from 0.5 to 30.0 mg/L for phenolic aldehydes and from 0.5 to 20.0 mg/L for phenolic acids.

Analyses using HPLC had two mobile phases composed of water/acetic acid (98/2, by volume) and methanol/water/ acetic acid (70/28/2, by volume) at a flow rate of 1.25 mL/ min. A pre-column Shimadzu VP-ODS (1 cm × 4.6 μm) and a C18 reversed-phase column model Shimpack VPODS (4.6 mm, 25 cm × 5 μm) thermostabilised at 40 °C were used. The UV detector was programmed to operate at variable wavelengths. The samples were filtered through a Millex-HV filter with polyvinylidene difluoride membrane (13 mm diameter, 0.45 μm pore size) prior to analysis.

Aldehydes, esters, methanol, acetic acid and higher alcohols (1-propanol, 2-methyl-1-propanol and 3-methyl-1-butanol) were analysed using gas chromatography with a flame ionisation detector (GC-FID) [1]. Aliquots of 1.0 μL were automatically injected into the chromatographic system (Shimadzu, QP-2010 PLUS, Tokyo, Japan) equipped with a Stabilwax-DA column (crossbond carbowax polyethylene glycol, 30 m × 0.18 mm × 0.18 μm film thickness). The analyses were performed at a 1:20 split ratio. Nitrogen was used as the carrier gas (flow rate of 1.5 mL/min, total flow of 27 mL/min and pressure of 252.4 kPa). The temperatures of both the injector and the detector were set at 240 °C. The oven temperature program was 40 °C for 4 min, followed by an increase to 120 °C at 20 °C/ min, kept for 1 min, and then up to 180 °C at 30 °C/min, and maintained for 4 min.

The determination of the colour intensity was carried out at 420 nm [17], in a Spekol 1300 UV-Vis single beam spectrophotometer (Analytik Jena GmbH, Jena, Germany) using quartz cuvettes.

Sensory assessment of sugar cane spirits was carried out using the Buxbaum TABLE 1 Retention time (RT), limit of detection (LOD) and limit of quantification (LOQ) of agemodel of positive ranking-marker compounds and correlation coefficients (a, b, r2) of the calibration curves for cachaça aged for ing [18,19]. Aged cachaça 36 months in new barrels made from tropical wood and oak. samples and control were diluted to 40% (by volume) AGEING-MARKER RT LOD LOQ a b r2 before sensory analysis.

COMPOUND (min) (mg/L) (mg/L) Statistical analyses were Gallic acid 6.37 0.03 0.10 1821.48 55.92 0.991 performed applying analVanillic acid 24.01 0.04 0.17 1263.18 256.33 0.997 ysis of variance (ANOVA) and the Statistical Analysis Syringic acid 26.62 0.03 0.11 2428.26 -102.89 0.998 System, version 9.3. The Vanillin 27.08 0.03 0.08 3110.11 -87.12 0.998 means of the results obSyringaldehyde 29.15 0.05 0.16 1079.89 341.11 0.997 tained from triplicate analConiferaldehyde 34.83 0.03 0.07 4548.14 146.96 0.997 yses were compared using the Tukey’s test (p ≥ 0.05).

Synapaldehyde 35.85 0.03 0.10 3217.87 102.27 0.996

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Castro et al.

RESULTS AND DISCUSSION PHENOLIC COMPOUNDS

A significant effect of wood species was observed on all the studied phenolic compounds. Syringaldehyde and benzoic acids were the main low molecular weight compounds in aged cachaça (Table 2).

Coniferaldehyde was found at low concentrations in cachaça aged in castanheira and European oak barrels and at the highest contents in the beverage matured in barrels made from amburana and cabreúva. Cachaça aged in castanheira and cabreúva barrels exhibited the lowest concentrations of sinapaldehyde, whereas the one aged in amburana barrels had the highest content of this compound, as well as the highest concentration of both cinnamic aldehydes added together.

The highest content of vanillin was registered in the distilled spirits aged in barrels made from amburana and both oak species. Syringaldehyde was found at the lowest concentration in cachaça aged in castanheira barrels and at the highest content in that matured in amburana barrels.

Cachaça aged in barrels made from American oak exhibited the second highest concentration of syringaldehyde. The beverages aged in barrels made from cabreúva and castanheira displayed the lowest concentration of both benzoic aldehydes added together.

The analysis of vanillic acid revealed the lowest concentrations in cachaça aged in European oak barrels, approximately half of the content found in the one aged in barrels made from all the other wood species. The content of syringic acid in cachaça aged in amburana barrels was the highest, whereas the distilled spirits aged in barrels made from castanheira and European oak exhibited the lowest content of this benzoic acid.

Some studies have reported the predominance of vanillin, syringaldehyde, vanillic acid and syringic acid in cachaça aged in oak barrels [17,20,21]. Cachaça aged in amburana barrels exhibited high contents of sinapaldehyde and vanillic acid. Cabreúva barrels imparted high contents of syringaldehyde and syringic acid to aged cachaça [1,8,17,22,23].

The results of low-molecular lignin-derived phenolic compounds obtained for cachaça aged in European oak barrels (Table 2) are in reasonable agreement with those

TABLE 2 Colour (%T at 420 nm) and content (mg/L) of ageing-marker compounds derived from lignin and some ratios among them in cachaça aged for 36 months in new barrels from tropical wood and oak.

AMBURANA ( Amburana cearensis)

CABREÚVA ( Myrocarpus frondosus)

CASTANHEIRA (Bertholletia excelsa)

EUROPEAN OAK (Quercus petraea)

AMERICAN OAK (Quercus alba)

Coniferaldehyde

< LOD1

4.60±0.13a

5.29±0.12a

1.39±0.05c

1.29±0.03c

2.16±0.05b

Sinapaldehyde

< LOD1

12.15±0.33a

1.66±0.04c

0.95±0.02c

2.37±0.08b

3.71±0.10b

16.75±0.46a

6.95±0.16b

2.33±0.07c

3.66±0.11c

5.87±0.15b

AGEING-MARKER COMPOUND

Cinnamic aldehydes Benzoic aldehydes

Benzoic acids

AGED CACHAÇA

CONTROL (non-aged cachaça)

Total Vanillin

< LOD1

7.60±0.18a

2.16±0.07b

1.93±0.07b

6.14±0.12a

6.12±0.14a

Syringaldehyde

< LOD1

41.27±1.51a

17.36±0.43c

6.20±0.16e

11.81±0.29d

29.95±0.67b

48.87±1.69a

19.52±0.50c

8.13±0.23d

17.95±0.41c

36.07±0.81b

Total Vanillic acid

< LOD1

12.45±0.29a

14.32±0.31a

14.10±0.33a

6.34±0.15b

13.89±0.39a

Syringic acid

< LOD1

33.25±0.60a

8.89±0.21b

4.66±0.11c

6.33±0.11c

10.64±0.33b

45.70±0.89a

23.21±0.52b

18.76±0.44bc

12.67±0.26c

24.53±0.72b

6.60±0.19c

3.23±0.09d

40.61±0.79a

22.82±0.48b

8.00±0.21c

0.87c

1.49c

21.04a

3.72b

1.31c

Total Gallic acid

< LOD1

Gallic acid/Vanillin Syringaldehyde/Vanillin

–

5.43b

8.04a

3.21c

1.92d

4.89b

Guaiacyl-type compounds

–

24.65±0.60a

21.77ab

17.42b

13.77c

22.17a

Syringyl-type compounds

–

86.67±2.44a

27.91cd

11.81e

20.51d

44.30b

99.8±0.1a

74.6±0.7c

62.4±0.6d

81.2±0.8b

73.5±0.8c

74.9±0.7c

Colour LOD: limit of detection

1

Means (x±standard deviation) in rows with different superscript lowercase letters are significantly different (p < 0.05) as analysed by ANOVA and the Tukey’s test.

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Aromatic aldehydes and acids (mg/L)

50 observed in commercial extra old wine distillates: 2.4 mg/L coniferaldehyde, 1.6 mg/L sinapaldehyde, 40 16.7 mg/L vanillin, 27.8 mg/L syringaldehyde, 4.5 mg/L vanillic acid and 8.3 mg/L syringic acid 30 [13]. The following concentrations of the studied phenolics were found in five to six year old commer20 cial grape mark distillates aged in 225L barrels made from European oak: 9.1 mg/L coniferaldehyde, 6.3 10 mg/L sinapaldehyde, 3.8 mg/L vanillin, 8.2 mg/L syringaldehyde, 2.4 vanillic acid and 3.6 mg/L syringic 0 Amburana Cabreúva Castanheira European oak American oak acid. In contrast, for the commer(Quercus alba) (Amburana (Myrocarpus (Bertholletia (Quercus petraea) cial grape mark distillates matured cearensis) frondosus) excelsa) in American oak 225-L barrels for Wood species 6 years, the values were: 2.0 mg/L coniferaldehyde, 4.2 mg/L sinapalCinnamic aldehydes Benzoic aldehydes Benzoic acids dehyde, 3.8 mg/L vanillin, 8.2 mg/L FIGURE 2 Cinnamic aldehydes (coniferaldehyde and sinapaldehyde), benzoic aldehydes (vanillin syringaldehyde, 2.1 vanillic acid and syringaldehyde) and benzoic acids (vanillic and syringic acids) contents in cachaça aged for 36 and 3.1 mg/L syringic acid [12]. months in new barrels made from tropical wood and oak.

All the phenolic families under study were measured at higher concentrations in cachaça aged in ratio of syringaldehyde to vanillin (8.04), whereas in the amburana barrels (Figure 2). Cachaça aged in barrels made distilled spirits aged in amburana and castanheira barrels from American oak showed the second highest contents of this ratio was 5.43 and 3.21, respectively. benzoic aldehydes and benzoic acids, while the beverage In addition, the relationship between benzoic aldehydes matured in cabreúva barrels exhibited the second highest (vanillin and syringaldehyde) and cinnamic aldehydes (cocontents of cinnamic aldehydes. The distilled spirits aged niferaldehyde and sinapaldehyde) allowed differentiating in castanheira barrels had the lowest contents of cinnamic the type of wood (chestnut or oak) used in the ageing proand benzoic aldehydes. Cachaça aged in barrels made from cess of wine brandies [28]. Orujo aged in American oak European oak exhibited the lowest contents of benzoic acbarrels had the highest ratio of benzoic aldehydes to cinids. namic aldehydes (1.95), whereas orujo aged in European The ratio of gallic acid to vanillin is influenced by the oak barrels this ratio was 0.79. Similar values were observed species of wood [24] and a higher ratio may contribute to in orujos aged in barrels made from French oak of different high quality brandy [25]. In the present study, this ratio was origins, ranging from 0.84 to 1.07 [12]. In the present study, significantly higher in cachaça aged in castanheira barrels the relationship between both aldehyde families was higher (21.0) compared to the distilled spirits matured in barrels for cachaça aged in American oak barrels (6.14), as shown made from all the other wood species (Table 2). Cachaça in Table 2. Cachaça aged in cabreúva and amburana barrels aged in European oak barrels exhibited the second highdisplayed the lowest ratio of benzoic aldehydes to cinnamic est gallic acid/vanillin (3.1), while in the distilled beverages aldehydes, 2.81 and 2.92, respectively. In the distilled spirits aged in barrels made from all the other wood species this matured in castanheira and European oak barrels this ratio ratio was near one. was 3.49 and 4.90, respectively.

The ratio of syringaldehyde to vanillin may indicate the Phenolic acids have been considered the main quality level of maturation of aged distillates [24,26], and for most markers for aged wine distillates [13]. In wine distillates of those aged in oak barrels it is near two, ranging from 1.4 aged for more than 20 years, the concentration of benzoic to 2.5 [12,13,27-29]. In this study, this ratio was 1.92 and acids increased continuously, reaching concentrations of 4.89 for cachaça aged in European oak and American oak 8.3 mg/L syringic acid and 4.5 mg/L vanillic acid, totalizing barrels, respectively. Among the tropical woods, cachaça 12.8 mg/L benzoic acids [13]. Similar values of total benzoaged in barrels made from cabreúva exhibited the highest ic acids were found in cachaça aged in barrels made from THE JOURNAL OF DISTILLING SCIENCE

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European oak in the present study (12.67 mg/L), and the highest content of these compounds was found in cachaça aged in amburana barrels, 45.70 mg/L. Cachaça matured in castanheira barrels exhibited 18.76 mg/L benzoic acids, while in that aged in cabreúva and American oak barrels the content of these compounds was near 24 mg/L. The ratio of syringic acid to vanillic acid can be taken into consideration for characterising the age of distillates. Cognacs aged for up to two years in used six-year-old European oak barrels (350L) displayed a prevalence of vanillic acid (syringic acid/vanillic acid from 0.67 to 0.89). Conversely, cognacs aged for 10 to 30 years at the same conditions exhibited higher content of syringic than vanillic acid (syringic acid/vanillic acid between 1.19 and 1.40) [30]. This ratio was 1.50 in commercial grape marc distillates aged for four years in 225L barrels made from European oak [12].

In this study, cachaça aged in American oak, cabreúva and castanheira barrels exhibited higher concentration of vanillic than syringic acid (syringic acid/vanillic acid from 0.33 to 0.77). In cachaça aged in barrels made from European oak, this ratio was near 1.0, while that matured in amburana barrels had a prevalence of syringic over vanillic acid (syringic acid/vanillic acid 2.67).

The highest concentration of syringyl-type compounds was found in cachaça aged in amburana barrels, followed by the beverages matured in barrels made from American oak, cabreúva and European oak. Cachaça aged in castanheira barrels displayed the lowest content of syringyl-type 90

compounds (Figure 3).

Furthermore, cachaça matured in amburana barrels also exhibited the highest concentration of guaiacyl-type compounds, followed by the distilled spirits aged in barrels made from American oak, cabreúva and castanheira.

Cachaça aged in European oak barrels had the lowest content of guaiacyl-type compounds.

Toasted powder extracts from Imburana (Amburana cearensis), oak (Quercus alba) and Balm (Myrocarpus frondosus) were obtained by solvent reflux (1.5 g of powder extracts in 50 mL of 50% (v/v) ethanol/water solution). The extract from Imburana exhibited the highest concentration of syringyl-type compounds compared to powder extracts from the woods balm and oak [31].

Except for cachaça matured in castanheira barrels, the distilled spirits aged in barrels made from all the other wood species showed higher contents of syringyl-type compounds than guaiacyl-type compounds (syringyl-type compounds/guaiacyl-type compounds ranging from 1.28 to 3.52).

A prevalence of syringyl-type compounds over guaiacyl-type compounds was observed in wine distillates aged for up to 50 years (syringyl-type compounds/guaiacyl-type compounds ranging from 1.60 to 2.09) [13]. Similarly, the contents of syringyl-type compounds were higher than those of guaiacyl-type compounds in grape mark distillates aged for 5–6 years in European oak and in American oak barrels, since the ratios of syringyl-type to guaiacyl-type compounds were 1.18 and 1.96, respectively [12].

Aromatic compounds (mg/L)

80

MAJOR VOLATILE COMPOUNDS

70 60 50 40 30 20 10 0 Amburana (Amburana cearensis)

Cabreúva (Myrocarpus frondosus)

Castanheira (Bertholletia excelsa)

European oak (Quercus petraea)

American oak (Quercus alba)

Wood species Guaicyl-type compounds

Syringyl-type compounds

FIGURE 3 Total content of guaiacol-type compounds (coniferaldehyde, vanillin and vanillic acid) and syringol-type compounds (sinapaldehyde, syringaldehyde and syringic acid) in cachaça aged for 36 months in new barrels made from tropical wood and oak.

22

The major volatile compounds in aged cachaça were 3-methyl-1-butanol and isoamyl alcohol (~160 mg/100 mL anhydrous ethanol, AE), followed by acetic acid (Table 3). Isoamyl alcohol was the most abundant higher alcohol present in cachaça [32], approximately 193 mg/100 mL AE. The contents of the two other higher alcohols, 1-propanol and 2-methyl-1-propanol (isobutanol), were similar in all aged spirits (~50 mg/100 mL alcohol). Higher alcohols, such as propanol, isobutanol and isoamyl alcohol, are related to yeast metabolism of sugars and amino acids during ethanolic fermentation [33]. Specific contents

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TABLE 3 Major volatile compounds (mg/100 mL anhydrous ethanol) in cachaça aged for 36 months in new barrels from tropical wood and oak.

AGED CACHAÇA

CONTROL (non-aged cachaça)

AMBURANA ( Amburana cearensis)

CABREÚVA (Myrocarpus frondosus)

CASTANHEIRA (Bertholletia excelsa)

EUROPEAN OAK (Quercus petraea)

AMERICAN OAK ( Quercus alba)

Acetaldehyde

10.31±0.55a

10.13±0.32a

10.30±0.58a

10.37±0.55a

10.38±0.64a

10.47±0.27a

Ethyl acetate

15.51±0.22d

15.59±0.13d

21.81±0.66c

23.51±0.72c

32.72±0.47b

47.22±1.92a

1-Propanol

51.56±2.45a

50.07±4.21a

48.48±3.85a

49.41±2,56a

50.81±2.53a

59.39±2.92a

2-Methyl-1-propanol

55.12±1.91a

54.37±2.73a

47.09±0.94a

49.51±2.66a

45.98±2.65a

56.62±3.54a

AGEING-MARKER COMPOUND

3-Methyl-1-butanol

164.43±21.80a 161.41±19.80a 155.22±19.50a 159.40±17.05a 160.69±15.40a 165.42±14.19a

Acetic acid

60.95±1.96d

70.92±1.95c

93.26±1.51b

51.01±0.99e

120.31±3.17a

110.25±1.86a

Methanol

7.43±0.19a

7.08±0.09a

7.79±0.21a

7.07±0.11a

7.30±0.17a

7.70±0.11a

Higher alcohols

271.11±23.50a 265.85±16.90a 250.79±21.40a 258.32±21.66a 257.48±25.70a 281.43±10.69a

Means (x±standard deviation) in rows with different superscript lowercase letters are significantly different (p < 0.05) as analysed by ANOVA and the Tukey’s test.

and proportions of higher alcohols positively influence the aroma of distillates. Conversely, when present in concentrations higher than 350 mg/100 mL AE, higher alcohols are often associated with poor quality distillates [33]. The contents of higher alcohol in aged cachaça ranged from 257 to 281 mg/100 mL AE. Similarly to what was observed for higher alcohols (1-propanol, 2-methyl-1-propanol and 3-methyl-1-butanol), negligible variations were also found for acetaldehyde (± 1.7% between the control and any of the aged spirits) and methanol (± 4.9% between the control and any of the aged spirits). In contrast, ethyl acetate was present at markedly higher levels (compared to the control) in cachaça aged in oak barrels (Table 3), with a 3-fold average increase for American oak and a 2-fold average increase for European oak. Cachaça aged in cabreúva and castanheira exhibited an average increase over 50% in ethyl acetate content. The content of ethyl acetate in cachaça aged in amburana was

similar to the control. Ethyl-acetate is the most common ester in cachaça [32]. Since the cachaça used as control in this study was produced using double distillation, it contained only 15.5 mg/100 mL AE ethyl-acetate, lower than the average of Brazilian sugar cane spirits (88 mg/100 mL AE) [32]. A significant increase in acetic acid content was observed in cachaça aged in oak barrels (+100% for European oak and +80% for American oak). Although cachaça aged in cabreúva and amburana barrels also had a significant increase in acetic acid content, it was smaller than that found in the cachaça aged in oak barrels, +50% and +16%, respectively. In contrast, cachaça aged in castanheira barrels showed a decrease by 16% in acetic acid content.

SENSORY EVALUATION

The differences in chemical composition of aged cachaça resulted in statistically significant differences (p < 0.05) in the sensory assessment (Table 4). Cachaça aged in

TABLE 4 Sensory assessment of cachaça aged for 36 months in new barrels from tropical wood and oak.

AGED CACHAÇA

CONTROL (non-aged cachaça)

AMBURANA ( Amburana cearensis)

CABREÚVA (Myrocarpus frondosus)

CASTANHEIRA (Bertholletia excelsa)

EUROPEAN OAK (Quercus petraea)

AMERICAN OAK ( Quercus alba)

Colour (max 2 points)

1.5±0.2b

2.0±0.0a

1.6±0.1b

2.0±0.0a

2.0±0.0a

2.0±0.0a

Clearness (max 2 points)

2.0±0.0a

2.0±0.0a

2.0±0.0a

2.0±0.0a

2.0±0.0a

2.0±0.0a

Odour (max 4 points)

2.5±0.7c

3.8±0.1a

2.8±0.4bc

3.2±0.2b

3.5±0.3a

3.6±0.2a

Taste (max 12 points)

7.0±0.5d

11.5±0.4a

8.5±0.4c

9.8±0.5b

11.1±0.3a

10.8±0.4a

Total (max 20 points)

13.0±0.4c

19.3±0.3a

14.9±0.4c

17.0±0.2b

18.6±0.2a

18.3±0.2a

AGEING-MARKER COMPOUND

Means (x±standard deviation) in rows with different superscript lowercase letters are significantly different (p < 0.05) as analysed by ANOVA and the Tukey’s test.

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European and American oak and amburana barrels obtained the highest total score and were characterized as smooth and pleasant in aroma and taste. Cachaça aged in castanheira barrels was also considered pleasant and the taste and smell were acceptable. Cachaça aged in cabreúva barrels received the lowest total score, similar to the control (non-aged cachaça), and the panelists described its aroma and taste as sharp, acid, pungent and burning. The common sensory descriptors associated with the cachaças aged in the different woods species were: European oak (roasted almonds, ripe fruits and spices), American oak (vanilla, coconut and caramel), amburana (vanilla, sweet and spices), castanheira (vanilla, chocolate, caramel and chestnuts), cabreúva (herbaceous, bitter and astringent).

CONCLUSION The results obtained in this study contributed to the knowledge of phenolic composition of cachaça aged in new barrels made from tropical wood species. Amburana showed great potential to provide lignin-derived phenolic compounds to cachaça during the ageing process. Cachaça samples aged in oak and amburana barrels received the highest scores in the sensory assessment. Tropical wood species, singly or complementarily to oak, enhanced flavour complexity of aged cachaça and simultaneously broadened and diversified its taste and aroma profiles.

FUNDING STATEMENT

The present study is part of the project ‘Characterization of the level of maturation of aged sugar cane spirit (cachaça): evaluation of maturation-related phenolic compounds’, Grant 2016/23211-5, funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), São Paulo, SP, Brazil and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) - Finance Code 001, Brasília, DF, Brazil.

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

ORCID IDs

M.C. Castro https://orcid.org/0000-0002-4159-0727 G.C. Silvello https://orcid.org/0000-0002-4672-4800 A.R. Alcarde https://orcid.org/0000-0002-9319-7847 CRediT CONTRIBUTOR

ARA conceived the idea and the design of the work.

MCC performed the analysis. GCS carried out data analysis and interpretation. ARA and MCC drafted the article.

ARA critically revised the article. All authors approved the 24

final version of the manuscript for publication and have agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

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[32] Bortoletto, A.M.; Alcarde, A.R. Assessment of Chemical

Maturation Related Phenolic Compounds in Cachaça Aged in New Oak Barrels. J. Inst. Brew. 2021, 127, 70–77. doi: 10.1002/jib.629.

New Approach for Barrel-Aged Distillates Classification Based on Maturation Level and Machine Learning: A Study of Cachaça. LWT. 2021, 140, 110836. doi: 10.1016/j. lwt.2020.110836.

[22] Zacaroni, L.M.; Cardoso, M.G.; Saczk, A.A.; Moraes, A.R.;

Anjos, J.P.; Machado, A.M.R.; Nelson, D.L. Determination of Phenolic Compounds and Coumarins in Sugar Cane Spirit Aged in Different Species of Wood. Anal. Lett. 2011, 44, 2061–2073. doi: 10.1080/00032719.2010.546017.

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A.R.S. Phenolic Compounds in Imburana (Amburana cearensis) Powder Extracts. Eur. Food Res. Technol. 2005, 221,739–745. doi: 10.1007/s00217-005-0065-3.

Quality of Brazilian Sugar Cane Spirits and Cachaças. Food Control. 2015, 54, 1–6. doi: 10.1016/j.foodcont.2015.01.030.

[33] Rodríguez-Solana, R.; Galego, L.R.; Pérez-Santín, E.;

Romano, A. Production Method and Varietal Source Influence the Volatile Profiles of Spirits Prepared from Fig fruits (Ficus carica L.). Eur. Food Res. Technol. 2018, 244, 2213–2229. doi: 10.1007/s00217-018-3131-3.

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ORIGINAL PAPER

Investigation of Appropriate Cleaning Solutions for Removal of Denatonium Benzoate from Distillery Equipment Lauren E. Mehanna1, Kara A. Davis1, Shankar C. Miller-Murthy1, Tracy A. Gastineau-Stevens2, Bert C. Lynn2,3, and Brad J. Berron1,3* 1 Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY, USA 2 Department of Chemistry, University of Kentucky, Lexington, KY, USA 3 James B. Beam Institute for Kentucky Spirits, University of Kentucky, Lexington, KY, USA

KEYWORDS COVID-19 hand sanitizer denatonium benzoate distillery cleaning compatibility

RECEIVED: April 22, 2021 ACCEPTED: June 14, 2021 * CORRESPONDING AUTHOR: Brad J. Berron E-MAIL: brad.berron@uky.edu © 2021 BY THE SOCIETY OF DISTILLING SCIENTISTS AND TECHNOLOGISTS

During the COVID-19 pandemic, alcohol distilleries pivoted their production lines to manufacture hand sanitizer. Denatonium benzoate is a bittering agent and denaturant in hand sanitizer and is detectable in trace amounts. As a result, transitioning between hand sanitizer back to distilled spirits creates products with bitter flavors. Several cleaning methods were studied to determine their effectiveness in removing denatonium benzoate from materials in distillery equipment. Hydrogen peroxide and activated carbon were most effective in removing denatonium benzoate in the solution phase, with more than 40% removed compared to the original solution concentration. Strong acidic and basic cleaners were ineffective, with less than 10% of the original compound removed. When tested as cleaners on the distillery materials, hydrogen peroxide and activated carbon methods were no more effective than other rinsing (water, glycerol) or extraction (pure ethanol) cleaners for removing denatonium benzoate. Chemical compatibility, specifically with concentrated ethanol, plays a large role in the permeation of denatonium benzoate into and out of some materials. Hard materials, such as metals and rigid polymers, have good compatibility with ethanol, resulting in little swelling and denatonium benzoate penetration when soaked with sanitizer. Since they retained little denatonium benzoate, they are cleaned by simple rising. However, elastomeric materials vary greatly in their compatibility with high proof ethanol, leading to swelling or breakdown in the presence of hand sanitizer and a greater amount of denatonium benzoate leaching into the material. While ethanol effectively extracts denatonium benzoate out of the elastomers, it damages the material, requiring more frequent replacement.

INTRODUCTION The Coronavirus Disease 2019 (COVID-19) created a global pandemic resulting in unprecedented challenges to businesses and personal lifestyles.

While this was first and foremost a public health crisis, it also brought about many changes in how people functioned in their daily lives. Businesses shut down, and the majority of people were forced to work from home, leading to a greater demand on many common household goods [1]. As government health agencies repeatedly marketed the message of frequent hand washing and sanitization as the universal strategy to help stay healthy, hand sanitizer became a key target of 26

consumer demand. The beverage industry, particularly distilleries, were granted U.S. Food and Drug Administration (FDA) allowances for the production of hand sanitizer to increase supply [2]. These distilleries were already accustomed to distilling and blending high proof ethanol for human consumption. In pivoting to hand sanitizer, distilleries needed only to blend ethanol with a few additives to produce the sanitizer product.

Their facilities were already equipped for ethanol distillation, blending, and bottling, not only combating the supply shortage but also helping to speed up the time to market.

For distilleries to bottle their product as hand sanitizer, a few additives had to be mixed with the ethanol THE JOURNAL OF DISTILLING SCIENCE

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prior to bottling in accordance with FDA and Alcohol and Tobacco Tax and Trade Bureau (TTB) guidelines [3,4].

These included hydrogen peroxide (antiseptic), glycerol (gentle on hands and reduces evaporation), denatonium benzoate (bittering agent), and water (dilutant) [2]. Denatonium benzoate, sold commercially as Bitrex, is a compound that has been used for many years as a denaturing agent in many commercial products, including but not limited to alcohol, cleaning fluids, cosmetics, pesticides, and other household items [5]. It is known for being an extremely bitter tasting compound and is added to these often toxic products to deter consumption and prevent bodily harm [6]. Uniquely, denatonium benzoate can be detected by taste at ppb levels [7].

Due to the potency of denatonium benzoate at such low concentrations, it is extremely difficult to remove from distillery production lines. As distilleries transitioned from manufacturing hand sanitizer back to spirit production, many found that their products were tainted by the characteristic bitter taste of denatonium benzoate. This became an unintended consequence for distilleries producing hand sanitizer, and there was no scientific research available to offer guidance on appropriate cleaning methods for the many materials of construction used in a modern distillery. Now that the role of a distillery firmly includes public health response, it is crucial to develop successful cleaning methods for removing denatonium benzoate from manufacturing equipment. Hydrogen peroxide cleaning is suggested by manufacturers of denatonium benzoate, but some materials that are common in a distillery are not compatible with hydrogen peroxide [8-14].

Distilleries may be called upon once again to assist with hand sanitizer production for public health emergencies, and we seek to better support a transition between sanitizer and premium spirits with an essential flavor profile. Without appropriate and proven cleaning methods in place, the distiller takes significant risks in transitioning back to beverage production. To help provide guidance to these distilleries, we investigated the efficacy of conventional cleaning techniques on the removal of denatonium benzoate from materials used in distillery equipment. We focused on cleaning techniques and materials common in the distillery setting, including solutions of caustic soda (sodium hydroxide), citric acid, hot water, and ethanol. We also looked at cleaning with the glycerol and hydrogen peroxide used in sanitizer production. Finally, distillers frequently use activated carbon before bottling, and we determined the effectiveness of activated carbon as an adsorbent to remove denatonium benzoate from solution.

Our approach to studying the cleaning methods in a distillery first focused on the materials compatibility between the proposed cleaning solutions and a broad cross section THE JOURNAL OF DISTILLING SCIENCE

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of materials used in a distillery (copper, stainless steel, elastomer gaskets, IBC totes, etc.). We then looked at the potential for cleaning solutions to capture or react with denatonium benzoate in solution, which describes the effectiveness at denatonium benzoate reduction within pooled solutions in tanks and piping dead legs. To determine the efficacy of removing residual denatonium benzoate from the surface of process equipment, we tested a range of solutions on materials that have been submerged in sanitizer for prolonged periods. In all, these cleaning studies provide guidance to the distiller on the removal of denatonium benzoate from their facilities during the transition back to flavor sensitive products.

MATERIALS AND METHODS Cleaning methods chosen for testing were categorized as dilution, extraction, reactive, or adsorption methods.

Cleaning reagents were obtained from VWR International (Radnor, PA) and Northern Brewer (Roseville, MN).

The distillery materials tested in these studies mimic those present in actual production lines and were requested for testing by local distillers. All materials for these studies were obtained from Grainger (Lake Forest, IL).

MEASURING DENATONIUM BENZOATE CONCENTRATION

The concentration of denatonium benzoate in solution for all studies was determined using LC-ESI-MS analysis.

Separations were performed using a Shimadzu Nexera X2 modular UHPLC system (Torrance, CA) consisting of the following modules: SIL-30AC, LC-30AD, CTO-20A, CBM-20A, and DGU-20A equipped with a Kromasil ExternityXT C18 UHPLC column (2.1 x 50 mm, 2.5 µm particles (Supelco, Bellefonte, PA)). The mobile phase gradient used for the separation was initialized at 85% water:15% acetonitrile, held for two minutes, then increased to 15% water:85% acetonitrile at 6.5 minutes, held at this percentage for two minutes and returned to starting conditions at nine minutes. The UHPLC effluent was coupled to a QExactive orbitrap mass spectrometer (ThermoScientific, Waltham, MA) equipped with a HESI source. Data was acquired in the full scan mode at 140,000 mass resolution.

Reconstructed high-resolution accurate mass ion chromatograms were used to quantify the denatonium cation.

Initial standards of denatonium benzoate diluted in deionized (DI) water were prepared and a linear dynamic range of ~200 fg – 50 ng was determined using LC-MS. DISTILLERY MATERIALS MASS CHANGE IN ETHANOL

Metals (304 stainless steel and 122 copper), elastomers 27

Investigation of Appropriate Cleaning Solutions for Removal of Denatonium Benzoate from Distillery Equipment

(EPDM, Nitrile, 70 Shore A Silicone, Viton FKM), and rigid polymers (polypropylene (PP) and ultra high molecular weight polyethylene (UHMWPE)) were tested in various ethanol solutions to determine any changes in structure and mass. The surface area and volume of each sample is provided in start Table 1. The initial mass of each material was first measured and recorded. The materials were then submerged in 140 proof ethanol, 200 proof ethanol, or hand sanitizer for three days. The hand sanitizer was a liquid solution prepared with 77% v/v of 200 proof ethanol, 1.4% v/v of pure glycerol, 0.38% v/v of 30% hydrogen peroxide, 0.00037% w/v of denatonium benzoate, and 17% v/v of deionized (DI) water according to FDA and TTB guidance. After three days, the materials were removed from their respective solution, dried completely, and the final mass was recorded. The percent change in mass was calculated by comparing the final mass to the initial mass of the material. Three replicates were prepared for each of the materials in each of the ethanol solutions.

SOLUTION PHASE INTERACTIONS WITH DENATONIUM BENZOATE

Initial studies tested the solution phase activity of various cleaning approaches to consuming denatonium benzoate. Six cleaning methods were chosen for testing based on cleaning products readily available or easily attainable in distilleries that are producing hand sanitizer. Cleaning solutions were prepared in DI water according to literature to an appropriate concentration used in the industry.

Solutions of 0.5 mg/mL denatonium benzoate in DI water were prepared. Each solution was mixed with a respective

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cleaning solution to achieve a final denatonium benzoate concentration of 0.05 mg/mL. The solutions were stirred continuously, and the treatment proceeded for 15 minutes.

An appropriate neutralization strategy was used for each solution to halt the activity and adjust the pH to 7. Table 1 details the cleaning methods investigated, appropriate concentrations, and neutralization strategies used in this study.

LC-MS analysis was used to determine the remaining concentration of denatonium benzoate in solution. Three replicates were performed per cleaning method. The remaining denatonium benzoate concentration in each solution was compared to controls of 0.05 mg/mL denatonium benzoate in DI water to determine the ratio of denatonium benzoate remaining in solution relative to the initial amount.

DENATONIUM BENZOATE REMOVAL FROM DISTILLERY MATERIALS USING CLEANING METHODS

Results from the solution phase reaction studies were used in determining appropriate cleaning solutions for extracting denatonium benzoate from the distillery materials. Only cleaning solutions that were promising for denatonium benzoate removal were continued for further testing. Table 2 lists the cleaning methods that were used for testing with distillery materials. Hot water was prepared by heating DI water to 70˚C. All other solutions were prepared with room temperature DI water at 24˚C. The same distillery materials were used as in the previous mass change experiments. Materials were submerged in hand sanitizer solution for three days. The materials were

TABLE 1 Cleaning methods tested for the removal of denatonium benzoate in solution with concentrations used in distilling applications. Neutralization strategies were specific to each cleaning method to adjust the pH. Activated carbon NORIT GAC 12-40 Mesh corresponds to when 90% of granules are captured with a sieve size between 0.42 to 1.70 mm.

CLEANING METHOD

CONCENTRATION FROM LITERATURE

CONCENTRATION USED IN SOLUTION PREPARATION

NEUTRALIZATION STRATEGY

Citric Acid

1 tbsp/gal [15]

3.78x10-3 g/mL

Addition of 1N Sodium Hydroxide

3% v/v [16]

5.00 mL of 30% H2O2 diluted in 45.00 mL DI H2O

Addition of 1M Sodium Metabisulfite, then 1N Sodium Hydroxide

Hydrogen Peroxide

Bleach

200 μL/L [5]

0.200 μL/mL

Addition of 1M Sodium Metabisulfite, then 1N Sodium Hydroxide

Sodium Hydroxide

1% w/v [17]

1.00 g/mL

Addition of 37% Hydrochloric Acid

Powdered Brewery Wash

1 oz/gal [18]

7.49x10 g/mL

Addition of 37% Hydrochloric Acid

Activated Carbon

150 mg/L (NORIT GAC 12-40 Mesh)[19]

1.50x10-4 g/mL

Filter 12-40 Mesh Activated Carbon Particles from Solution

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-3

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then removed from solution and submerged in a respective cleaning solution, which was continuously stirred for 15 minutes. The materials were removed from the cleaner and submerged into 200 proof ethanol for 24 hours to extract the remaining denatonium benzoate from the material. The liquid extracts were then analyzed using LC-MS analysis to determine the relative amount of denatonium benzoate remaining. Three replicates were prepared for each of the materials using each cleaning solution. Control samples of 0.05 mg/mL denatonium benzoate in DI water were prepared for each batch of samples and LC-MS analysis was used to determine the original concentration of denatonium benzoate in solution before treatment. The concentration of denatonium benzoate in each of the sample extracts after cleaning was compared to the concentration of denatonium benzoate in the control samples to

determine the percentage of denatonium benzoate remaining after treatment.

OUTLIER TESTING

Materials extraction studies were repeated for polypropylene (PP) in glycerol and ethanol rinsing methods, as they produced large standard deviations with three replicates. Once replicates were analyzed, the 6 data points were combined for outliers to be determined using the method based on the interquartile range. Upper and lower limits for outlier determination were calculated using the 25th (Q1) and 75th (Q3) percentiles and the interquartile range (IQR = Q3 – Q1) of the data set. Any outliers detected in the data set were noted, but not included when graphing.

TABLE 2 Cleaning solutions tested and their method of removing denatonium benzoate from various distillery materials.

CLEANING SOLUTION

METHOD OF CLEANING

Hot Water

Dilution

Pure Ethanol

Extraction/Dilution

25% v/v Glycerol

Extraction/Dilution

Upper Outlier Limit = Q3 + 1.5 * IQR

[Equation 1]

Lower Outlier Limit = Q1 − 1.5 * IQR

[Equation 2]

SIGNIFICANCE TESTING

For statistical analysis, two-group t-testing was used to compare the percent change in mass between materials when placed in various ethanol solutions. Two-group t-testing was also used to relate the relative amount of denatonium benzoate remaining in solution from various cleaning methods in the initial reaction and materials extraction studies. Differences in the samples were considered significant if p<0.05.

3% v/v Hydrogen Peroxide Reactive & Dilution Activated Carbon

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Adsorption & Dilution

TABLE 3 Chemical compatibility of common distillery materials with various cleaning methods tested for denatonium benzoate removal. Each pairing was rated from A-D for resistance to chemical attack, and 1 for thermal dependence if applicable [8-13]. PBW is a powdered brewery wash with main ingredients sodium metasilicate and sodium percarbonate. EPDM is ethylene propylene diene monomer and UHMWPE is ultra-high-molecular-weight polyethylene.

HOT WATER

ETHANOL

GLYCEROL

CITRIC ACID

HYDROGEN PEROXIDE

BLEACH

SODIUM HYDROXIDE

PBW

ACTIVATED CARBON

EPDM

A

A

A

A

B1

A1

A1

A

A

Nitrile

A

D

A

A

B1

C1

B1

D

A

Viton (FKM)

A

D

A

A

A

A

B

D

A

Silicone

B

B

A1

A1

B1

B1

A1

C

A

UHMWPE

A

A

A

A

A

A

A

A

A

Polypropylene

A

A

A

A

A

A1

A

A

A

304 Stainless Steel

A

A

A

A

B

D

A

A

A

122 Copper

A

A

A

C

D

D

D

B

A

MATERIAL

A

Excellent – Resistance to chemical attack. No change to material structure.

B

Good – Slight chemical attack occurs and only minor effects to material structure, including slight corrosion or discoloration.

C

Fair – Some chemical attack occurs and moderate effects to material structure, including loss of strength and swelling. Not recommended for continuous use.

D

Poor – No resistance to chemical attack and severe effects to material structure. Immediate damage may occur. Not recommended for any use.

1

Compatible to 72˚F (22˚C)

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RESULTS AND DISCUSSION Manufacturing facilities specifically choose equipment materials that will be compatible with the liquids and solids that they will be in contact with during production.

For example, distillery lines are designed to transport the liquids to and from the distillation columns; additionally, the columns themselves are designed to withstand high heat and the transport of concentrated ethanol. However, when these distilleries transitioned their systems to hand sanitizer production during the COVID-19 FIGURE 1 Percent change in mass of each distillery material after being soaked in 140 proof ethanol, 200 proof ethanol, and hand sanitizer solutions for three days. EPDM is ethylene pandemic, these materials were in propylene diene monomer, UHMWPE is ultra-high-molecular-weight polyethylene, and PP is contact with many reagents that polypropylene. Error bars represent the standard deviation of n=3 sample replicates. were outside of their original purpose, including the bittering agent denatonium benzoate. allow permeation of denatonium benzoate than the other When deciding on cleaning aptested materials. Additionally, it is expected that 200 proof proaches to remove trace amounts of denatonium benzoate ethanol will be able to permeate into these elastomers to within the production lines, it was important to determine extract out any denatonium benzoate that penetrates the if these cleaning chemicals would be compatible with each original material. of the equipment materials. The materials were categorized The cleaning methods studied were liquid solutions exinto three groups – metals, elastomers, and rigid polycept for activated carbon, which contains solid particles mers. All materials were chemically compatible with hot that are free-flowing in DI water. Activated carbon is a water, glycerol, and activated carbon. The metals and rigid powdered derivative of charcoal that has been oxidized and polymeric materials were also compatible with ethanol, alcontains a large number of micropores, increasing the surthough some elastomers were prone to chemical damage face area available to adsorb various chemicals or pollutants (Table 3). in solution, in this case denatonium benzoate [20]. ActivatThe chemical compatibility of each material with ethanol ed carbon with 12-40 mesh was used in these studies, as it played a key role in whether denatonium benzoate could is one of the most common sizes used in industrial systems permeate into or out of the material. The metal and rigid [21]. This 12-40 mesh relates to a sieve size in which most polymeric materials all have excellent compatibility with of the activated carbon granules are retained, and in this ethanol, and as expected, had little changes in their original case corresponds to 90% of granules being retained with mass when placed in any ethanol solution for prolonged sieve sizes of 0.42 – 1.70 mm. Because it is considered an periods of time (Figure 1, SI Table 2). inert solid material, activated carbon is not chemically reIn comparison, there was more variability in the mass active and will not damage the chemical nature or cause change of the elastomeric materials relative to their original swelling of any of the distillery materials. However, due mass, corresponding to differences in the compatibility of to its nature as a granular solid, there are concerns with these materials with ethanol (Table 3). This was especially abrasion as well as becoming trapped in tubing and pump evident when the elastomers were placed in hand sanitizsystems [22]. This makes the long-term uses of activated er and 200 proof ethanol, as their percent change in mass carbon cleaning less desirable, as it could erode the matewas significantly different than all the metal and rigid polyrials over time. To combat these issues, it is recommended mers, indicating that at high proof, these materials were to install an appropriate filter system to remove the solid more prone to either swelling or structural break down (SI particles once used or to create a mesh/membrane setup Tables 3-4). As a result of having greater percent changes within the equipment to contain the activated carbon, so in their mass, the elastomeric materials are more likely to liquids that need denatonium benzoate removal could flow 30

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through the mesh instead of allowing free-floating particles in solution. Since all other cleaners are liquid solutions, they are not a concern for abrasion.

All metals and most rubbers were poorly compatible with bleach. It is known that bleach is corrosive to stainless steel and copper alloys, as the hypochlorite ions undergo a redox reaction to form free chloride ions, which adsorbs on the metal’s surface and initiates local pitting corrosion [23-25]. Bleach is concerning to many elastomer seals and gaskets for the same reason, as it is incompatible with three of the four elastomer materials used in these studies (Table 3). Additionally, chlorine cleaners, such as bleach, are not recommended in distillery settings, as they can form TCA FIGURE 2 A) Schematic representation of solution phase interactions of various cleaning methods (2,4,6-trichloroanisole). TCA with denatonium benzoate. Cleaning solutions were prepared according to Table 1. B) Percentage of is concerning because it credenatonium benzoate remaining in solution compared to the original concentration after treatment with various cleaning methods. PBW (powdered brewery wash) is a non-caustic oxidizing alkaline cleaner. ates mold within production Significance was determined using 2-group t-testing with p<0.05(*) and p<0.01(**). Error bars represent lines and aging barrels which the standard deviation of n=3 sample replicates. is very difficult to remove and can change the flavors of the alcohol products [26]. may be better depending on the classification of the mateTherefore, bleach should be avoided and only be used as a rial (metal, elastomer, rigid polymer) and would still be aplast resort for cleaning distillery equipment. Instead, many propriate for use if certain materials could be isolated from distilleries use dilute citric acid to clean copper stills, as it others through system disassembly. is a chelating agent that binds to ions to prevent precipitate deposits on the surface of the copper [27,28]. However, increasing the citric acid concentration or using it excessively ELIMINATION OF DENATONIUM is known to shorten the lifespan of the still, and therefore BENZOATE VIA SOLUTION PHASE should only be used sparingly at or below 1 tbsp/gal [15].

INTERACTIONS From a general chemical compatibility standpoint, the We first determined if there were any common distillery safest universal cleaners for distillery equipment would be cleaning methods that would be effective in reducing the hot water, glycerol, and activated carbon. All listed mateconcentration of denatonium benzoate in solution prior to rials are good at resisting chemical attack from these soltesting on the distillery materials. Cleaning solutions (Tavents. This is because these three cleaning methods are ble 1) were reacted with a solution of denatonium benzoate better for binding to or rinsing denatonium benzoate away in DI water for 15 minutes, then neutralized to pH 7. Using from a material rather than chemically reacting with it.

LC-MS, the percentage of denatonium benzoate remaining These methods are low risk for systems with unknown or in solution was determined, which gave insight into which highly susceptible materials of construction. However, the cleaning methods performed best (Figure 2A). other cleaning methods should not be overlooked, as some THE JOURNAL OF DISTILLING SCIENCE

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Initial testing determined that hydrogen peroxide and activated carbon were promising in lowering the denatonium benzoate concentration in solution, as they had the lowest average percent of denatonium benzoate remaining compared to the starting concentration. Hydrogen peroxide was effective in removing 79% of the denatonium benzoate present in the original solution, outperforming all other tested cleaners. It is used in many cleaning products as an antiseptic and can also be used individually as a stand-alone cleaner as well. Hydrogen peroxide is a strong oxidizing agent. In solution, hydrogen peroxide can dissociate to produce hydroxyl and hydroperoxyl free radicals that react with organic compounds and convert these organic compounds into more soluble materials that are easily removed in water [29, 30]. Therefore, it is successful at cleaning many larger organic compounds, such as denatonium benzoate. Activated carbon, which uses adsorption, was the second-best cleaning method, removing 42% of the denatonium benzoate from the original solution. As activated carbon is inert, it did not require neutralization, only filtering out the solid particles from solution. Statistical analysis revealed that these two cleaning methods were the only ones with significant differences in the percentage of denatonium benzoate remaining after treatment when compared to at least one other method (Figure 2B).

Denatonium benzoate is an effective bittering agent in a wide pH range, supporting its effectiveness at rendering the alcohol unpalatable even when mixed with a variety of beverages [31]. There are a variety of cleaning methods used for removal of denatonium benzoate based on acid or base interactions. Acidic reaction systems are used to clean mineral deposits, rust, oxides, and tarnish from surfaces [32]. They usually contain chelants, which bind to metal ions and prevent them from forming precipitates on their surface. Citric acid is an excellent chelating agent, and is widely known to bind with copper ions, making it a popular cleaner for stills and other equipment. Basic (high pH) reaction systems are more commonly used to remove oils and fats from surfaces [32,33]. When using a basic cleaner, a chemical reaction called saponification occurs, converting organic fats and oils into a soap that is water soluble and can easily be removed from the surface [34]. However, high pH cleaners are generally more corrosive to metals and milder alkaline cleaners are recommended to avoid surface damage.

The remaining cleaning methods tested were extremely acidic (citric acid) or basic (bleach, powdered brewery wash (PBW), sodium hydroxide), but were not as effective in removing denatonium benzoate. Each method had on average greater than 80% denatonium benzoate remaining in solution after treatment. In addition to bleach having poor chemical compatibility with the materials used in 32

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distillery equipment (Table 3), bleach performed poorly in the reaction studies, with the final solution retaining 97% of the denatonium benzoate from the original solution.

PBW had large variability in the sample data, making it difficult to determine its efficacy in removing denatonium benzoate from solution. Since PBW is an alkaline cleaner, with primary ingredients sodium percarbonate and sodium metasilicate, it was expected to perform similarly to other alkaline cleaners.

Overall, hydrogen peroxide and activated carbon were the only compounds that effectively reduced the level of denatonium benzoate through solution phase neutralization. The pH stability noted in the literature is supported here, where reactions with acids and bases failed to reduce the solution level of denatonium benzoate.

REMOVAL OF DENATONIUM BENZOATE FROM DISTILLERY MATERIALS Since residual amounts of denatonium benzoate left on distillery equipment can cause extremely bitter flavors in bourbon products or other spirits, it is important to determine appropriate cleaning methods that will not only be compatible with the equipment materials, but also be successful at denatonium benzoate removal. Here, we simulate the cleaning of several distillery materials that have been exposed to a controlled dose of a denatonium benzoate through hand sanitizer. Effective cleaning methods determined from the solution phase studies (Figure 2B), as well as a few additional rinsing approaches, were tested for removing denatonium benzoate from distillery materials. These materials were soaked in hand sanitizer solution containing denatonium benzoate for three days, transitioned to an appropriate cleaning method for 15 minutes, then placed in pure ethanol for 24 hours to extract any remaining denatonium benzoate from the materials (Figure 3A).

METALS

Metals are generally impermeable to water and other chemicals [35]. As a result, copper and stainless steel are two common metals used in manufacturing equipment, such as distillation columns, stills, and storage vessels [36].

We expected that when soaked in hand sanitizer, denatonium benzoate would not absorb into copper or stainless steel and would instead remain in the surrounding solution; as a result, only remnants of the denatonium benzoate would remain when the hand sanitizer was removed and replaced with a cleaning method. The results in Figure 3B support this hypothesis, as the two metals tested had the THE JOURNAL OF DISTILLING SCIENCE

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lowest percent of denatonium benzoate remaining after treatment when compared to the other elastomers and rigid polymeric materials. Additionally, there is no significant difference between the effectiveness of any of the cleaning solutions with these two metals, so these systems are effectively cleaned with any form of rinsing.

ELASTOMERS

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backbone, resulting in increased permeation of ethanol and swelling of the elastomer (Figure 1) [41]. A type of Viton FKM, the form of Viton with a lower fluorine content, was used in these studies rather than Viton ETP, which contains a higher fluorine content. This explains why Viton in these studies enabled swelling in ethanol (Figure 1) and most of the denatonium benzoate was removed from the material during the cleaning step (Figure 3).

Because both nitrile and Viton have poor chemical resistance to ethanol, the denatonium benzoate likely leaches into these materials with the hand sanitizer soak [12,13].

Then when cleaned with 200 proof ethanol, the ethanol facilitates an improved removal of denatonium benzoate compared to other cleaning methods in Figure 3B. However, both materials have good resistance to the permeation of water, hydrogen peroxide, and glycerol solutions [12,13].

This indicates that none of these methods will work well in extracting out the denatonium benzoate once it is leached into the material. A two-group t-test revealed that there is a significant difference for nitrile and Viton cleaned with hydrogen peroxide compared to all other materials tested

The elastomer materials tested (EPDM, nitrile, Viton, and silicone) are commonly found in pipe fittings, such as gaskets and O-rings, that can withstand high pressure and prevent liquid or gas permeation into important downstream equipment [37]. These materials had the highest variability in the removal of denatonium benzoate after exposure to the hand sanitizer solution and cleaning treatment.

Nitrile and Viton in particular, had the lowest percentage of denatonium benzoate remaining when rinsed with 200 proof ethanol, likely due to their high swelling in ethanol (Figure 1) and poor chemical compatibility with polar solvents [38]. Since ethanol is polar due to its hydroxyl group, it can undergo a substitution reaction with the polar groups in the elastomer backbone; as a result, it is prone to an increase in swelling and permeation of ethanol into the material, compromising the structural integrity of the elastomer with longterm exposure [38].

Viton is a brand of fluorocarbon elastomer (FKM) used in many sealing applications. There are various specialty types of Viton products, varying between 60-70% fluorine content, which in turn varies the chemical and temperature resistance of the material [38,39]. Viton products have improved performance and resistance to ethanol and other reagents by increasing the fluorine content because the bulky fluorine atoms can help shield the polymer backbone from attack FIGURE 3 A) Schematic representation of the interaction and removal of denatonium benzoate with [40]. Conversely, a lower various distillery materials. B) Percent of denatonium benzoate remaining in solution following a soak fluorine content reduces in hand sanitizer solution, cleaning, and extraction with ethanol compared to the original concentration (control). EPDM is ethylene propylene diene monomer, UHMWPE is ultra-high-molecular-weight this shielding and increaspolyethylene, and PP is polypropylene. es attack to the polymer THE JOURNAL OF DISTILLING SCIENCE

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Investigation of Appropriate Cleaning Solutions for Removal of Denatonium Benzoate from Distillery Equipment

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TABLE 4 Hydrogen peroxide cleaning effectiveness across material types. A) P-values obtained from significance testing using a two-group t-test comparing hydrogen peroxide cleaning of Nitrile and Viton to all other materials with this same cleaner. B) P-values comparing ethanol and hydrogen peroxide cleaning methods for Nitrile and Viton materials. Comparisons were considered significant if p<0.05. EPDM is ethylene propylene diene monomer, UHMWPE is ultra-high-molecular-weight polyethylene, and PP is polypropylene.

A)

Nitrile Viton B)

VITON

EPDM

SILICONE

PP

UHMWPE

1.63E-03

6.60E-04

1.15E-04

9.45E-05

6.60E-05

5.70E-05

7.86E-05

3.27E-04

2.32E-04

1.81E-04

1.88E-04

2.04E-04

2.07E-04

ETHANOL vs HYDROGEN PEROXIDE

Nitrile

2.52E-05

Viton

5.17E-04

with this cleaner (Table 4A), where the peroxide is particularly poor at removing the large amounts of denatonium benzoate imbibed in these materials. In sharp contrast to solution phase experiments (Figure 2), the hydrogen peroxide is significantly less effective at removing denatonium benzoate from the nitrile and Viton than ethanol rinsing (Table 4B). This is likely due to these elastomers having high swelling and poor chemical resistance to ethanol, while having good compatibility with hydrogen peroxide (Table 1).

While ethanol can effectively remove denatonium benzoate from these two materials, it is only recommended as a short-cycle cleaner and not as a long-term cleaning solution, as it can cause swelling and damage the material backbone, weakening the elastomeric structure with time (Figure 1). If 200 proof ethanol is used routinely as a cleaning method for denatonium benzoate, these elastomers will need replacement more frequently.

As EPDM and silicone rubbers have a fair chemical resistance to ethanol, it is likely that the denatonium benzoate in the hand sanitizer solution was not able to diffuse into these materials as easily as nitrile and Viton, therefore, not as much was retained within the material to be removed by a cleaner [11,13]. The percentage of denatonium benzoate extracted out of the EPDM and silicone materials was much lower than that of nitrile and Viton and was much more comparable to the rigid polymer and metal materials.

EPDM rubber also has a low absorption of water, acting as a barrier to not absorb water-based liquids, which often leads to its use in outdoor applications [42,43]. Both EPDM and silicone are more resistant to chemical attack by polar solvents [44,45]. This suggests why the hand sanitizer containing ethanol and denatonium benzoate did not diffuse into these materials as much as the other elastomers (Figure 1). Consequently, there was less denatonium benzoate to be extracted out of the material and an overall 34

304 STAINLESS STEEL 122 COPPER

lower percentage of denatonium benzoate remaining in the final extracts (Figure 3B). However, both materials are still prone to swelling and structural changes in the presence of 200 proof ethanol, used in extracting out any denatonium benzoate after the hand sanitizer soak, so they will still need constant monitoring and replacement if cleaned frequently with high proof ethanol (Figure 1).

RIGID POLYMERS

More rigid polymeric materials, such as plastics (UHMWPE and PP), are found in distilleries in totes, storage containers, and components in pumps and valves. They are considered porous plastics and are similar in terms of mechanical strength and chemical resistance [46,47]. Both plastics offer good chemical resistance to the components in the hand sanitizer solution and minimal swelling (Figure 1), so we expect that little denatonium benzoate would permeate into the material when placed in hand sanitizer solution [8,9,48,49]. From the findings in Figure 3B, this hypothesis is supported, as both had low levels of denatonium benzoate extracted from the materials. See SI Table 5 for outlier sample analysis. The low magnitude of extracted denatonium benzoate was comparable to the impermeable copper and stainless steel samples.

CONCLUSION Material and chemical compatibility plays a large role in selection of a cleaning approach in a given processing facility. From the initial solution phase interactions, many cleaners, particularly those extremely acidic or basic, were ruled out for being ineffective in removing the denatonium benzoate or for having poor compatibility with several of the materials tested. Of the cleaners studied in the solution interactions for pooled liquids, only hydrogen peroxide THE JOURNAL OF DISTILLING SCIENCE

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and activated carbon were effective in eliminating a significant amount of denatonium benzoate while still being compatible with most common distillery materials. Therefore, it recommended to use hydrogen peroxide or activated carbon in a distillery setting where dead legs or pooled liquids are present.

We also observed a significant role of material of construction in the persistence of denatonium benzoate. All cleaners performed similarly in removing denatonium benzoate from the surface of metals and rigid polymers.

For these cases where little pooled liquid is expected, it would be recommended to use cleaners categorized as rinsing approaches with relatively neutral pH, such as water, glycerol, or ethanol, as they are readily available and would not cause issues when transitioning back to spirits production. Activated carbon performed similarly to the rinsing approaches. Activated carbon is unique in that it is an inert adsorption method that will not cause a chemical reaction with any of the materials or species. However, due to being a solid material, it is more likely to cause abrasive damages and cause clogging in equipment with use.

Elastomer materials used in seals and gaskets are the most challenging for the elimination of denatonium benzoate that has been in contact with the materials for 24 hours.

While very little denatonium benzoate in the hand sanitizer solution is being leached into the metal or rigid polymer materials, there was a large percentage of denatonium benzoate remaining after cleaning in some elastomer materials.

All elastomers had noticeable mass changes in the presence of hand sanitizer and 200 proof ethanol solutions, indicating that denatonium benzoate can permeate into these swollen materials. Only 200 proof ethanol worked well in removing denatonium benzoate from the elastomeric materials, likely due to these materials having high swelling and poor compatibility with high proof ethanol. There is a tradeoff between removing the denatonium benzoate from the elastomers and maintaining the structural integrity of the material. Because of this poor chemical resistance, ethanol will cause these materials to swell or break down, so it is only recommended for removing the denatonium benzoate if this degradation can be tolerated or the elastomer components may be replaced. If larger quantities of ethanol are to be run through the system, it is recommended to use another cleaning method on these materials that would not cause swelling and damage its structural integrity. If 200 proof ethanol is used often for cleaning, it is likely that all of the elastomers will need more frequent replacement.

FUNDING STATEMENT

This publication was supported in part by the University of Kentucky’s COVID-19 Unified Research Experts (CURE) Alliance. This publication was supported in part THE JOURNAL OF DISTILLING SCIENCE

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Mehanna et al.

by the National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health, through Grant UL1TR001998.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. This material is based upon work supported by the National Science Foundation Graduate Research Fellowship under Grant No. 1839289. Any opinion, findings, and conclusions or recommendations expressed in this material are those of the authors(s) and do not necessarily reflect the views of the National Science Foundation.

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

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ORIGINAL PAPER

Chinese Baijiu - Finding a channel to design a defined starter culture

Bowen Wang1, Huiyi Hao1, Hehe Li1, Jinyuan Sun1*, and Baoguo Sun1 1 Beijing Laboratory for Food Quality and Safety, Key Laboratory of Brewing Molecular Engineering of China Light Industry, School of Light Industry, Beijing Technology and Business University, Beijing, China

KEYWORDS Baijiu defined starter enzyme microbiota Qu synthetic microbiota

Qu (a starter composed of multiple microbes, enzymes, and nutrients) is essential for initiating Chinese baijiu fermentation and is usually prepared in an open system to enrich the starter complex with microorganisms from the local environment. However, with the challenge of increasing manufacturing and labor costs, traditional spontaneous fermentation cannot meet the growing industrial needs for standardization and modernization. Nowadays, the development of a synthetic microbiota built up from selected and cultured microorganisms enables the repeatable, standardized production of fermented foods. The use of such synthetic microbiota to convert raw materials into foods can hopefully reproduce the smells and tastes of traditional products. This review critically summarizes the properties of traditional qu and discusses the potential of a defined synthetic microbiota to revolutionize the production of such fermentation starters for future baijiu production. The prospects and challenges in dealing with the identification, selection, cultivation, and incorporation of microbes into such synthetic microbiota (or new ecological complexes) are specifically related to developing a fully defined and effective mixed-starter culture for use in traditional fermented food production are detailed.

1. INTRODUCTION

* CORRESPONDING AUTHOR: Jinyuan Sun E-MAIL: sunjinyuan@btbu.edu.cn © 2021 BY THE SOCIETY OF DISTILLING SCIENTISTS AND TECHNOLOGISTS

ha lle n

al qu

ncy icie eff In ity

on and standardis isati atio ern no d o ff m oo o Traditional manufacturing t d ge

Spontaneous

Undefined

Qu Defined

nd

es

tro

us

l

ta

in a

b il

Modern industry it y

and c

o n sist e n c y o f

foo

d

a qu

lit

y

a

M

ov pr

on

f

Im

c re

la v or or ee ffic ie n c

y

Control

Mo

VOLUME 1 NUMBER 1

Unc on tro l C

Unconsistency

Baijiu is the national liquor of China and is warmly welcomed by consumers in the Orient [1,2]. Chinese people manufacture baijiu via solid-state fermentation and use qu (a sort of equivalence to koji) as a starter [3]. The preparation of qu is partly a spontaneous process, relying on an enriched collection of microorganisms drawn in from the local environment. The microbes release enzymes to convert substrates into fermentable sugars and other nutrients required for efficient fermentation [4].

However, this spontaneous process faces challenges related to climate change, raw materials consistency, and technical adjustments, which ultimately affect the consistency and stability of resultant products [1-3]. These drawbacks need to be overcome by introducing modern standards and developing a defined qu via a validated, sustainable, and better-controlled industrial process [5,6] (Figure 1). It should be possible to provide a better definition of qu when THE JOURNAL OF DISTILLING SCIENCE

RECEIVED: April 19, 2021 ACCEPTED: August 10, 2021

More stability FIGURE 1 Schematic diagram from traditional spontaneous fermented Qu to modern defined starter.

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Wang et al.

the responsible microbes, critical enzymes, and formative mechanisms are identified in the traditional qu starters currently in use. For a long time, researchers explored the definition of qu employing traditional culture-dependent and modern culture-independent approaches [1-4]. However, defined qu can only be realized when the following posed questions are answered; “What is the composition and function of the microorganisms and enzymes that are present?”, “What is the formative mechanism of microbial communities in the traditional process?” and “How do we construct the community to recreate the metabolism of autochthonous microbiota?” Resolving these questions would provide a significant advance in creating a defined qu for baijiu fermentation and present a knowledge-based improvement to developing defined starters for other food fermentations.

Herein, we critically address the research progress related to traditional qu and illustrate the feasibility of realizing a defined qu for modern industrial production. Moreover, we discuss the potential of synthetic microbiota (new and defined ecological populations) to revolutionize the future of a designated starter for Chinese baijiu fermentation.

2. QU: A STARTER FOR BAIJIU FERMENTATION

38

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Chinese baijiu generally uses traditional qu (incorporating a multitude of generally undefined microbial species) as a fermentation starter, unlike western fermented foods driven by defined starters (composed of a single species or a few microbiological strains) [4,5]. Meanwhile, qu is also used to ferment other traditional Oriental fermented foods, like vinegar, soy sauce, rice wine, etc.[5]. This typical starter is mainly made of cereals (wheat, rice, soybean, etc.), and produced under a spontaneous solid-state fermentation in an open system [1]. Moreover, qu is also assigned the names, Daqu, Xiaoqu, and Fuqu, based on raw materials, inoculations, fermented parameters, and their utilization in different liquors [1-4]. For example, daqu, in the shape of a brick with a larger size, is mainly made of raw wheat and produced under a spontaneous fermentation without specific inoculations. Daqu is always classified into three types by the fermented temperature: high-temperature daqu (60-70 ºC), medium-temperature daqu (50-60 ºC), and low-temperature daqu (40-50 ºC) [3,4]. Meanwhile, xiaoqu, in the shape of a ball of relatively small size, is always made of steamed rice and inoculated with functional strains, wheat steamed rice then fermented in an open sysBaijiu tem [1]. In comparison, fuqu is fermentation composed of a known set of pure cultures produced under modern sorghum or rice mechanized conditions by inoculating functional strains in brans squeezing [1]. Overall, the traditional promicrobes & duction of qu includes ingredient matured inoculation enzymes pretreatment, shaping, incubaQu tion in a qu-making house, and maturation during storage (Figmixing ure 2). During preparation, qu storage gradually becomes a matured microbial ecosystem with abundant microorganisms, enzymes, and metabolites [2]. This starter then moulding provides the functional microorganisms and their products (enzymes, flavors, or precursors) to fermentation to shape the flavor fermentation of the different baijiu[3]. Thus, an understanding of the microbiota in the starter is a key to the quality-controlled use of the qu and Daqu Xiaoqu to developing and characterizing fully defined forms of the other qu-type starters.

FIGURE 2 Schema of traditional Qu production for Chinese baijiu fermentation.

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Chinese Baijiu - Finding a channel to design a defined starter culture

3. MICROBIOTA IN TRADITIONAL QU 3.1. MICROORGANISMS

The spontaneous fermentation of qu is a process that propagates microorganisms from the raw materials and the local environment. Therefore, the composition of the microbial community determines the quality of the matured qu [7]. Thus, understanding the compositions of microbial communities in traditional qu provides the basis for improving quality by subsequently adjusting the structure of the microbial community and developing fully defined starter formulations. Since the 20th century, microbiologists have applied culture-dependent methods to isolate and identify strains from qu samples [8-12]. Up to now, hundreds of microorganisms have been cultured by scientists, with the presence of bacteria, molds, and yeasts [8-12]. Bacillus spps, lactic acid bacteria (Lactobacillus, Pediococcus and Weissella), Acetobacter, Clostridium, Pantoea, Enterobacter, and Acinetobacter species always appear in the bacterial communities, whereas the molds (Aspergillus, Mucor, Rhizopus, Monascus and Trichoderma species) and the yeasts (like Saccharomyces, Pichia, Candida, Saccharomycopsis, Wickerhamomyces and Schizosaccharomyces) are dominant [8-12]. In addition, researchers have succeeded in isolating various thermophilic or acidic-,

Wang et al.

alcoholic-tolerant strains from the preparations of qu, under environmental extremes, including high temperature, moisture, acidity, and alcoholic conditions [13-17]. A new species of Thermoactinomyces daqus was isolated from a high-temperature daqu used for a typical strong-flavor baijiu production, and its genomic information was well-studied to illustrate the thermophilic nature and mechanisms of stress tolerance of this unique strain [13]. Many unique microbes, including a thermophilic bacterium (Scopulibacillus daqui sp nov.), a facultatively alkaliphilic species (Franconibacter daqui sp nov.), heat resistant Enterobacteriaceae and Bacilli, have been isolated from qu samples, and the interactions between these strains and the local environment have been well-studied [14-17].

This knowledge of the strains found to be present contributes to understanding the formation of special microbiota in traditional qu and could be developed and utilized in food fermentations and other industries. As knowledge of the growing number of strains isolated and identified grows, it becomes more promising that the mystery behind traditional qu can be unraveled. However, culture-dependent analysis is not enough to reveal a complete understanding of microbial communities, owing to the complexity and species diversity of the microbial ecosystem in traditional qu [1-4].

In facing this challenge, culture-independent methods (PLFA, PCR-DGGE, PCR-SSCP, RISA, and high

TABLE 1 Microbial community members in traditional Qu. MAIN MICROBES NAME

CLASS

MOLDS

BACTERIA

YEASTS

Aspergillus, Lichtheimia, Mucor, Rhizomucor, Rhizopus, Thermoascus, Thermomucor, Thermomyces

Bacillus, Brevibacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Weissella, Kroppenstedtia, Pediococcus, Microbacterium, Rubellimicrobium, Saccharopolyspora, Staphylococcus, Streptomyces, Thermoactinomyces

Candida, Hanseniaspora, Hansenula, Pichia, Saccharomyces

Medium-temperature Daqu (50-60 ºC) [22-28]

Absidia, Aspergillus, Lichtheimia, Rhizopus, Rhizomucor

Bacillus, Enterobacter, Lactobacillus, Leuconostoc, Kroppenstedtia, Staphylococcus, Saccharopolyspora, Pantoea, Pediococcus, Weissella

Candida, Hyphopichia, Pichia, Saccharomyces, Saccharomycopsis, Trichosporon

Low-temperature Daqu (40-50 ºC) [29-31]

Absidia, Aspergillus, Rhizomucor, Rhizopus

Acetic acid bacteria, Bacillus, Enterobacteriales, Lactobacillus, Staphylococcus, Streptomyces, Weissella

Pichia, Saccharomyces, Saccharomycopsis, Wickerhamomyces, Zygosaccharomyces

Absidia, Aspergillus, Rhizopus

Acetic acid bacteria, Acinetobacter, Corynebacterium, Deinococcus, Lactobacillus, Pediococcus, Streptococcus, Weissella, Xanthomonas

Hansenula, Pichia, Saccharomyces, Saccharomycopsis

Rhizopus, Aspergillus niger, Aspergillus oryzae, Aspergillus albicans

Bacillus licheniformis

Saccharomyces cerevisiae, ester yeasts

High-temperature Daqu (60-70 ºC) [18-21]

Daqu

Xiaoqu [32-36]

Fuqu [66-70] THE JOURNAL OF DISTILLING SCIENCE

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Wang et al.

throughput sequencing analysis) have provided an efficient set of approaches to aid in discovering the global features of microbial communities, and now an increasing number of species have been identified in the community of qu [1836].

Different qu exhibits unique characteristics in the composition of the microbial community, based upon the regulations of the manufacturing process, the fermentation ecology, and geographical features (Table 1, Figure 3). The microbial responses to the local environment may be the driving force behind the formation of the microbiota [37].

For example, microbial dispersion on substrates could be used to distinguish the microbial assembly of daqu or xiaoqu. The dispersals of plant-commensal microbes (like Bacillaceae, Enterobacteriaceae, and plant-associated lactic acid bacteria) on raw materials, could drive the microbial formation of daqu as its preparation originates from the use of raw grains. On the other hand, the selection and inoculation of competitive microbes, which are adapted to the host, should shape the community of xiaoqu [38]. In addition, the adaptability to extreme environments may direct the selection of microbiota in the same qu. For example, the Bacillus and Aspergillus strains (which are tolerant of high-temperature and low moisture conditions) can survive within such extreme conditions and are thus selected to become the dominant species within the community as compared with the community populations in low-temperature to high-temperature daqu [18-31]. Therefore, understanding the correlations between microbial populations as adapted to specific local environments may also provide a potential avenue to regulate microbiota formation for developing a defined qu.

In the preparations of qu, enriched microorganisms release abundant enzymes via their respective metabolic activities[1,3]. Researchers also utilize culture-dependent or culture-independent methods to reveal the enzyme profiles of qu [1-5]. In short, current knowledge indicates the complexity and diversity of enzyme profiles in this special bio-system of qu [39]. Previous studies generally isolated functional strains from qu samples, and then examined the characteristics of enzymes released under such culture-dependent conditions [40-42]. The isolated filamentous fungi always present vigorous activity with respect to various glucosidases, hydrolyzing polysaccharides to release oligosaccharides, monosaccharides (the fermentable sugars included), or flavors [40-42]. For example, an isolated Aspergillus sydowii F5 strain exhibits high activity of α-galactosidase under optimal conditions [43]. A Rhizopus microsporus var. tuberosus strain (isolated from daqu) can release extracellular fibrinolytic enzymes, and the purified enzyme

has been well-studied [44]. Besides filamentous fungi, the plant-commensal bacteria also release hydrolases as the requirement for their penetration into raw materials [37].

Bacillus and Streptomyces species, isolated from low-temperature daqu used for light-style baijiu fermentation, can produce extracellular alpha-amylase and glucoamylase, like Bacillus cereus H17 and Bacillus licheniformis H55 [11].

Given the complexity and diversity of enzyme profiles, the culture-dependent analysis of single strains has not yet provided a comprehensive description of the full spectrum of enzymatic profiles and potential of qu.

Furthermore, the profile of the metabolic activities of microbial species isolated and studied under cultured conditions cannot represent the characteristics of particular strains present in situ [7,38]. To resolve this dilemma, modern metatranscriptomic and metaproteomic approaches provide an efficient approach to reveal the global feature of enzyme profiles within qu matrices[7,38,39,45,46]. For example, relevant metatranscriptomic studies indicate that approximately 1,000 carbohydrate-active enzymes are potentially expressed in the medium-temperature daqu, and the active Mucorales and Bacillales are responsible for the expression of key enzymes associated with polysaccharide hydrolysis and flavor generation [39,46]. In addition, the respective high-temperature stage contributes to the expression of thermostable enzymes and promotes the activity of such enzymes for flavor production [39]. Moreover, several thermostable enzymes (like fungal alpha-amylase, endoglucanase) have been purified from daqu [47-49].

These studies reveal the enzyme profiles and their interactions with the local environment in the preparations of qu.

On this basis, the metaproteomic studies further strengthen our understanding of the essential features of the enzyme profiles in the qu bio-system [7,38]. So far, more than 2,000 enzymes have been identified in samples of qu, with these enzymes being expressed by about 200 fungal or bacterial genera, including Lactobacillus, Aspergillus, Pichia, Saccharomyces, Rhizopus, and so on [7,38,46]. According to annotations in GO, KEGG or other databases, these identified enzymes are associated with the hydrolysis of polysaccharides (allowing for a controlled release of fermentable sugars), ethanol metabolism, and flavor generation [7,38,46].

In short, current knowledge indicates that glycosidases are always the most abundant enzymes in the qu starters and determine the efficiency of polysaccharide hydrolysis and metabolite production during fermentation [38]. The expression of enzymes may be determined by the selection of materials and the control of parameters. For example, the diversity of glycosidases in daqu is significantly higher than that of xiaoqu, as the raw materials for daqu production present a more complex mixture of substrates[39,46].

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FIGURE 3 Microbial distribution in traditional Qu. (A) Microbial structure in high temperature Daqu, medium temperature Daqu, low temperature Daqu and Xiaoqu. (B) Dominant functional microorganisms in high temperature Daqu, medium temperature Daqu, low temperature Daqu and Xiaoqu.

A)

High-temperature Daqu

Low-temperature Daqu

Medium-temperature Daqu a: b: c: d: e: f: g: h: i: j: k:

a: b: c: d: e: f: g: h:

Aspergillus Trichocomaceae Eurotiales Pichia Pichiaceae Saccharomyces Saccharomycetaceae Saccharomycopsis

i: j: k: l: m: n: o:

Streptomyces Streptomycetaceae Streptomycetales Bacillus Lentibacillus Bacillaceae Thermoactinomycetaceae Bacillales Lactobacillus Pediococcus Lactobacillaceae

l: m: n: o: p: q: r: s: t: u: v:

Xiaoqu

Leuconostoc Weissella Leuconostocaceae Lactococcus Streptococcaceae Lactobacillales Enterobacteriaceae Enerobacteriales Acinetobacter Moraxellaceae Pseudomonadales

Saccharomycopsidaceae Saccharomycetales Rhizomucor Lichtheimiaceae Rhizopus Rhizopodaceae Mucorales

B)

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The abundant glycosidases are mainly expressed by the temperature- and desiccation-tolerant strains (like Aspergillus, Bacillus, etc.) [39,46]. By comparison, xiaoqu contains specific hydrolases (like α-amylase, glucoamylase, etc.), and the steaming process exposes more starch sites for hydrolysis[7,38]. These enzymes are mainly expressed by the inoculated and competitive strains (like Rhizopus, Aspergillus, Rhizomucor, etc.) in xiaoqu [7,38]. Besides the species, the cooperation of glycosidases is identified as a key to sustaining efficient saccharification and fermentation in the baijiu fermentation [38]. For example, the synergistic effect of α-amylase and glucoamylase could enhance starch hydrolysis and ethanol production by regulating fermentable sugars' formation to affect the core strains' metabolism in the baijiu fermentation [38,50]. Thus, this active mode could help baijiu producers better understand and manage the enzyme profile of qu or develop a defined qu for enhancing ethanol and flavor production in baijiu fermentation.

Although, as noted above, thousands of enzymes are identified already in the qu starters, there are still lots of unidentified enzymes, which hampers us in the deeper exploration of microbial resources in various fermented foods. The emergence of a specific database for other traditional fermented foods would provide for a more efficient approach to explore and utilize the outstanding resources available concerning such foods. This could enable the application of such knowledge towards defined qu production, as will the development of new and superior biotechnology techniques.

3.3. FORMATION OF MICROBIOTA

respective enzymes by regulating the moisture transfer and acidity variations within the system [7]. Besides the manufacturing parameters, the key environmental factors (temperature, moisture, acidity, etc.) also drive the microbiota dynamics in qu fermentation [52-54]. For instance, the accumulation of bioheat results in a peak inner temperature, affecting moisture transfer and the metabolic activity of microbes [52,53]. The peak temperature thus plays a vital role in the diversity of the microbial community and contributes to the abundance of thermotolerant microbes (like Bacillus, Aspergillus, etc.) [54].

Thus, all these noted studies indicate the key factors associated with the formation of microbiota in the preparations of qu. Moreover, modeling of microbial communities, with details of environmental factors included, has been implemented to try and predict the dynamics of the activities of the various microbes and the formation of their metabolites in the solid-state fermentation of qu. With the reminder that the production of qu starters themselves are subject to substantial fermentation activity to render them suitable for subsequent baijiu production [55-57]. It provides a new perspective to study the correlations between microbial formation and environmental changes in the traditional production of qu. Therefore, understanding the effects of multiple factors on the formation of the microbiota would facilitate better control and improvement of the overall microbial consortium for developing defined qu starters.

4. EFFECTS OF QU ON BAIJIU FERMENTATION

The solid-state fermentation of qu is a spontaneous process in an open environment, and the formation of its microbiota is shaped by microbes from the raw materials and the local environment [3-5]. Raw materials mainly contribute bacterial communities (like lactic acid bacteria, Bacillales, etc.) to the qu community. In contrast, the local environment is the primary contributing source of the fungal communities (like Mucorales, Saccharomycetales, etc.) for the qu ecological community [51]. In the preparation of qu, different factors drive the formation of the microbiota and the enzyme profiles in this unique solid-state fermentation; manufacturing parameters (raw material, inoculation, process-control, etc.) and environmental factors (season, weather, location, transport, and storage) [3]. For example, the selections and pretreatment (raw or steaming) of substrate materials (wheat, rice, etc.) determine the compositions of the community, especially the species of lactic acid bacteria, Bacillales, and Mucorales (Figure 3A).

In addition, the shape and size of qu also drive the dynamics of microbial communities and the production of their

Qu is both a starter and a part of the raw materials for Chinese baijiu fermentation. The microorganisms from qu contribute to alcoholic fermentation and flavor generation in the primary baijiu fermentation process [35,36]. Qu provides a large part of the fungal communities (more than 60%) and a small part of the bacterial communities (approximately 20%) to fermentation [58]. These microorganisms from qu, therefore, play crucial roles in the metabolic activities important to baijiu production: (1) filamentous fungi can secrete abundant hydrolases, which act to release fermentable sugars, amino acids, and fatty acids from nutritional macromolecular components (such as starch, protein, and fat), (2) Saccharomyces cerevisiae and non-Saccharomyces cerevisiae strains contribute to the generation of alcohols and esters during baijiu fermentation, (3) bacteria (like Bacillales, lactic acid bacteria, etc.) are the main contributors to the production of acids, alcohols, ketones, and various other aroma compounds [1-5].

Besides microorganisms, qu itself, by nature of its constituents, is also a main contributor of enzymes to the baijiu fermentation. These enzymes play essential roles in the

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solid-state fermentation process of baijiu production [1].

Qu thus also provides a large contribution of enzymes (more than 60%, from the various microbial species) such as glycosidases to the baijiu fermentation mashes. The optimal combination of glycosidases could enhance ethanol production in the fermentation process [38]. These facts strengthen the understanding of the effects of glycosidases on baijiu fermentation. However, more attention should be paid to the effects of other enzymes from qu upon baijiu fermentation activities.

In summary, conclusions regarding the effects of qu on baijiu fermentation have been made based solely on an understanding of the relative abundance of microorganisms and enzymes present. Full knowledge of the quantification of the qu starter contributions on baijiu fermentation is still needed. With the development of newer analytical techniques, researchers could better qualify and quantify the contributions to baijiu fermentations and utilize such information as the criteria to realize a fully defined starter.

5. PROGRESS TO A DEFINED STARTER As a further reminder, the solid-state fermentation of traditional qu occurs under an open environment system.

Thus, baijiu producers always face the challenge in maintaining stability and achieving quality consistency in batch production [59]. As noted, previous studies indicate that the compositions of microorganisms and enzymes are associated with the quality and function of qu [7,60]. Therefore, it is now more important to monitor and control the formation of qu within modern baijiu production facilities and the distilling communities where the spirit products are made. This will then answer our question as to how to develop a defined qu starter best and most efficiently and how it might apply to the different styles or types of baijiu produced.

5.1. FORTIFIED QU

Poorly controlled conditions, microbes present within raw materials, and the environment currently drive the formation of the ecological populations of microorganisms during the spontaneous fermentation production stage of qu starter formation. The deliberate inoculation of some of the typical autochthonous strains may thus be efficient for improving the quality of traditional qu by optimizing the starter microbiome [61-65]. In developing better-defined starters, isolated strains of Bacillales, Lactobacillales and Saccharomycetales are always selected as the candidates to build a fortified qu[61-63]. In practice, fortified qu could partially improve the structure of the microbial community and the efficiency of ethanol production in THE JOURNAL OF DISTILLING SCIENCE

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baijiu fermentation [64,65]. However, the formation of the microbial community in the preparation of fortified qu, is regulated by the ecological constraints and correlations between the inoculated strains and the autochthonous microbes, and via local environmental conditions [62]. Thus, this process of fortification by implementing the addition of functional strains faces the challenge of uncertainties of other variables involved regarding regional or seasonal differences in the populations of the localized organisms, to possible instability or a lack of robustness with respect to the autochthonous microbiota - synergistic and antagonistic considerations in play, and to potentially poorly controlled fermentations [61-62]. Thus, the consistent construction of a starter qu of defined population and quality will require implementing a more efficient and refined approach under more appropriate and stringent standards of microbiological quality control to prevent unwanted microorganisms from gaining access to the qu starter matrix.

5.2. PURE CULTURES

The term fuqu is applied to pure cultures, yet these are still only partially defined starters. They are made via the inoculation of functional strains into brans and prepared using mechanized equipment [66,67]. The unique species of Mucorales, Bacillales, and Saccharomycetales have been selected to develop a pure culture (Table 1). In the baijiu fermentation, these pure cultures provide outstanding ethanol production and efficient utilization of raw materials [68-70]. However, the liquors produced by pure cultures are still imperfect in both taste and aromatic qualities due partly to the loss of metabolites from poorly active, undesirable, and possibly decaying microorganisms in the fermentation [67,68]. Meanwhile, the current combined community is susceptible to being disturbed by the presence of the “ecologically new” microorganisms within the preparations of pure cultures due to an overall decreased microbial diversity [7].

Invasion by undesired microorganisms creates competition with endogenous microorganisms and dramatically affects the structure and function of pure cultures [70].

Therefore, constructing a stable and sustainable community (enough to resist the disturbing effects of the undesired microorganisms) is a goal of developing an efficient defined starter.

6. PERSPECTIVES AND CONCLUSIONS Chinese baijiu and other traditional fermented foods are part of our daily life, although their production is based on practical experience and skills passed down through the generations [71, 72]. Qu and relevant starters are the 43

Chinese Baijiu - Finding a channel to design a defined starter culture

Fermentation

S1

E1

fermentable sugars microbiota

function strains

Ethanol

S3

Flavors

Microbial network strains

Design

substrate

Build

isolation

Back-slopped fermentation

Candidates selection

Test

pu

Learn

community

ut

in

enzymes

t

modeling

compounds parameter

fermentation

Machine learning

C1 C2 C3

optimal strategy

Process control

starter baijiu

Function evaluation

FIGURE 4 Schema of synthetic microbiota to design a defined Qu.

Culture-dependent approaches have led to the isolation of a vast number of strains from traditional sources, and these strains have been well-studied under optimal cultured conditions. Moreover, meta-omics analyses can shed light on the metabolism of specific strains within in situ systems. Therefore, the combinations of culture-dependent and culture-independent analyses lead to the proposals for selecting the outstanding strains needed to develop pure cultures. However, the current attempts at using pure cultures limit the selection to one or several functional strains but neglect the role of enzymes that are indispensable for baijiu fermentations. The recent studies reveal that the synergistic effects of multiple enzymes or the combination of strains and enzymes may enhance saccharification, ethanol production, and flavor metabolism in baijiu fermentation.

Thus, the future development of defined starters may be selecting the combinations of multiple elements (strains, enzymes, or substrates) as a basis for production.

44

S2

Starch

tp

6.1. ISOLATION, SELECTION, AND COMBINATION OF CANDIDATE STRAINS OR ENZYMES

Qu

ou

essential ingredients to improve the quality of spontaneous solid-state fermentation. However, the traditional production of qu represents a poorly controlled process with the inevitable fluctuations of quality and productivity. Therefore, industrial production of a defined starter would increase the ability to improve the quality and safety of baijiu or fermented foods in the future. Herein, with the advances of biotechnology and research, this review proposes a roadmap to establish a rational and controlled process for developing a defined qu or starter in the industry (Figure 4). The goal of constructing a synthetic microbiota is to realize a defined qu for baijiu with the desired flavor profile.

Wang et al.

6.2. IDENTIFICATION, MONITORING AND THE CONTROL KEY DRIVING FORCES

As already illustrated, traditional preparations of qu are generally developed under an uncontrolled environment, and the poorly controlled conditions (like temperature, moisture, acidity, etc.) affect the assembly of the microbial community in this spontaneous process. With the application of systems biology methods, the correlations between environmental factors and microbiota have been established as key driving forces that determine microbiota formation in traditional qu production. Furthermore, this knowledge will potentially regulate the critical factors for further improving and developing a defined starter.

6.3. SYNTHETIC MICROBIOTA TO DESIGN A DEFINED STARTER

Designed synthetic microbiota serve as an efficient approach to develop a defined qu or pure culture mixes [73].

This method has succeeded in allowing the construction THE JOURNAL OF DISTILLING SCIENCE

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of a tractable microbiota in vitro system for optimizing traditional food fermentations, like cheese, light-style baijiu, and soybean sauce [73-76]. The tractable microbiota is constructed through strain selection (based on structure, function, and correlations with the local environment), flora combinations, and function evaluation [73-76]. Additionally, the production of these foods may be likened to the production of traditional qu, through the metabolic activities of similar microbiota, the raw materials used, processing and type of end products made. Therefore, this progress in our understanding opens a channel to construct a reproducible and tractable microbiota driving towards standardization and modernization of defined fermentation starters. First, for the design, we select candidate strains based on their roles in the baijiu fermentation and combine the community with/without enzymes to reduce the redundancy of specific pathways. Second, we rely on back-slopped fermentation to domesticate the combined community in selected materials or substances. Third, we monitor the metabolism of the synthetic microbiota (biomass, enzymes, metabolites, etc.) and gather the process parameters (temperature, moisture, acidity, etc.). Fourth, we build the model of the fermentation of designed microbiota to estimate and evaluate the productivity and function of defined starters. Fifth, we attain the optimum combinations of synthetic microbiota from the modeling and develop the group of desired starters. Then we study the effects of desired starters on baijiu fermentation to verify and acquire a more efficient and competitive starter. Sixth, we expect to build a broader database about specific synthetic microbiota and the desired baijiu, to assist in other traditional fermented food production (Figure 4). Therefore, the idea of building qu by a synthetic microbiota approach is worth developing to provide a defined starter, and such starters then need to be proven in practice.

of modern technology, a defined qu could be realized and overcome traditional technical limitations, resulting in better quality and consistency of baijiu spirit.

FUNDING STATEMENT

We gratefully acknowledge the National Natural Science Foundation of China (NSFC) (32102119, 31972193), National Key R&D Program of China (2016YFD0400500) and Postgraduate research capability improvement program of BTBU (19008021082).

CONFLICTS OF INTEREST

The authors declare that there are no conflicts of interest.

ORCID IDs

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[45] Zhang, B.; Kong, L. Q.; Cao, Y.; Xie, G. F.; Guan, Z. B.; Lu,

[58] Wang, X. S.; Du, H.; Zhang, Y.; Xu, Y. Environmental

microbiome and exogenous enzyme production in Moutaiflavor Daqu. J. I. Brew. 2018, 124 (1), 91-99.

Aspergillus sydowii F5 Producing α-Galactosidase and Statistical Optimization for the Enzyme Production. Asian J. Chem. 2012, 24 (2), 541-545.

X.; Wang, H. Purification and Characterisation of a Fibrinolytic Enzyme from Rhizopus microsporus var. tuberosus. Food Technol. Biotech. 2015, 53 (2), 243-248.

J. Metaproteomic characterisation of a Shaoxing rice wine “wheat Qu” extract. Food Chem. 2012, 134 (1), 387-391.

[46] Fan, W.; Zhao, X.; Du, G.; Chen, J.; Li, J.; Zheng, J.; Qiao, Z.;

Zhao, D. Metaproteomic analysis of enzymatic composition in Baobaoqu fermentation starter for Wuliangye baijiu. Int. J. Food Sci. Tech. 2021.

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Jin, Y.; Zhao, H. Directly mining a fungal thermostable alpha-amylase from Chinese Nong-flavor liquor starter.

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Luo, H.; Zhao, D.; Zheng, J.; He, H.; Jin, Y.; Zhao, H. Characterization of a fungal thermostable endoglucanase from Chinese Nong-flavor daqu by metatranscriptomic method. Int. J. Biol. Macromol. 2019, 121, 183-190.

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D.; Luo, H.; He, H.; Sun, Q.; Zhao, H. Biochemical and synergistic properties of a novel alpha-amylase from Chinese nong-flavor Daqu. Microb. Cell Fact. 2021, 20 (1): 80.

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promote microbial interactions in Chinese baijiu fermentation. LWT-Food Sci. Technol. 2021, 138. doi. org/10.1016/j.lwt.2020.110631.

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of raw materials and environments on the microbiota in Chinese Daqu starter. Int. J. Food Microb. 2019, 297, 32–40.

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Predicting the moisture content of Daqu with hyperspectral imaging. Int. J. Food Eng. 2021, 17 (1), 37-47.

A. Modeling of industrial-scale anaerobic solid-state fermentation for Chinese liquor production. Chem. Eng. J. 2020, 394: 124942.

Y. Water dynamics during solid-state fermentation by Aspergillus oryzae YH6. Bioresource Technol. 2019, 277: 68-76.

Microbiota Drives Microbial Succession and Metabolic Profiles during Chinese Liquor Fermentation. Appl.

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Shen, C.; Shi, J.; Xu, Z. Daqu microbiota exhibits speciesspecific and periodic succession features in Chinese baijiu fermentation process. Food Microbiol. 2021, 98, doi: 10.1016/j.fm.2021.103766.

[61] Li, P.; Lin, W.; Liu, X.; Wang, X.; Gan, X.; Luo, L.; Lin,

W. T. Effect of bioaugmented inoculation on microbiota dynamics during solid-state fermentation of Daqu starter using autochthonous of Bacillus, Pediococcus, Wickerhamomyces and Saccharomycopsis. Food Microbiol.

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of the Quality of Mixiang Baijiu Produced by Handmade Jiuqu/Mechanical Jiuqu. Liquor-Making Sci. Technol. 2019, (3), 90-93. (in Chinese)

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trace components in sesame-aroma type of baijiu. Food Res. Int. 2020, 137, 109695-109695.

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the profile of trace components in Baijiu. Food Rev. Int. 2021, 1-27. doi: 10.1080/87559129.2021.1936001.

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2008, (2), 65-66. (in Chinese)

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Would you like to see your work reviewed and published in the Journal of Distilling Science?

Guides to publication format and how your works are to be reviewed begin on the next page.

Following the guidelines and seeing how your manuscripts will be reviewed can ensure faster publication of your important works and materials.

Contact us immediately to be published in the next issue.

Email our Lead Science Editor, Gary Spedding, Ph.D. at gspedding@jdsed.com

EDITORIAL

GENERAL GUIDE FOR CONTRIBUTORS/AUTHORS EDITION 2021

CALL FOR PAPERS The call for papers is thus now on open invite with the lead science editor appointed. Authors should contact Gary Spedding, Ph.D. gspedding@jdsed.com in all matters concerning manuscript submission and addressing suitable areas of coverage.

Other documents and guides for authors will also be found on the journal website. Such materials include notes on Ethical Publishing in the 21st Century, Guidelines for Contributors/Authors — Manuscript Formatting, and Preparation, and a Manuscript Submission: Conflict of Interest Declaration and Author Agreement Form. Peer review guides are also available.

Manuscripts must be submitted in the English language, and, unlike many other journals, the JDS team will accept carefully cited authentic references published in other languages. Most papers today will carry an English title and often an abstract in English even if the main text is written in a different language. The journal is open to worldwide authorship with the aim of leaving no relevant work missed or inaccessible to the distilling community. All relevant translations of titles must be made and the non-English language speaking author will be encouraged to make use of professional translation services to convey all meaningful facts, data and truly expressed interpretation of all the material conveyed in their carefully written manuscripts. Peer reviewers will then be able to concentrate on the interpretation and significance of the science and results, and not on rewriting the paper.

SUBMISSION, ACCEPTANCE AND REVIEW PROCESS As for many journals, authors will submit articles to the lead science editor (noted above) for an initial screening.

Based on content and subject matter, in relation to the general scope of the journal, manuscripts will be either be rejected after this initial screening or, if suitable, will then be sent on for peer review.

The peer review process here is the single-blind model.

Manuscripts will be reviewed by three independent, anonymous expert referees. Reviewer reports, with comments and recommendations will be returned to the lead science editor within three weeks of their receiving the manuscripts and reviewer forms. Final editor assessments will be made and announcements issued to authors based on their acceptance or rejection decisions regarding their manuscripts.

Occasionally manuscripts will be accepted with no 50

revisions required. Reviewers are to supply notes and recommendations for any revisions needed — minor or extensive, and authors will be directed to address those prior to final acceptance and preparation for publication of their works. The process will follow with publication layout and final proofing of documents. Final proof copies sent to authors to be returned within one week of receipt to ensure timely publication.

Final publication will appear according to the timeline set for the next issue of the journal. Authors will be notified as to which issue their article has met the deadline for. For 2021 only one issue is planned with a quarterly issue plan set forth thereafter. Consult with the lead editor for ongoing details in this regard and be sure to lookout for the 2022 edition of this and other guides to authors and reviewers.

MANUSCRIPT SUBMISSION NOTES, PLUS CONFLICT OF INTEREST DECLARATION AND CONTRIBUTOR/AUTHOR AGREEMENT FORM Authors are responsible for their own content as submitted and will be allowed to use their writings and data elsewhere at their discretion. However, the layout of their work in the journal remains the copyright property of the publisher. The publisher will issue the necessary transfer of copyright documents after manuscripts have been accepted, peer reviewed, revised as necessary, and when being set for layout. If any doubts arise about this copyright process, the editor and publisher will be happy to explain what is involved and the details of your rights to use some or all of the material elsewhere. To summarize: in general you will retain all rights and responsibilities to material as expressed in your initial manuscript, however, US copyright rules will dictate as to what is owned by the journal based on their responsibilities in publishing the work.

No page charges will be incurred, nor fees instituted for the request to have items appear in print in color. Any extended rework needed will be discussed with authors, editor and publisher as necessary.

Manuscripts will not be accepted by the lead science editor unless accompanied by the signed form: Manuscript Submission: Conflict of Interest Declaration & Author Agreement Form.

That form covers the topic of conflicts of interest (COI), for the group and individual authors. The lead or corresponding author will ultimately assume and accept global responsibility for submission of the paper. However, it is THE JOURNAL OF DISTILLING SCIENCE

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General Guide for Contributors/Authors

expected that all authors listed as contributing to the work will have signed off on any disclosure statements under the authority and signature of the lead/corresponding author or team leader. Full terms and conditions are expressed in that agreement document.

In addition to the above statements, declarations as to the nature of funding for the research, or the writing (in the case of review papers) will need to be made.

Any copyright material submitted as part of a manuscript belonging to or owned by other parties must be accompanied by the appropriate end use agreement and release authorization documentation. Authors will be asked to sign a “No Conflict of Interest” statement.

All authors will, furthermore, respect any and all final decisions concerning publication by the editorial staff, the reviewers, SDST and the publisher of the JDS. Any complaints made by any party at any time will, however, be carefully and fairly considered with all due recognition of respected and accepted international scientific publishing guidelines taken into account. The JDS aiming only to publish the finest and most ethically prepared and reviewed scientific papers, reviews, notes and reports.

All guides and materials of interest can be found at http:// artisanspiritmag.com/journalds/.

A SUMMARY OF KEY POINTS FOR AUTHORS PUBLISHING IN THE JDS ETHICAL STANDARDS

The JDS publisher and editor aim to publish the finest scientific papers possible within its pages. Manuscripts will be peer reviewed — three reviewers per paper.

The highest ethical standards only will be tolerated. Authors and reviewers and all JDS staff will put forth their best efforts and aim to eliminate all biases and to report any and all conflicts of interest.

PUBLICATION STYLE GUIDANCE

Instructions to Author Guides and other documents are to be viewed and signed off on for successful publishing within the journal. The guides will assist in the author(s) setting the right style for publication.

The lead science editor will make the initial decision as to suitability of manuscripts for the journal and send on those passing initial inspection to three reviewers. All manuscripts will be published in English. JDS recommends you to seek translation services when necessary to help ensure manuscripts pass initial selection criteria.

SINGLE-BLIND PEER REVIEW METHOD

A NOTE ON CONFLICTS OF INTEREST Affiliations with or involvement in any organization or entity with any financial interest (such as honoraria, educational grants, supplier/manufacturer funding/supplies, participation in speaker’s bureaus, membership, employment, fellowships, consultancies, stock ownership, or other equity interest, and expert testimony or patent-licensing arrangements), or non financial interest (including personal or professional relationships, affiliations, knowledge, biased views or beliefs) in the subject matter or materials discussed/ presented in the manuscript are considered under the terms of actual or potential conflicts of interest.

Were sponsors or others involved at any stage of the process — research and or writing et cetera.? Acknowledge or reference any further contributions to this paper or review — such as data analysis, statistical analysis, data collection, data management or data storage services, professional language translation services (writing/editorial assistance) or any other assistance or support. [See details on the CRediT process, dealing with manuscript and research associated contributions, under the main instructions for contributors/authors document for more on this important new development in publishing.]

Three leading experts will be chosen from the editorial review board to single-blind review your manuscripts. Authors’ names will be known to the reviewers — authors will not know the identities of their reviewer “committee”. The three reviewers will also not know who their “team members” are.

The single-blind peer review method is in use for many scientific and medical journals. Like the alternatives there are advantages and disadvantages to this model. Reviewers will be chosen by the editor. Reviewers must attest to eligibility and without bias as to authors or topic before they will receive the actual manuscript and supporting documents.

Additional commentary can be found in the Guides to Reviewers.

AUTHORSHIP OF THE PAPER

All listed authors must have contributed to the work. To the conception, design or the acquisition, analysis or interpretation of the data, drafting the paper, adding to its value via revision and or giving final approval.

All authors will agree to be accountable for all aspects of the work. And in ensuring that any questions raised, related to the accuracy or integrity of any part of the work, are appropriately investigated and resolved.

REFERENCING THE WORKS OF OTHERS

Credit must be given to all sources of information used in the study and as expressed in the text. Details of THE JOURNAL OF DISTILLING SCIENCE

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General Guide for Contributors/Authors

methodology and results should not be withheld or data points removed without reason. Failing to present data in contradiction of your prior published data is unacceptable.

Manuscripts are often screened today for similarity or for detecting acts of plagiarism. Unauthorized reproduction of the work/writings of others is theft and may be identified as copyright infringement.

CONFLICTS OF INTEREST

All authors must disclose any conflicts of interest (COI) related to their intended publication. The manuscript must not be submitted or published elsewhere at the time it is undergoing JDS review nor upon its acceptance without JDS agreement.

A COI can arise if authors are paid by any commercial entity to write an article, to do the research for that article or compile a review. Ghosting/ghostwriting, whereby a third party writes an article submitted by others must be stated upon forwarding the manuscript for consideration. Other details provided above under “A note concerning conflicts of interest”.

Materials submitted remain the property and responsibility of the authors but the presentation/ layout will be copyright assigned to the Publisher.

Further details on these points will be found in the Manuscript Submission: Conflict of Interest Declaration & Author Agreement Form. Manuscripts will not be accepted by the lead science editor unless accompanied by the signed form. Legible manuscripts only accepted if they are set in the style described in the JDS Guidelines for Contributors/Authors — Manuscript Formatting and Preparation — Edition 2021 (or later year revision).

Manuscripts for consideration should be sent electronically to the Lead Science Editor.

Gary Spedding, Ph.D. gspedding@jdsed.com

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EDITORIAL

GUIDELINES AND NOTES FOR REVIEWERS

EDITION 2021

OVERVIEW AND SCOPE Full instructions to authors are provided in a separate document. This guide is primarily concerned with the journal’s manuscript reviewing system and providing guidance for the reviewer. Reviewers should also see the JDS Ethical Publishing in the 21st Century guide.

The lead science editor — currently Gary Spedding, Ph.D. — thanks each and everyone of you who have signed up for the very responsible role of reviewing manuscripts and providing your expert and considered opinion on the merits of the works supplied for publication in the JDS. The advice supplied here will guide reviewers in the responsibilities of providing unbiased, educated and informed opinions on the merits of work done by authors from around the world. This is one reason we have an internationally recognized body of experts from around the globe to oversee manuscript submissions. Most countries have groups involved in potable distilled spirits research with much to offer the growing distilling community and in raising the safety and quality levels of potable beverages.

While we do not expect our reviewers to translate or interpret for the journal, a team with members whose primary language is not English will offer our authors a benefit not found in many other journal editorial/review groups.

We have seasoned reviewers aboard — those having served such a role before or whom are currently serving on other boards. Many of our reviewers also having an established and solid record of publication. As a new journal we also have those less familiar with the process. Many works dealing with ethical and quality publishing have appeared on line and in PDF downloadable documents. A quick presentation of such materials will help ease our newer reviewers into the process and/or provide some reminders or even fresh advice for our veterans.

All may benefit from the advice presented here — especially in view of the concerns of the scientific community of late in seeing a decline in the reputation of science.

The decline is based in part on poorer publishing practices, biases and fraudulent publication, and the retraction of publications sometimes associated with even very famous names. The JDS must accept only the finest that our scientific and technically trained colleagues can supply. Journals today make use of “similarity searches” to ensure the authenticity and freshness of the material. Such searches often performed by means of seeking out acts of plagiarism or to see if authors have published such material elsewhere, and are thus claiming multiple publication credit. Editorial review systems that have moved to totally electronic access THE JOURNAL OF DISTILLING SCIENCE

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platforms have also been hacked of late and fraudulent papers published. Financial and non-financial rewards being earned by those getting away with these behaviors. Authors appearing on the title roster not having made any contribution to the research or the review are regularly called out.

The JDS will only accept manuscripts signed off by all authors, except in those rare circumstances when an author is no longer able to do so. The lead author or the team leader will then need to sign-off on any subsequent changes to the authorship. Authorship of publications currently the topic of debate within scientific circles (1-4).

IS REVIEWING RIGHT FOR YOU? We appreciate that the invitation to review for the JDS puts a lot of responsibility upon our collective team members. However, we thank you and believe your responsible actions and dedicated care to the task at hand will raise the bar on the quality of distilling publications and the broadening of knowledge. Knowledge and actionable data that is quite clearly needed and in a more central location by the distilling community. Searching through perhaps the only journal in the world with distilling in it’s title and at its core mission to find much needed information will prove of enormous value for the industry as a whole. We indeed hope this important responsibility is right for you.

THE REVIEW PROCESS AT JDS The review process is to be a single-blind model. Author’s names will be understood by the reviewers but authors will not know who reviewed their manuscripts. All reviewer models are not without their issues and inherent biases. The single-blind model seeing favor with pure science-based journals. See the references in the JDS Ethical Publishing in the 21st Century guide.

The JDS will field articles to three reviewers. Reviewers will receive a two-page Electronic Form as a PDF, along with the manuscript. The form will indicate reviewer #; 1, 2 or 3. These numbers fully meaningful only to the lead science editor. In certain cases this will relate to a hierarchical distribution based on the expertise of the reviewers chosen, or based on their indicated requested topic/subject areas for review. In other situations each reviewer will be on a level playing field. All reviewers’ comments and decisions will be important to making the final rejection or publish decision.

The reviewer form is largely self-explanatory with radio buttons for the final decision (accept, with or without revision, or reject) and two sections for commentary and 53

Guidelines and notes for reviewers

revision suggestions. One “Confidential” section will be seen only by the editor and the other, separated from the returned document, forwarded to the authors for them to make any necessary final changes prior to publication or for them to understand why their work has not met publication criteria.

The courtesy of a return of the completed reviewer form within three weeks of acceptance will greatly assist the speed to publication of all articles. Any reviewer who feels unqualified to review the research reported in the manuscript forwarded to them, or who knows its prompt review will be impossible must notify the lead editor or editorial office immediately and excuse themselves from the review process. Due to the confidential nature of the peer review process, the manuscript PDF or other files or documents should promptly be deleted or destroyed in such cases.

Certain journals allow authors to suggest reviewers — assuming no bias, and ensuring confidentiality — this possibility may occasionally be entertained. Likewise a member of our review board might suggest another person deemed suitable for the task. Decisions made to use alternates will only be known to the lead science editor, so no assumptions should be made as to employment of such persons suggested by author or fellow reviewers. For the coverage of papers from all countries, the reviewer body will grow with additional members suitably qualified as drawn from many countries across the globe.

In regards to all this, the lead science editor must also endeavor to avoid any biases and Gary Spedding (initial lead editor) hereby makes a promise to all authors, reviewers and team members of the Society of Distilling Scientists and Technologists (SDST), the JDS, and the publisher, and all others, that he will indeed do all in his power to avoid the biases and potential pitfalls at play in all such activities.

Upon formation of the SDST and the full incorporation of the journal into its jurisdiction the editorial committee to be formally approved will assume the greater role of governance over the editorial process.

THE ROLES AND RESPONSIBILITIES OF REVIEWERS AND CONFLICTS OF INTEREST Authors will be asked to declare any conflicts of interest when they submit their manuscript. As reviewers will be asked to scrutinize otherwise confidential documents they too must eliminate bias and also declare if they see any conflict of interest whatsoever. The editorial review board members being potential authors of manuscripts for the journal must not unfairly criticize or reject work based on their or their research group pursuing similar interests. This is one critique against the single-blind, as opposed to double- or even triple-blind reviewing. See the article on peer review — The Good, the Bad, and the Ugly (5) and the review — Reform and Renewal in Scientific Publishing (6) on 54

this matter.

Authors are referred to the American Chemical Society’s (ACS) Style Guide for a general discussion of the principles and practices of scientific publishing. The American Medical Association’s AMA Manual of Style, and similar works, are also recommended. Moreover, those authors’ whose primary language is not English are encouraged to seek out translation services whenever possible or needed. The JDS team not currently set up for such translation activities.

THE FLOWCHARTS OF INFORMATION To aid all authors and our reviewers across the globe we note the availability of flowcharts describing the publication principles and ethics involved in publication — including duplication of published materials, plagiarism, fabricated data, changes in authorship, ghost writing, the retraction of publications and biases issues. These guides, available in Chinese, Croatian, English, French, Italian, Japanese, Persian, Polish, Spanish and Turkish, are available from COPE (publicationethics.org). These charts will assist our reviewers to better understand all aspects of scientific publication and their duties as critical components in the process of publishing in the JDS and indeed elsewhere in the global scientific arena.

The Voice of Young Science (VoYS) research network discusses the ins and outs, advantages, and limitations of the peer-review process. A series of interviews with editors and reviewers provides a Q-and-A segment of interest (7). The VoYS publication also discussing the three main models of peer review in use today (7).

HOW REVIEWERS CAN AID IN THE ADVANCEMENT OF DISTILLING SCIENCE AND TECHNOLOGY INFORMATION AND KNOWLEDGE Peer reviewers are essentially gatekeepers — they determine which papers should be accepted for publication and which thereby become an important part of the body of knowledge accepted by the field. They may also influence, through suggested revisions, the overall appearance of the data and the overall picture portrayed to the world at large.

In so doing they must not alter the data, or bias the story in any significant way that takes away the voice of the authors, unless it genuinely corrects errors, mistakes or misinterpretations which the authors must address and deal with in revision. The process must also avoid reviewer-based biased interpretation. It is also to be hoped that nothing is lost in the translation of voices in one language to that of the publication (English in this case). Peer reviewers of course have come to be accepted as the appropriately qualified and unbiased experts in their respective fields.

Reviewers should always keep in mind that they are THE JOURNAL OF DISTILLING SCIENCE

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assessing the work and not the author. Reviewers will summarize for the author what they believe the paper aimed to achieve and if they succeeded in that overall aim. A reviewer will highlight the major findings, strengths and significance of the manuscript and point out any deficiencies.

Helping the author fully understand the larger significance of the work and potentially pointing out ways to express their ideas a little more clearly. Furthermore, reviewers will dissect the methods, results and discussion sections and try to ensure that all relevant background resources and references have been included within the arguments made in defense of the research. Reviewers should identify relevant published work that has not been cited by the authors. Any statement that an observation, derivation, or argument had been previously reported, but missed by the authors, should be accompanied by the relevant citation. Reviewers must also call to the lead science editor’s attention any substantial similarity or overlap between the manuscript under their consideration and any other published paper of which they have personal knowledge. Such manuscripts can be subject to similarity searches. An infographic: “Reviewer Evaluation Considerations” will be available to JDS reviewers covering concepts derived and adapted from Peer Review: Reform and Renewal (6); many points above and below expounded upon in that graphic.

Reviews should be honest, objective and free from personal prejudice. Personal criticism of the author(s) is inappropriate. Reviewers should express their views clearly and with supporting arguments.

A SUMMARY OF CRITERIA TO LOOK FOR IN A MANUSCRIPT Assessment benchmarks and their leading questions will often include (6): ORIGINALITY: Is the data new, and are concepts novel in

their nature?

VALIDITY: Are the results testable and reproducible?

CONTEXT: Do the authors seem to be aware of other similar work in the field? This will be seen through the introduction and cited references. [Editor: Today we are seeing more reinventions of the wheel with similar data and ideas having seen the light of day in decades old issues of journals. As big data collection catches up and more journal archives are completely recorded this issue may diminish. It is also hoped that the three-reviewer process will eliminate many cases of missed earlier works of a comparable nature to the one currently being reviewed.]

CLAIMS: Do the authors seem to overplay their hand with

respect to their tone and conclusions in relation to their actual findings?

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ACCURACY: Is the paper free of obvious errors? Are units, significant digits, algorithms and or statistical evaluations sound? Are references cited completely and correctly? Are there any ambiguous terms — especially important in understanding sensory descriptors. Different languages may have many terms for the sensory acuities and sensory attributes, or have only a few terms to describe the same things, and confusion can arise easily in this regard.

In reviewing experimental design and analysis protocols, a paper dealing with the mathematical and statistical rigor needed for publication in the British Journal of Pharmacology will be of use to authors and reviewers of materials to be published elsewhere, including in the JDS (8).

Is the context for the data collection described in sufficient detail? Are any exclusion or inclusion criteria covered (for data, or subjects in sensory trials for example)? How was the data analyzed? Are variables and control strategies discussed? Where are the experimental errors? How are/ were errors avoided, reduced or eliminated?

SYNTHESIS: If the article is a review of a specific topic or field of investigation, does it appear to be sound, comprehensive and balanced and, moreover, representative of a complete evaluation of the current literature. Does it appear to be unfairly weighted/biased towards the authors own findings or beliefs — or to others maybe exerting pressure on the authors? Articles in the JDS must not be overly commercial or advertorial in nature. [See A note on Review Papers below.]

LIMITATIONS: Do there appear to be limitations to the research or findings as presented and have the authors comprehended this and addressed them? Could other experiments or additional information improve the science?

Would the presentation be improved if extra work was to be recommended? What would that extra research work involve?

TECHNIQUES AND EVALUATION TOOLS: Were the appropriate methods, techniques, instrumentation, calculations and statistical tools applied and implemented correctly?

Were the right controls and number of replicates etc. used.

[See also under Accuracy above.]

ETHICAL ISSUES: Was everything done to and within accepted ethical standards and protocols? Are there any apparent conflicts of interest?

IMPLICATIONS: Does the work advance our understanding of the field? In what way is it contributing to our knowledge and how applicable are its findings to the industry at large?

Are the findings of a positive or a negative nature? Do they confirm or refute previous findings and concepts?

As an aside. Scientific publications have been accused of failing to report findings of a negative nature. This topic would form a lengthy treatise in its own right. Experiments 55

Guidelines and notes for reviewers

and research that genuinely and soundly conclude that systems do not work can be as valid as those that are workable concepts. While science must always be able to attempt to prove published results the presentation of negative findings can save others pursuing the same “dead end” leads.

A NOTE ON REVIEW PAPERS Review papers can form important contributions to the scientific literature but have been regarded sometimes as a quick way to publication, benefiting both author and publisher. The JDS will feature reviews, both invited and unsolicited — the call for papers covering original research topics, reviews and other types of technical reports. All materials will be peer-reviewed.

The hallmarks of a good review according to one author (8) seem relevant here. “There are two classes of useful reviews: (i) comprehensive and well-ordered literature surveys and (ii) (even better) surveys providing, in addition, novel aspects and views resulting from the synopsis of the original papers within a field.” (8) Authors and reviewers should both ensure that the JDS publishes only first-class reviews in furtherance of the dissemination of world-class quality knowledge applicable to the mission of the creation and appreciation of distilled spirits and related alcoholic beverages.

EXAMPLES OF LITERATURE CITATION/REFERENCE STYLE [1] Von Bergen, C.W.; Bressler, M.S. Volume 32. Academe’s

unspoken ethical dilemma: author inflation in higher education. The Research in Higher Education Journal. 32, 1-17. (No date provided.) https://files.eric.ed.gov/fulltext/ EJ1148909.pdf

[2] Tilak, G.; Prasad, V.; Jena, A. B. Authorship Inflation

in Medical Publications Inquiry. 2015, Jan-Dec; 52: 0046958015598311. Published online 2015 Jul 29. doi:10.1177/0046958015598311 https://www.ncbi.nlm. nih.gov/pmc/articles/PMC4943864/

[3] Is there an inflation in the number of authors per paper?

https://academia.stackexchange.com/questions/16759/isthere-an-inflation-in-the-number-of-authors-per-paper

[4] Wren, J.D.; Kozak, K.Z.; Johnson, K.R.; Deakyne, S.J.;

Schilling, L.M.; Dellavalle, R.P. The write position. A survey of perceived contributions to papers based on byline position and number of authors. EMBO Reports.

2007, 8, 988-991 https://www.ncbi.nlm.nih.gov/pmc/ articles/PMC2247376/pdf/7401095.pdf

[6] Etkin, A.; Gaston, T.; Roberts, J. Peer Review: Reform and

Renewal in Scientific Publishing. Charleston Briefings.

Against the Grain (Media), LLC. 2017. doi.org/10.3998/ mpub.9944026. https://quod.lib.umich.edu/c/cb/ mpub9944026/--peer-review-reform-and-renewal-inscientific-publishing?view=toc

[7] Standing up for Science 3. Peer Review: The nuts and

bolts. A guide for early career researchers. 2012. Voice of Young Science. https://senseaboutscience.org/wpcontent/uploads/2016/09/peer-review-the-nuts-andbolts.pdf

[8] Curtis, M.J.; Alexander, S.; Cirino, G.; Docherty, J.A.;

George, C.H.; Giembycz, M.A.; et al. Experimental Design and Analysis and Their Reporting II: Updated and Simplified Guidance for Authors and Peer Reviewers.

British Journal of Pharmacology. 2018. 175, 987-993. https://www.researchgate.net/publication/323665515_ Experimental_design_and_analysis_and_their_ reporting_II_updated_and_simplified_guidance_for_ authors_and_peer_reviewers_Editorial

[9] Schubert, I. We Have an Inflation of Review

Papers-for What Are Reviews Good? Frontiers in Plant Science. 2016. 7, Article 88. doi: 10.3389/ fpls.2016.00088. https://pdfs.semanticscholar. org/722c/d408f9feee9c3440d3e3e1668efcb6698d05. pdf?_a=2.159432381.1233659581.1612141289904542928.1612141289

[All links last accessed 2-04-2021.]

In formulating the policies and directives for publishing in the JDS, the above quoted references have proven useful guides to the current scientific publishing scene. The rules set forth by the Publisher and the Editor not limited to the advice derived therefrom. With all rights reserved, policies, activities and actions may change over time but will always be implemented to ensure the best possible practices of fair, truthful and unbiased publishing.

As in all matters of concern, or where further advice is needed, the lead science editor stands ready to assist. gspedding@jdsed.com

Terms and conditions and formatting guidelines may change from time to time, so please ensure you are reading the latest versions of the JDS guides for publishing within its pages and on-line.

Other materials, infographics and guides are available in relation to publishing in the Journal of Distilling Science.

[5] Research Solutions Marketing Team. Peer Review:

The Good, the Bad, and the Ugly. 2019. https://www. researchsolutions.com/blog/peer-review-the-good-thebad-and-the-ugly

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EDITORIAL

MANUSCRIPT FORMATTING & PREPARATION EDITION 2021

OVERVIEW AND SCOPE Full instructions to authors are provided in a separate document. The Manuscript Formatting and Preparation guide appearing separately here.

We encourage authors to refer to the American Chemical Society’s — The ACS Style Guide for a general discussion of the principles and practices of scientific publishing. The American Medical Association’s AMA Manual of Style, and similar works, are also recommended especially for “new to publishing” authors. Manuscripts will be subject to similarity check services to verify authenticity and originality. All manuscripts should be submitted in English (UK or American spelling accepted).

All cited references should carry an English title. Important contributions to the field are lost to us by journals not accepting genuine and legitimate references written in other languages. If the article was published in another language, the original title should also be supplied, along with a statement of the language it is written in (see 60). Reviewers will do all in their power to avoid or resolve ambiguity or misinterpretation of terms and meaning for authors whose primary language is not English. All authors are, however, encouraged to seek out the advice of those with both a grasp of the science and the English language (translation services) to aid in resolving any issues that may arise during their translation. The editor will follow up as needed. Manuscripts will be returned if illegible or incomprehensible.

MANUSCRIPT LENGTH Scientific articles are usually contained to shorter length pieces, by nature of the materials covered, though there are exceptions to all rules.

Typically the JDS expects to receive articles dealing with scientific discovery, and application of findings to process development, of about 6-10 pages (maximum 12) in American National Standards Institute letter size. Measuring 8.5 by 11 inches (215.9 by 279.4 mm) this being similar to “A4 paper standard” as adopted by many countries and as approved by the International Organization for Standardization and defined as ISO 216. Submitted manuscripts might be in different-sized format with the expectations that the final revised text will fit within the typical specifications listed above and below. Certain reports such as technical briefs, might of course be much shorter in length.

Documents fitting within the specifications noted above will vary in word count depending upon the thesis descriptive text, figures and tables, acknowledgments and THE JOURNAL OF DISTILLING SCIENCE

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references, but as a guide will be between 5000-10,000 words in scope. Times, Arial and the Symbol Font are commonly used, but the use of other suitable open access fonts should not in most cases pose any problems for submitted review copy. We suggest 12 point font size for main text purposes.

Reviews might conform to different specifications, and authors whose papers are expected to be longer or outside the limits of the specifications noted above may discuss this issue with the lead editor prior to submission of their work.

Manuscripts may be rejected on submission without review if they do not conform to these general guidelines.

Manuscripts will be rejected on submission without review if they lack scientific or technical merit — the aim of the journal being to present only exemplary research findings and views that best support the safe and efficient production practices, the quality production of, and the appreciation of the highest quality potable distilled spirits and other medium- to higher-strength alcoholic beverages.

MANUSCRIPT LAYOUT AND FORMATTING Classically the title of the work will be followed by the names of all authors with the lead or corresponding author noted with an email address supplied for subsequent outreach. All current affiliations for all authors must be illustrated with the submission (a form to accompany each manuscript — available online).

Manuscripts should be submitted in a line-numbered documentary format for ease of editing, with double-space paragraph formatting used (to aid in review and revision).

As noted above, it is suggested that the use of 12-point font size be used for main body text for clarity. Title headings will be formatted by level 1, 2, 3 headings to style by the publisher — authors may set their titles and headings in bold type to larger point font sizes. Italics should only be used within convention — such as for genus, species classification and genetic codification.

(Manuscript page numbers will of course be typically greater than the final layout page number. See notes above regarding article length, and anticipated word counts, and consult with the editor when preparing to submit manuscripts that will potentially produce lengthier articles.)

STRUCTURE Structural organization will vary depending on type of paper — review, original research, technical report, etc. For the research-style paper, the main document will generally 57

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consist of title, list of authors, abstract, an introduction to the topic, materials and methods (or experimental design and approach), results and discussion sections, conclusions, author contributions, acknowledgments and references (cited literature). Publisher-based mockups are available to act as a guide as needed.

TITLE Titles should be short and to the point. Articles are expected to be stand alone and not series pieces as such, nor, usually, as posed questions; so, should not be noted as part 1, part A, etc., and contain no quotation marks. Titles will be set in bold font with any taxonomic names in bold italics.

(See Reference Style, 60 for further guidance.)

AUTHORS AND AFFILIATIONS Authors are listed with the format: First name, initials (for middle names as required) and surname. Accented letters are allowed. Indicate the authors affiliation with a superscript number (1, 2, 3 etc) after each surname. Provide the address of each affiliation, with the superscript number preceding it. Identify the ‘corresponding author’ and provide the email address for this author only. Such information will also be provided by the authors in the agreement document that must accompany the submission of the manuscript.

Title Gary Spedding1, Heléne E. Copperstill2, Kashime Polk1 and George R.S. Pepperpots3.

(Addresses — this example would be three.)

(Corresponding Author: Gary Spedding. Email) The publisher will set details for the Corresponding author and author affiliations in the proof layout.

ABSTRACT Each article will have an abstract describing the work.

This abstract will only appear in the English language at this time. Abstracts will be of about 250 words in length and typically appear as a single paragraph of text. However, as modern practice allows for headings such as Aim/Scope, Methods, Results and Conclusion in short statements, the authors may consider such a format — keeping within the 250 word limit. Call outs to figures, tables, supporting information and references should not appear in the abstract.

RUNNING-HEAD KEYWORDS For search and indexing purposes the title abstract should be accompanied by four to six key words conveying the main terms or theme of the article.

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INTRODUCTION Typically all papers including reviews will contain an Introduction. An introduction should provide sufficient background information for the reader to understand the reasoning for the work, and the significance surrounding the topic area, then follow the flow of experimental results and subsequent discussion. Cited references should thus be specific, and place in context the work that is being reported. The introduction should not include sub-headings.

MATERIALS AND METHODS Experimental details will appear under a Materials and Methods heading. Facilities, instrumentation, and steps needed to perform the research will be covered in the necessary detail here — the publisher assuming no responsibility for, nor accepting any endorsement of specific company names or products. Articles appearing in the JDS are not to be of an advertorial nature, however, for the scientific tenet of the ability to reproduce experimental findings, sufficient detail must be supplied or referenced accordingly.

Theoretical, engineering, cooperage, still operations, warehousing/maturation, and sensory analysis papers etc., may need to include different style result or discussion headings/terms for the approaches taken by the authors.

Such considerations may involve unique or common terms such as blueprints, regulatory approval/safety notices, ethical approvals, noise/pollution control, lexicon/language/ descriptive terms, threshold limits, panel number, training and validation systems, scale, process, replicates and data analysis, and more according to specific discipline.

RESULTS AND DISCUSSION The preferred format for many journals is for a combined Results and Discussion section. Though flexibility is deemed important here based on the nature of topics to be covered, so separate Results and Discussion sections are permissible. For example, “Results, Methodology, Assay Development” or “Principles for Establishing Safety and Quality Standards”, “Sensory Analysis”, or, “Theoretical Modeling/ design”, etc., might be appropriate Results Heading Terms.

DISCUSSION AND CONCLUSION SECTIONS In view of the above, regarding results and discussions, if a separate Discussion section is covered by the authors they may consider adding in a final Conclusion subheading or a succinct closing Conclusion heading by way of summarizing their work and pointing to future actions, research and activities needed for the further advancement of the field.

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ACKNOWLEDGMENTS, COMPLIANCE WITH ETHICAL STANDARDS, FUNDING STATEMENTS, ETHICAL APPROVAL Specific Author Contribution statements/sections will also be included as appropriate (see below).

Funding and competing interests information does NOT belong under the Acknowledgments heading.

CONFLICTS OF INTEREST All articles will carry a statement conveyed by the lead/ corresponding author that, “The authors declare no conflict of interest” if that should be the case. Otherwise wording should be in place to describe any allowable interests, which do not preclude their publishing of their article, based on rules of the publisher and ethical practices. All contributors and authors understanding that, via agreement with their corresponding/lead author/contributor, they are signing off on a conflict of agreement statement prior to their manuscript being accepted for review. The agreement statement discussing varying conflicts of interest which may apply to publishing original content.

ORCID If applicable, authors may optionally include their ORCID ID (Open Researcher and Contributor ID — persistent digital identifier) to link readers to their professional information and gain credit for their contributions.

REFERENCES References should be cited as discussed below.

SUPPLEMENTARY MATERIALS If online and linked supplementary material is to be included, a statement illustrating this fact with a link to that material will be provided by the publisher. Supporting material should follow the rules of structure as noted herein and be supplied at the time of the main manuscript submission. Supplementary material is considered as supporting evidence that would dilute the storyline of the main article but helps interested readers delve deeper into the evidence presented. Such matter may include additional experimental details, chromatographic profiles, engineering diagrams, data storage account access links, and so on. Guidance from the editor, and via the reviewers will play a role here. Any such supporting information will be delivered to the editor as separately uploaded files.

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OTHER ARTICLES AND STYLE Review and short report-type manuscripts will entail a different structure but should follow general rules of writing practice and headings, subheadings, referencing, et cetera.

FIGURES AND TABLES Figures and tables, charts and diagrams will appear in manuscripts. These items should be used sparingly and only to enhance the coverage of the material when text alone will not convey the full story, or when long strings of text would lead to a cluttered array of terminology or organism species names etc. Think clarity of expression!

Figures and tables should be cited in order as “Figure 1”, Figure 2”, “Table 1”, et cetera. When citing as a group or multiples: “Figures 1 and 2”, “Figures 1-3”, “Figure 1a-1d”, and so on. Figure captions are to appear directly after the paragraph in which they are first cited. Tables cited in text do not appear with captions. “Specifically, as Table 1 and Figure 1 show...”, “Figure 1. Flow diagram of distillation process.” Figure 1. (a) (b) (c) etc., used for multi-diagram call out text (appears below the actual Figure). Example: “Figure 1. Title in bold text — legend description non-bold text”.

Tables will carry short clear headings (bold text) above the tabulated data and may include table footnotes (non-bolded text). Tables should be cell-based with no padding or spacing rows.

FIGURES: FILE SUBMISSION Typically GIF, PNG, and JPG file formats are used for online publishing with EPS (preferred) and TIFF files reserved for print publishing. An EPS file is a vector file of a graphic, text or illustration and, as it is a vector image, it can easily be resized to any size it needs to be. Files submitted with manuscripts will be titled “Fig1.tiff ”, Fig2.eps” etc. These files will be submitted separately as individual files in most cases. If there are any concerns about file types or processing please consult with the lead editor for further detail and advice.

Resolution of images should be 300-600 dpi (dots per inch). Dimensions on width: 789-2250 (max) pixels (at 300 dpi resolution) by 2625 pixels for max height.

Size ranges here will be from 2.63 inches (6.68 cm) to 7.5 inches (19.05 cm) width by 8.75 inches (22.23 cm) 4 inches in height. Note: max. height is full page and allows no room for captions. As for the body text the safest fonts to use will rely on End User License Agreements (EULA) and standard publishing practices.

Figures will be grayscale or, if color, adhere to the RGB color profile at 8 bits/channel. Be aware of different RGB profiles and color rendering.

Figure files should, moreover, be presented with the 59

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embedded figure or diagram/schematic in the actual orientation in which it should appear in the body article. Regardless as to desired orientation, figures must fit within the page size as noted earlier — no spanning of pages will be permitted for figures. Contact the editor first for any possible exceptions.

The publisher may send specific instructions prior to final layout of the manuscript.

EQUATIONS Chemical equations will follow standard practice and may make use of equation formatting programs. Mathematical and engineering equations and formulations should be formatted with suitable (Mathtype or Equation) tools and not use graphic objects. This will apply to both display/numbered equations and also to in-line text equations (though in-line may also be prepared using regular text formating tools). The symbol font should NOT be used for equations.

SI nomenclature, symbols, mathematical terms and units and significant figure expression and statistical rigor should apply in all cases.

REFERENCE CITATIONS All references cited in the text must be in the reference list and cited in order at first mention. Cited in brackets by number. “[1]”, “[2-5]”, “[3,7,9,11]” “[1-5,7,9,11-15,17]” and so on.

Reference details should provide complete publication information. Several styles and formats are seen in the literature. For the JDS, the following example formatting will be used.

For journal articles: all authors’ surnames and initials, year of publication, full title of the article, name of the journal, volume number, and page range of the article will be required.

For books: all authors’ surnames and initials, year of publication, title and page range of the book chapter (if in an edited book), title of book, editors’ names (if any), and the publisher’s name and location (city, state/country) will be needed.

For published proceedings (considered books: provide the publisher’s name and location (city, state/country), not the date and location of the meeting. In some journals the date (days) of the conference itself will be noted. The JDS considers it more important to state the publication date of the resulting Proceedings or Symposium volume.

EXAMPLES OF LITERATURE CITATION/REFERENCE STYLE JOURNAL ARTICLES [1] Abernathy, D.G.; Spedding, G.; Starcher, B. Analysis of

Protein and Total Usable Nitrogen in Beer and Wine Using a Microwell Ninhydrin Assay. J. Inst. Brew. 2009, 115,122-127.

[2] Curtin, C. D.; Langhans, G.; Henschke, P. A.; Grbin, P. R.

Impact of Australian Dekkera bruxellensis Strains Grown under Oxygen-Limited Conditions on Model Wine Composition and Aroma. Food Microbiol. 2013, 36, 241–247. doi: 10.1016/j.fm.2013.06.008.

[3] Marchal, A.; Prida, A.; Dubourdieu, D. New Approach

for Differentiating Sessile and Pedunculate Oak: Development of a LC-HRMS Method to Quantitate Triterpenoids in Wood. J. Agric. Food Chem. 2016, 64, 618–626. doi: 10.1021/acs.jafc.5b05056.

[4] Tian, Y.; Kong, X.; Fang, F. Microbial n-propanol

synthesis during Luzhou-flavor liquor Fermentation.

Acta Microbiol. Sin. 2020, 60, 1421-1432. (in Chinese).

BOOKS, DISSERTATIONS AND CHAPTERS IN BOOKS [5] Boothroyd, E. L.; Jack, F.; Harrison, B.; Cook, D. J. The

Impact of Increased Wash Fatty Acid Levels on the Nutty/ Cereal Aroma Volatile Composition of New Make Malt Spirit. In Distilled Spirits: New Horizons: Energy, Environmental and Enlightenment; Walker, G, Hughes, P., Eds.; Nottingham University Press: Nottingham, 2010, pp 167–173.

[6] Fukuyo, S.; Myojo, Y. Japanese Whisky. In Whisky:

Technology, Production and Marketing; Russell, I., Stewart, G.G., Eds.; Elsevier: Amsterdam, 2014, pp 17–26.

[7] Simpson, K. L.; Priest, F. G. Characterization of Some

Lactic Acid Bacteria from Scotch Whisky Distilleries.

Proceedings of the Fifth Aviemore Conference on Malting, Brewing and Distilling, The Institute of Brewing, London, 1999, pp 275–278.

[8] Merizalde Carillo, J.C. Feasibility Testing of Chill

Filtration of Brown Spirits to Increase Product Stability.

Masters Thesis, University of Louisville, USA. 2015.

ONLINE JOURNALS AND WEBSITES [9] Scotch Whisky Associations. Fact and Figures. https://

www.scotch-whisky.org.uk/insights/facts-figures/ (accessed May 18, 2020).

[10] Staub, T. (2001). Induced disease resistance in crop

health management. Online. Plant Health Progress. doi:10.1094/PHP-2001-0913-01-PS.

[11] National Institute for Occupation Safety and Health.

(2005). Carbon dioxide. In: NIOSH Pocket Guide to Chemical Hazards. Published online at www.cdc.gov/ niosh/npg/npgd0103.html. Centers for Disease Control, Atlanta.

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[12] Kyoto Encyclopedia of Genes and Genomes.

KEGG PATHWAY Database, 2020. https://www.kegg.jp/kegg-bin/highlight_ pathway?scale=1.0&map=sce00290&keyword=

(Due to the ephemeral nature of websites — such should be referenced sparingly in the journal. If in doubt about longevity it may be best not to cite such materials.)

UNPUBLISHED DATA AND PERSONAL COMMUNICATIONS

References that are only cited in the text. Websites, unpublished data, submitted manuscripts, personal communications and patent applications should only be quoted parenthetically in the text, with the initial(s) and last name(s) of all authors. We again note that online references should be used only sparingly if at all — as a last resort. Web links can be ephemeral and often not of safe archival-quality.

ABBREVIATED JOURNAL NAMES

For standard abbreviations for most journals consult the NLM Catalog: Journals referenced in the NCBI Databases.

Italicize but do not add a full stop/period for each abbreviation. DOI. The Digital Object Identifier is required for all appropriate references. This facilitates direct access to the paper where available. It must be added in the form https:// doi.org/10.

ENDNOTE & ZOTERO BIBLIOGRAPHIC TOOLS

EndNote is a commercial reference management software package, used to manage bibliographies and references when writing essays and articles. It is currently produced

by Clarivate Analytics. The JDS publisher and editor will utilize this software to assist in manuscript preparation for the journal. An alternative tool to use to help collect, organize, cite and share research is Zotero. Used in academic settings, this program is from the Corporation for Digital Scholarship.

SUMMARY OF RULES, TERMS AND RECOMMENDATIONS Every figure, table, and reference must have a call out in the text. References must be sequentially numbered in the order that they appear the first time in the text. If your article includes reproduced or adapted versions of previously published tables, figures, illustrations, or extensive quotations from other sources, you must obtain appropriate written permission, and provide copies of the correspondence to the lead editor of the JDS. Authors themselves are allowed the right to retain their own submitted materials for subsequent use (though not impinging upon the “not submitted anywhere else at this time” rules), but the layout of their article in the JDS is copyright reserved by the THE JOURNAL OF DISTILLING SCIENCE

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publisher. If others wish to utilize materials presented in the JDS, written permission must be sought from and released by the publisher and authors with all due citations and references to the source made for any subsequent publication.

For notes on authorship and contributorship roles in manuscript preparation see below.

As in all matters of concern or where further advice is needed, the lead science editor stands ready to assist: gspedding@jdsed.com Terms and conditions and formatting guidelines may change from time to time so please ensure you are reading the latest versions of all relevant instructions to author documents online.

CONTRIBUTORSHIP VERSUS AUTHORSHIP? Recent developments in publishing include the need to be aware of Contributor Role Taxonomy (CRediT). This system of citing the contribution of scientists, associated staff, and assistance teams involved in the research, preparation of data and manuscripts and the submission of the research results, or reviews et cetera, goes a long way to ensure visibility and diversity in research contributions. The system also shows the roles, and sometimes the actual weighting of each contributor’s/author’s contribution to the work.

Several references are available for the reader to review to find out more about this new system. See below.

For the immediate future the JDS will keep this system simplified and provide only a few general rules and ideas for its use within the JDS. In essence, multiple contributory terms apply to the CRediT system (see references). These include the supervisor of the research (mentors etc), the hands-on staff doing the bench work or the research (graduate students or post-doctoral fellows), those conceptualizing the research, designing the methodology — seeking the funding, designing and running the software or the data mining work, statistical validation teams, those providing resources and writing drafts and more.

At this time, the current editor feels that the supervisor, those doing the research and writing the manuscript would be considered the authors of the actual manuscript. Their contributions will be noted on the manuscript submission agreement sheet. If extensive parts of the work were done by different individuals; complementary methodologies run by different members of the team, or the design by them, of those methods or techniques, then they can and should be noted.

The names of suppliers of materials, samples or equipment, the funding source persons or organizations, or personal communications of advice givers, etc., belong in acknowledgments, and declaration of conflicts of interests or funding sources addenda headings as noted above (and on the manuscript submission sheet). The JDS will not accept 61

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advertorial articles. However both industry and academic contributions will be considered as they should be for the advancement of the field.

For this journal, an exception on not including authorship-ownership by funding personnel covering the research costs and preparation of the manuscript might be a distillery owner or group funding the research, depending upon their actual involvement if the work is performed in another research facility or laboratory, and how the project was conceptualized, addressed and the significance of their findings. However, we feel they must also have been involved in the actual research in a more hands-on manner to consider an authorship role on their manuscript. This can be so noted on the manuscript submission agreement form and in the text of their submitted manuscript. Relying solely on third-party laboratory input or data should be noted. If any doubts persist here please consult with the editor.

The JDS and editorial team will, however, grow with the “Indicate Who Did What” CRediT process as it develops, and we encourage our contributors to be aware of this important newer program, and for them to consult some of the references below to learn more about this program development. Most importantly, however, we hope to encourage authors to better illustrate their responsibilities behind the work they submit for presentation to the JDS. This will ultimately help ensure transparency, visibility, removal of biases, ensure credit toward future career moves by scientists, and better illustrate potential conflicts of interest, or a lack thereof. Ensuring the validity and integrity of the scientific process and contributions towards distilling science and the production of high quality, flavorful, and safe to consume alcoholic beverages — thus, the mission of the JDS.

REFERENCES TO THE CRediT CONTRIBUTOR, NOT AUTHORSHIP PROCESS ON-LINE REFERENCES:

https://casrai.org/credit/#:~:text=CRediT%20 (Contributor%20Roles%20Taxonomy)%20is,contribution%20 to%20the%20scholarly%20output https://www.enago.com/academy/experts-take-giving-propercredit-in-multi-authored-publications/ https://www.editage.com/insights/how-journals-are-usingcredit-to-capture-author-contributions-in-editorial-manager https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3164109/ pdf/fpsyg-02-00196.pdf https://www.nature.com/news/publishing-credit-wherecredit-is-due-1.15033 https://us.sagepub.com/en-us/nam/credit (These links were last shown to be active in April 2021. As noted above, the use of web references which can be ephemeral should be used sparingly if at all to ensure archival-quality of information and data.)

PUBLISHED REFERENCES: [1] Tscharntke, T.; Hochberg M.E.; Rand, T.A.; Resh, V.H.;

Krauss, J. Author Sequence and Credit for Contributions in Multiauthored Publications. PLoS Biology. 2007, 5, Issue 1, e18. doi: 10.1371/journal.pbio.0050018.

[2] Eggert, L.D. Best Practices for Allocating Appropriate

Credit and Responsibility to Authors of Muli-authored Articles. Frontiers in Psychology. 2011, 2, Article 196. doi: 10.3389/fpsyg.2011.00196.

[3] Allen, L.; O’Connell, A.; Kiermer, V. How can we Ensure

Visibility and Diversity in Research Contributions? How the Contributor Role Taxonomy (CRediT) is Helping the Shift from Authorship to Contributorship. Learned Publishing. 2019, 32, 71-74.

[4] Holcombe, A.O. Contributorship, Not Authorship: Use

CRediT to Indicate Who Did What. Publications. 2019, 7, 48. doi: 10.3390/publications7030048.

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SPECIAL THANKS TO OUR SPONSORS

JOURNAL OF DISTILLING SCIENCE

THE OFFICIAL PUBLICATION OF THE SOCIETY OF DISTILLING SCIENTISTS AND TECHNOLOGISTS

JOURNAL OF DISTILLING SCIENCE THE OFFICIAL PUBLICATION OF THE SOCIETY OF DISTILLING SCIENTISTS AND TECHNOLOGISTS

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Winter 2021