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Vol 13 Issue 3 July 2021

AQUAFEED Advances in processing & formulation An Aquafeed.com publication

SOY IN AQUAFEEDS Odor control Natural carotenoids Aquafeed evaluation model Published by: Aquafeed.com LLC. Kailua, Hawaii 96734, USA www.aquafeed.com info@aquafeed.com

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Species-specific solutions for a sustainable and profitable aquaculture At Adisseo, we offer species-specific nutrition and health solutions to aquaculture customers around the world. There is a lot to gain by optimizing your feed additive strategy. Our aqua experts are passionate to help you find out how to increase your productivity and profitability. We look forward to sharing our vision with you!

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AQUAFEED

VOL 13 ISSUE 3 2021

Contents

FUNCTIONAL AND SUSTAINABLE SOY INGREDIENTS FOR AQUACULTURE RATIONS 21 Soy varieties with elevated and ultra-high protein content and very low levels of oligosaccharides are intended to function as an alternative product to soy protein concentrate.

ODOR CONTROL IN AQUAFEED PRODUCTION 17

A SUSTAINABLE FEED INGREDIENT FOR AQUACULTURE 27

CAROTENOIDS FOR SALMON AND SHRIMP 34

How to reduce the odor emission coming from the processes of aquafeed production through non-thermal plasma.

A high protein feed ingredient produced from the fermentation of sweet potato biomass could reduce the use of fish and soybean meals in aquafeeds.

Natural carotenoids are reliable pigments for salmon and shrimp, enhance animal health and meet consumer’s expectations.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

AQUAFEED

VOL 13 ISSUE 3 2021

Contents 6 Interview 11 News Review 14 Saving costs in vacuum coating in the production

of aquaculture feeds

Odor control in aquafeed production using non-thermal plasma

21 Better from the beginning: Using plant genetics and value chain integration to provide functional and sustainable soy ingredients for aquaculture rations

*Cover photo

24 Soy in aquaculture 27 Fermented sweet potato biomass:

a sustainable ingredient for aquafeeds

ctivated zinc oxide: An innovative nutritional approach A to reduce WSSV-induced mortality in shrimp

34 38

Natural source of carotenoids for salmonids and shrimps

50 52

The gut microbiota drives the successful replacement of fishmeal by microbial and processed animal proteins in gilthead seabream

Aquafeed evaluation under specific farming conditions: A RAS application

How to help the anti-infective immunity of European carp

How we used biosynthesis to produce the world’s strongest antioxidant

Columns 46 Albert Tacon – Disorders in mineral nutrition

Calendar of events

To read previous issues in digital format or to order print copies, visit: http://www.aquafeed.com/publications/aquafeed-magazine/

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

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Karim Kurmaly is Chief Executive Officer of Veramaris. Karim was with DSM for 16 years, most recently as Vice President Animal Nutrition & Health for the Asia Pacific and India region. He is a marine scientist who holds a PhD from the University of Wales.

INTERVIEW AQ: What has been Veramaris' journey in the feed industry? KK: Veramaris® is borne out of technology discovered over 35 years ago by NASA scientists who were asked to solve the challenge of growing food in space, where sustainability is a matter of survival. They discovered algae that produced high levels of omega-3, essential nutrients for life. This breakthrough led to the production of Veramaris’ algal oil as we know it today, the next-generation algal oil. It provides a rich source of both EPA and DHA omega-3 with a potency of 60%, the richest available on the market.

with Karim Kurmaly The company was founded in 2018 by two of the world’s largest science and chemicals companies, DSM and Evonik, as a joint venture. They recognized the potential for Veramaris’ algal oil technology to provide solutions to the challenges faced by the aquaculture industry including sustainable growth, fish health and human health. Thirty-five years of R&D have made us experts in algal technology. Since the company’s inception, Veramaris has won the Future of Fish Feed Challenge for sales of its omega3 algal oil. We have also become the first microalgae oil producer to achieve joint ASC-MSC certification, adding

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

an estimated 45% to the global supply of MSC certified EPA & DHA omega-3, and tripled production at our manufacturing plant in 2020. AQ: Veramaris oil is currently being used in salmon and trout feeds, but what are the perspectives on its utilization in other aquaculture species? KK: Veramaris is utilizing technology to optimally increase levels of both EPA and DHA omega-3 fatty acids in feed to ensure farmers can secure fish health and welfare, and deliver a quality product that is nutritiously rich in omega-3 to end consumers. Veramaris has the ability to substitute and ultimately replace fish oil for a variety of aquaculture species. Today, our algal oil is used to raise salmon, shrimp, trout, steelhead trout, yellowtail, largemouth bass, seabream and seabass. And there are many more possibilities on the horizon. There are also shrimp that are entirely raised on Veramaris algal oil – a “vegetarian shrimp” – with zero fishmeal and zero fish oil. As it stands, by using Veramaris EPA and DHA omega-3 algal oil in the salmon industry alone, aquaculture will be able to reach its target of becoming a net producer of fish – a substantial sustainability milestone for the industry. AQ: With your facility in Nebraska, USA, Veramaris has the capacity to meet 15% of the global omega-3 fatty acid demand for salmon aquaculture. Does the company have new investments scheduled? KK: Our $200 million state-of-the-art facility in Nebraska is run on renewable energy, produces zero waste and consistently delivers a highly potent natural marine algal oil that contains the same high levels of both EPA and DHA omega-3 - 24/7, 365 days a year. In 2020, production tripled. Using the F3’s own forage fish calculator, the use of Veramaris algae oil saved more than 8 billion forage fish from use in aquaculture feed. Our capacity to cover 15% of the global salmon industry’s annual demand of omega-3 is equivalent to the amount of EPA and DHA omega-3 derived from 1.2 million tons of wild-caught fish. To put this into perspective the annual wild catch of the Mediterranean Sea is 0.8 million tons. Our international R&D team is continually increasing the already high levels of both EPA and DHA omega-3 by improving yields, enhancing the fatty acid profile, and

Our international R&D team is continually increasing the already high levels of both EPA and DHA omega-3 by improving yields, enhancing the fatty acid profile, and increasing output to even higher levels. In fact, our algal oil is richer than ever before, with a potency level of 60% compared to 50% previously. increasing output to even higher levels. In fact, our algal oil is richer than ever before, with a potency level of 60% compared to 50% previously. Our algal pioneers have a reputation today which is unmatched. AQ: Veramaris oil became the first microalgae oil producer for feed to achieve joint ASC-MSC certification. What is the expected impact of this certification? KK: Being recognized as the first and only microalgae oil producer for feed to achieve joint ASC-MSC certification is a significant achievement for Veramaris and a genuine testament to the strength of our team. Importantly, the certification adds an estimated 45% more MSC-certified EPA and DHA omega-3 to global supply, supporting the industry to grow within planetary boundaries. The impact of ASC-MSC certification extends throughout the value chain reaching retailers, consumers, feed millers and fish farmers. Retailers are under intense scrutiny not just from customers but from shareholders and NGOs who are reviewing retail ownership of feed ingredients used in animal production. We know that large European retailers are concerned about the quality of feed ingredients and certification means that retailers can now demonstrate their commitment to responsible sourcing in a tangible way. We also know that food safety is a critical factor for consumers when making

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

In 2020, production tripled. Using the F3’s own forage fish calculator, the use of Veramaris algae oil saved more than 8 billion forage fish from use in aquaculture feed. seafood purchasing decisions and that consumers are putting pressure on retailers. Feed millers can now produce feed not only with the highest levels of both EPA and DHA omega-3 but also with an ingredient that is ASC-MSC certified. Farmers know they can produce healthy fish, with the higher levels of EPA and DHA omega-3 resulting in secure fish health and welfare, and a more robust fish. Ultimately, ASC-MSC certification results in tangible benefits for all stakeholders along the value chain allowing the industry to grow sustainably. AQ: Veramaris partnered with several retail groups in different countries to deliver Veramaris-fed salmon and trout. How are retailers and the foodservice sector perceiving Veramaris oil? KK: Today’s retailers are under severe public scrutiny to drive change throughout their food supply chains. We now see retailers taking accountability for the feed ingredients they use while consumers are asking more about the provenance of the food they consume. Veramaris offers many of the answers that retailers and consumers have been searching for, particularly around sustainability, fish health and human health. For example, salmon fed on Veramaris’ natural marine algal oil delivers the highest levels of EPA and DHA omega-3 in salmon fillets, levels not seen in decades. Consequently, it also offers the highest standards in fish health and food safety. Veramaris’ value chain approach has resulted in meaningful collaborations with retailers around the world providing Veramaris-fed salmon and trout, naturally rich in sustainable EPA and DHA omega-3, to consumers. Large European grocery retailers are already marketing substantial volumes of fish and supporting the use of algae-based feed in aquaculture.

French retailer Supermarché Match achieved over 12% category growth in its salmon category, following the introduction of salmon from Norwegian farmers Lingalaks and Lerøy, raised on a diet including Veramaris’ algal oil. Veramarisfed salmon is also on sale in European retailers including Cora, Auchan and Kaufland, as well as US retailers such as King’s Food, Baldacci’s and online retailer KnowSeafood. AQ: Veramaris won F3 Challenge in 2019. How do we grow aquaculture sustainably? KK: Feed ingredient suppliers and indeed the aquaculture industry as a whole face a common challenge: to help the industry grow within planetary boundaries while protecting fish health and human health. To quantify the challenge, 45 million MT of extra feed is required to feed the growing population. So, how do we grow aquaculture sustainably, meeting a growing demand? Veramaris’ technology allows farmers to optimally and consistently maintain levels of both EPA and DHA omega-3 fatty acids in feed to secure fish health and welfare, and deliver a quality product that is nutritiously rich in omega 3 to end consumers. Scientific research shows that high levels of EPA and DHA omega-3 in feed improves fillet quality and provides a high level of omega-3 in the fillet. And all this can be done without increasing FFDRoil or dependency on wild catch fish. Feed millers can now produce feed with the highest levels of both EPA and DHA omega-3. Because of the very high potency of our algal oil of over 60%, low volumes need to be transported across oceans and less tank space and extra handling is required. Retailers are facing increasing pressure from consumers, stakeholders and NGOs on fish health responsible sourcing and are now beginning to ask for sustainable, EPA and DHA omega-3 rich seafood. Our purpose is to expand the world’s access to sustainable and consistently EPA and DHA omega3 from natural marine algae, which allows us to contribute towards the continued sustainable growth of aquaculture, within planetary boundaries. Today, we see industry leaders using Veramaris natural marine algal oil because it’s a sustainable and affordable source of natural algal omega-3.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

AQ: And finally, where would you like to see the aquaculture feed business in 10 years? KK: Currently, three billion people worldwide rely on seafood as their main source of protein and aquaculture will need to increase production by 30-40 million tons by 2030 to keep up with the current rate of consumption. In 2020, 22 million tons of fish production were used for fishmeal and fish oil for the aquaculture industry. This has led to stocks of wild-captured forage fish, such as anchovies, herrings and sardines, being fished close to the maximum yield. It has also led to a decline in omega-3 levels in fish. This is clearly unsustainable and will not meet the demand of our growing population, which is expected to reach 9.7 billion by 2050. Only aquaculture can fill the gap, yet the constraint to the growth of aquaculture is the industry’s reliance on marine ingredients used in the feed. This is why we are helping reduce aquaculture’s reliance on marine resources with technology like Veramaris® and nutritional solutions that enable sustainable aquaculture.

Veramaris expands the world’s access to certified EPA and DHA omega-3 by 45%. Both fish and humans need a reliable source of both EPA and DHA omega-3 for optimum health and Veramaris’ natural marine algal oil provides a consistently rich source of both these essential nutrients compared to fish oil. Fish oil alone cannot provide fish and humans with the required levels of both EPA and DHA omega-3. A successful future is about collaboration. And, as an industry, we must work together to increase levels of EPA and DHA omega-3 that we see in feed, fish and fillets. By doing so, we can continue to deliver on the brand promise to our consumers, and raise fish that provides answers and do not raise questions. Today, we see industry leaders using Veramaris natural marine algal oil because it’s sustainable and an affordable source of natural algal omega-3. Ultimately, our mission is to increase the world’s access to EPA and DHA omega-3, and in ten years, I hope we are well on our way to achieving our goals.

CLEAN FEED. CLEAN WATER. Wenger Extrusion Solutions for RAS Feed Production Wenger innovative extrusion solutions deliver clean, durable, nutritional feeds specifically designed for the most efficient RAS operations. Feeds produced on Wenger systems maintain their integrity better and longer, for clean and clear water. So you feed the fish, not the filter. Learn more about the Wenger RAS advantage. Email us at aquafeed@wenger.com today. PHONE: 785.284.2133 | EMAIL: AQUAFEED@WENGER.COM | WENGER.COM USA

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Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

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NEWS REVIEW Highlights of recent news from Aquafeed.com Sign up at Aquafeed.com for our free weekly newsletter for up-to-the-minute industry news

Skretting introduces new water care solution for fish and shrimp ponds

Salmofood introduces a new nutritional supplement to protect gills The company launched a new nutritional supplement called Recover, a product that has been added to its line of CareBlocks. Recover prevents and reduces the damage caused by pathogens, parasites, contaminants and physical injuries in the gills.

Skretting is expanding its AquaCare portfolio with a new water care solution for fish and shrimp pond farming. AquaCare Mineral Balance is a mineral combination offering high bioavailability of macro and trace minerals. This practical, cost-effective solution ensures that pond systems have stable, nutrient-rich water that is also ionically balanced, thereby providing the platform necessary to optimize species’ growth and health.

ASC unveils its feed standard tackling unsustainable practices The standard requires that feed mills meet strict environmental and social requirements, source ingredients from socially responsible suppliers, and use environmentally responsible raw materials. In doing so, issues in both the supply chain and at raw material level are addressed. Requirements on reporting of performance will also improve the transparency of the industry, reward environmental sustainability, and assist future research into responsible feed.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

University of Illinois Feed Technology Center open for business After nearly two years of construction, the new Feed Technology Center at the University of Illinois is complete and open for business. The 12,000 square-foot facility is optimized with leading-edge equipment, including smart sensors to monitor feed ingredient quality in real-time, digital automation tools to ensure precise diet formulations, a standalone extrusion line to create high-quality pet and livestock feeds, current safety and sanitation features, and more.

BioMar’s new RAS feed targets the bottleneck in marine nurseries

BioMar unveiled a new RAS feed concept, LARVIVA ORBIT, that aims to improve the

efficiency of marine nurseries. It will support an industry move of a prolonged time at the hatchery for fry before they are transferred to sea. This will aid in the expected acceleration in the use of RAS technology in marine nurseries, and thereby support the development of hatchery businesses for marine fish species.

New feed facilities and expansions

Vinh Hoan started building a new aquafeed factory in My Hiep, Dong Thap, Vietnam expected to go into operation at the end of 2021. It will have a production capacity of 350,000 tons per year in a total area of more than 6 ha. Thai Union Feedmill Public Company Ltd. established a new joint venture named AMG-THAIUNION FEEDMILL (PRIVATE) Ltd. in Pakistan to manufacture and distribute fish and shrimp aquafeeds. Skretting plans to expand its production capacity through the purchase of Ridley’s extrusion facility in Westbury, Tasmania. Cargill started producing its fry starter feed AquaXcel® at its Serang plant with the aim of enabling aquaculture customers to thrive by achieving more sustainable growth.

Alltech Coppens develops sustainability scoring system for feeds Alltech Coppens Aqua Center developed a sustainability scoring system for feeds. This scoring system aims to create awareness

in the market and enable fish farmers to improve environmental performance. The resulting score reflects the carbon footprint,

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

as well as the impact on other aspects, such as water use, eutrophication and even marine resource sustainability.

NEW ON THE MARKET Probiotic additive to improve water quality in shrimp ponds Evonik, Vland developed AQUAVI® ProPond for improving water quality in aquaculture. It is tailored primarily to the requirements of shrimp farming in ponds. The new probiotic additive is the result of about two years of joint development work and is to be launched in China at the end of the year.

FOSS unveils fully-automated mycotoxin analyzer for rapid tests FOSS introduced MycoFoss™, a new mycotoxin analyzer for rapid tests for the presence of mycotoxins in grain and corn. It is operated at the push of a button. Users simply place a ground sample in the instrument and select the appropriate test on a touch screen display. Results for key mycotoxin risks including DON, FUM, AFLA, ZEN, OTA and T-2 are delivered within eight minutes. A multiplex option allows up to six mycotoxins to be tested in one go.

Evonik upgrades feed raw material database with new features Evonik introduced AMINODat® 6.0, a database for feed raw materials that is up to date at all times and more comprehensive than any other offer available to the feed industry. Based on this database, feeds for livestock can be formulated with even greater precision.

PEOPLE IN THE NEWS Helene Ziv-Douki Cargill named Helene Ziv-Douki president for Cargill’s Aqua Nutrition business. Ziv-Douki, who joined Cargill in 2003, succeeds Pilar Cruz who was recently promoted to Chief Sustainability Officer, a new role for the company.

Brett Glencross Professor Brett Glencross will take over as IFFO’s technical director. He has been an important contributor to the aquaculture sector and academic research work for the last 20 years.

Donald Lightner Shrimp industry veteran, Donald V. Lightner, passed away. Lightner put together a team of researchers at the University of Arizona that provided much of the basic knowledge on shrimp disease and health which enabled shrimp aquaculture to grow into a global industry.

Tietjen introduces new grinding solution The Crusher CR is a versatile machine for coarse crushing of bulk materials. It is suitable, for example, for crushing agglomerates or for pre-crushing coarse pieces of material for further

grinding. Pre-grinding with the Crusher CR reliably protects the sieves and thus ensures consistent product quality and less downtime and wear costs.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

Saving costs in vacuum coating in the production of aquaculture feeds Stefan Lungwitz, Amandus Kahl

For liquid addition in feed production, spraying at the end of the production line (post-pelleting application, PPA) has been established in recent years. In case a liquid quantity of greater than 7-8% has to be added to pellets, a vacuum application after pelleting or extrusion is inevitable. Compared to spraying pellets under normal atmosphere, 30-75% more liquid can be introduced into a product without it showing liquid on the surface. In the vacuum application, the liquid medium is not only bound to the surface but penetrates the core of the pellets at the porous fracture surfaces

and pores. Thus, fat additions of up to 30-40% are possible with extruded feed and 8-10% with pelleted feed. Enzymes and other active ingredients can also be added in small quantities, either directly or in a liquid mixture (blend). The vacuum coating process was developed in the 1990s for the salmon feed industry in Norway and is now, 30 years later, also used for the production of pet food, poultry feed and in the food industry. The most common types of vacuum coater to date are twin-shaft paddle mixers and conical screw mixers.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

Long-known points of criticism with these mixer designs are high fines formation due to aggressive mixer tools, insufficient hygiene (caking, dead spots) and high specific mechanical energy consumption. Due to these points and the resulting market requirements, the machine manufacturer, Amandus Kahl, has reconsidered this process and developed a new technology for vacuum coating. The new Gentle Vacuum Coater GVC consists of a rotating mixing drum that swivels around its own axis into different positions during the respective work cycles. The interior of the machine is equipped without separately rotating mechanical mixing tools, which offers maximum product protection and minimum breakage and abrasion. This is a crucial aspect, as resulting fines can lead to caking and, due to the higher surface area, would also bind more liquid and thus tend to overconcentrate. The mixer cover of the GVC contains both the spray nozzles for up to ten liquids and a special rubber lip seal for sealing during vacuum generation. After a sufficient vacuum of up to 50 mbar abs. has been generated in the machine, the liquid is sprayed onto the kidney-shaped product accumulation that forms during the mixing process. This then mixes gently according to the free-fall principle. The mixing drum is emptied by a vertical swiveling movement. Such a process with the machine type GVC 1000 was recently put into operation at the world's largest feed producer, among others. At the production site in Thailand, the machine is used to refine extruded fish feed. Continuous operation on site has confirmed that, in direct comparison to a twin-shaft paddle mixer, the Gentle Vacuum Coater GVC can achieve consistent product quality with the production of less pellet breakage and fines. An analysis of the operating data shows that this machine can achieve enormous potential savings in production costs.

Cost savings through the reduction of pellet breakage and fines From the perspective of plant operators, fines are a cost factor that should not be neglected. In the production of fish feed, the rule of thumb in Central Europe is €25 per ton (€/t) of recycled fines. The reason is that in so-called dry recycling, the fines have to be added again proportionally before grinding and thus have to pass through the entire process again. This means that the entire mechanical and thermal process energy is used again. On top of this, there are the wear, maintenance and personnel costs, as well as the loss of production capacity due to precisely these fines. Calculated on the basis of an example of a medium-sized fish feed company, an annual production of 80,000 tons per year results in about 1-3% fines that are screened out of finished pellets.

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Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

The causes of the fines are, apart from the stress on the pellets caused by mechanical conveying, mainly the mechanical stress during vacuum coating (e.g. with the twin-shaft paddle mixer). These 1-3% fines result in a total quantity of 800-2,400 tons per year in the aforementioned operating example. Calculated with the above-mentioned rule of thumb of €25 per ton of fines, recycling costs of €20,000-60,000 per year accrue for this production plant. It has been shown that by using the Gentle-VacuumCoater compared to a twin-shaft paddle mixer, the production of fines during vacuum coating can be reduced to less than 0.1% on average. Thus, the annual savings potential for a production plant is obvious. Another problem is the fines that are produced both during transport of the feed to the fish farm and during fish farming due to the use of automated feeding systems. Particularly in the case of pneumatic pressure conveying from the transport ship to the floating net cages, as is frequently used in Scandinavia, for example, the stress on the pellets is especially high. Excessive prestressing of the pellets in the vacuum coater can lead to "micro-cracks" in the pellet structure of the finished feed. These cracks then lead to a further break-up of the pellet structure after delivery to the fish farmer due to pneumatic pressure conveying (air speeds > 30 m/s). This results in pellet breakage and renewed fines formation. This fraction cannot be eaten by the fish and reduces the feed conversion ratio (FCR). In recirculating aquaculture systems (RAS), this additionally leads to excessive contamination of the filter systems in the water treatment. This loss of feed and the resulting loss costs for the farm are usually significantly higher than the example calculated above, depending on the size of the fish farm. Using the example of a medium-sized Scandinavian salmon farm consisting of 6 to 10 floating net cages, up to 40 tons of pellets worth approx. €60,000 are fed per day, depending on the water temperature. These feed costs give an idea of the dimensions in which savings can be achieved for the fish farmer by avoiding microcracks and the resulting fines.

Cost savings through the reduction of energy consumption Furthermore, the design of the new GVC provides potential savings in energy consumption. Compared to

other vacuum coater designs of the same size, the GVC, operated by a 4 kW drive motor, requires up to 80% less drive power. In addition, in the standard version, all drive motors in the entire system are operated with frequency converters (vacuum coater, vacuum pump, liquid pumps), which can save more than 40% energy per drive motor.

Cost savings through automatic cleaning (CIP) As vacuum coating takes place at the end of the production line, there are high requirements in terms of hygiene and cleanability to prevent contamination. Compared to the twin-shaft paddle and conical screw mixer designs, the GVC has a high degree of selfcleaning, as the entire mixing drum is permanently in contact with the product. This means that there are no dead spots. In the event that cleaning with a rinsing liquid is nevertheless necessary, for example after the use of medical additives or other critical additives, the system offers a fully automatic cleaning program according to CIP (cleaning in place). The special feature is that this cleaning process does not require any personnel to carry out the disassembly and cleaning work on the machine. A water-based cleaning liquid is added completely automatically, and after a short washing process, it is automatically drained into a separate outlet and disposed of. This results in significant savings for maintenance personnel as well as a reduction in downtimes. Summary Depending on the application example and the plant size, vacuum coating offers potential savings in operating costs in many respects. These can be easily calculated for each application.

More information: Stefan Lungwitz Process Engineer Amandus Kahl, Germany E: lungwitz@amandus-kahl-group.de

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

Odor control in aquafeed production using non-thermal plasma Merijn Appelman, Aerox

With the increasing amount of people living on earth and the higher demands that we have on our living environment, odor control is getting more and more important. In certain industries, odors cannot be avoided during processes like extruding, drying or cooling. Especially in aquafeed production where smelly ingredients are used like fishmeal, a lot of odor is emitted via the exhaust air of those processes. To have fresh air for your neighbors and/or comply with odor legislation, odor control is often a necessity. A common and effective used technique for odor control in the aquafeed industry is the Non-Thermal Plasma Injection technology (NTP).

What is non-thermal plasma? Plasma is commonly known as the fourth state of matter, alongside liquid, solid and gas. It is made up of ionized gas, which consists of a balanced mix of electrons, positive ions and neutral particles. Due to the highly energetic state of the constituents of plasma, it produces its own electromagnetic radiation. Aerox developed NTP-modules that generate NTP by electricity. Since NTP operates at room temperature, relatively little electricity is needed, ensuring a sustainable and cost-effective odor control solution. The NTP does not generate a significant temperature increase in process airflow. How does it work? Ambient air is cleaned using a three-stage filter (Fig. 2) and then pushed into the injector cabinet where it passes through the NTP-modules. Within the plasma modules, oxygen and water vapour molecules

Figure 1. Non-Thermal Plasma module.

are split, transforming them into active oxygen. This highly reactive mixture is then directly injected into the process air exhaust duct. The active oxygen in the injection air executes a high-speed oxidation reaction (< 0.5 seconds) with the offensive odor molecules inside the process air, creating less smelly or undetectable molecules, solving the odor problem.

Odor control in aquafeed production To reduce the odor emission coming from the processes of aquafeed production one can choose to treat the

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

most relevant odor emitting sources like the extruder, dryer and cooler or one can choose to treat all the odor sources. In most cases, the most relevant odor sources are treated by an odor abatement system like the NTPsystem. To establish the best way to solve the odor problem, these questions should be answered: How many production lines does the plant have? What is the airflow of each line/source? Are results of odor measurements available? After having these questions answered, we can estimate what capacity of the Aerox-Injector NTP system is needed. To verify this estimation a pilot test can be performed including independent odor measurements to check the odor reduction efficiency

Figure 2. Three-stage filter.

of the system. With the results of this pilot test, the optimum capacity of the NTP system can be chosen. The NTP technology is especially suitable for the aquafeed industry because the odorous components are adequately converted to less odorous components achieving an odor reduction of 75% - 95%.

Sustainability The design of NTP units makes it easy to recycle almost any part of the unit. The NTP modules need to be replaced after a certain period. The modules are made up of glass, metal and plastics which are, if correctly separated, easy to reuse or recycle. When the modules are to be replaced, we aim to reuse as many of the modules as possible. The parts that cannot be reused directly are recycled which means that not one part is wasted completely. The NTP unit does not need any fossil fuel, chemicals or water. All the unit needs is a relatively small amount of electricity to operate. Since no water and chemicals are used there will be no wastewater stream. In the current life, more and more things are automatically controlled. Implementing automated controlling by using the right sensors not only reduces time for the operators but can also contribute to energy

Figure 3. Typical set of an NTP-system treating 60.000 m3/h.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

Figure 4. Odor dispersion modelling graphics without odor reduction (left) and with 75% odor reduction using an Aerox-Injector unit (right).

savings. By using Internet of Things (IoT), it is possible to control the unit, e.g. using a windvane, the unit can be shut down when the wind direction is in the opposite direction of a nearby urban area. Using such tools even more energy can be saved. With increasing prices for tradable carbon emissions and fossil fuels, the use of sustainable solutions for odor control is getting more and more important. Using an NTP unit solely on sustainable energy the CO2 created by the unit can even be reduced to zero (calculation based on Dutch averages).

Odor control and environmental impact Since odor can be a significant nuisance for people living close to factories or is an environmental problem, it is high on the list of priorities within companies across many industries. Corporate image and social responsibility also contribute to the fact that companies are reducing their odor emissions. The odor impact (odor immission) at ground level can be calculated using a dispersion model. Figure 4 shows odor dispersion modelling graphics with a significant reduction in the odor contours by a 75% odor reduction on emission level. By reducing the odor emission with such a significant quantity, factories will reduce the risk of getting

odor complaints from their neighbors and are able to comply with the local odor legislations or environmental permits. Our aim is to run the Aerox®-Injector on as few resources as possible while keeping the installation as small as possible. Our environmentally friendly odur control unit runs completely on electricity and nothing else. It is, therefore, compared to units that use fuel, water, or chemicals, better for the environment. One of the biggest advantages of cold plasma technology is that the ionization process starts at room temperature, usually combustion is achieved at much higher temperatures. Therefore, relatively little electricity is required and the CO2 emission remains low. By continuous research, we keep trying to further lower its energy consumption as well as minimizing its size.

Advantages of NTP In addition to the benefits of sustainability the NTP unit has several other advantages. The Aerox-Injector NTP unit is a compact industrial odor reduction unit, converting only a small air volume into active oxygen. Nevertheless, direct injection allows treatment of high volumes of process air (up to 200,000 m3/h). The injection technology makes it easy to integrate

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

into the production process. Due to the injection technology, there is no direct contact between the process air and the generated plasma field and therefore the NTP-modules stay clean, reducing precious time which would otherwise be spent cleaning the odor abatement system. The unit can easily be switched off in case of a production stop.

Conclusion With the NTP unit, the exhaust air from extruders, dryers, coolers, hammermills and coating can be treated with a typical odor reduction achieved in the aquafeed industry of 75-95% and a typical volume flow rate to be treated with one unit of 60.000 m3/h. As an odor abatement solution provider Aerox wants to provide the best available solution for its clients. Due to our globally large installed base, the validation tests and pilot tests we have performed, a large database is available to give the best service and advice to our clients. Reference Mrs. A. Snik - van de Burg. Odour disperion modelling. Olfasense B.V. (2019, March 12).

More information: Merijn Appelman International Sales Manager Aerox, The Netherlands E: merijn.appelman@aerox.nl

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Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

MICRO

Better from the beginning: Using plant genetics and value chain integration to provide functional and sustainable soy ingredients for aquaculture rations Dan Northrup, Hannah Lucas, Benson Hill

Just as aquaculture is critical to sustainably nourish the world’s population, aquaculture feed is vital to produce a nutritious and environmentally sustainable product. Salmon, trout and other marine finfish have traditionally relied on feed ingredients such as fishmeal and plant

protein concentrates to achieve high protein and fat requirements and avoid diseases such as enteritis – an inflammatory condition of the gut caused by anti-nutritional factors present in soybeans*. These legacy plant protein concentrates are created

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

Specialty soybean meals are now offered under the Bright Day™ brand as a fully defatted hexane-extracted product (<1% fat), or as an expeller-pressed product (5% fat) (Table 2). With continuous improvement of the genetics and custom agronomic practice, Bright Day™ protein levels now reach as high as 58% on an as-is basis.

Table 1. Feed conversion results in Bright Day™ feeding trials*.

Table 2. Typical values for Bright Day specialty soy meals (as-is).

from commodity, high-yielding soy crop varieties which were developed to service the scale needed for livestock feeds, not aquaculture. That doesn’t have to be the case. In the same way we can tap the genetic diversity of plants to optimize harvest quality, disease resistance, and yields, today we can leverage tools such as AI and predictive breeding to design a soybean that better meets the dietary needs of aquaculture rations.

Better from the beginning The natural genetic diversity of plants offers a host of options to create ingredients that can enter rations with minimal processing – lending a more sustainable profile while simultaneously lowering production costs. We like to think of this as letting mother nature do the upfront work. Benson Hill is commercializing soy varieties with elevated protein content (HP), and varieties with ultra-high protein content (UHP) and very low levels of oligosaccharides. Ingredients derived from these soy varieties are intended to function as an alternative product to soy protein concentrate (SPC). The high protein, low anti-nutrient trait package has been tested in several aquaculture species including trout, pacific white shrimp, yellowtail and cobia. In all studies to date, meals provided comparable protein function to SPC without causing disease (Table 1)*. Benson Hill is actively testing new material in trout and salmon and welcomes inquiries about testing in new species.

Industry adoption Riverence is a vertically integrated family of farms producing steelhead trout in the pacific northwest United States. Rangen Feeds, a manufacturer of salmon, trout and shrimp feeds located in Buhl, Idaho uses Bright Day™ as a protein and fat source in Riverence diets. “Maintaining a high standard of quality to the customer’s plate requires continued focus on fish diet and nutrition. Benson Hill’s ingredients function well in our diets and are aligned with our sustainability strategy,” said Jason Mann, Director of Nutrition for Riverence. “Partnering with companies like Riverence and Benson Hill, who share our vision for sustainable growth of US aquaculture, results in efficiencies, value and healthy sources of protein for consumers. High quality, domestic ingredient sources play a big role in achieving this,” noted David Brock, Nutritionist for Rangen Feeds. Sustainability spotlight: Processing Aquaculture’s nutrition density and feed conversion efficiency make the industry a strong sustainability story from the start, with the potential to do even more to enhance its positive environmental impacts. Feed is a major component of the environmental footprint of a farmed fish, making it an attractive target for improved stewardship. The ability to eliminate processing steps means less energy and water is required to produce the end product, inherently improving the sustainability profile of Benson Hill’s ingredients. In a life-cycle assessment comparing UHP Bright Day™ meals to Brazilian-sourced SPC, the ingredient was found to

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

Figure 1. An Integrated approach to higher quality, more sustainable feed and fish.

require up to 70% less water and up to 50% less CO2 per kg of product*. All grain is produced in North America, where there is little risk of deforestation or land conversion.

Sustainability spotlight: Environmental stewardship A large portion of the environmental footprint of feed comes from crop production practices. A lack of traceability in existing commodity supply chains means that aquaculture producers are limited in their ability to drive improved management practices on specific farms. In contrast, identity-preserved supply chains have increased potential to change on-farm management practices especially in a closed loop or integrated models. Higher levels of traceability enable direct tracking of farm production practices. Through partnership with soy growers, the adoption of on-farm technology and agronomic practices can be promoted to further sequester carbon within the supply chain. As aquaculture farmers promote land and water stewardship on their own farms, sourcing through companies like Benson Hill enables the expression of the same values at the beginning of their supply chain. Sustainable, high-quality protein options are needed to enable aquaculture’s continued growth. Crop genetics have the potential to help meet this demand, directly generating attributes that are commonly achieved through grain processing. The high protein, low oligosaccharide trait package was designed with aquaculture in mind, and Benson Hill is developing additional traits to further customize the genetics. Through collaboration, we can support the sustainable

growth of the aquaculture industry through protein ingredient innovation. Benson Hill, Inc. moves food forward with the CropOS® platform, a cutting-edge food innovation engine that combines data science and machine learning with biology and genetics. The company empowers innovators to unlock nature’s genetic diversity from plant to plate, with the purpose of creating healthier, great-tasting food and ingredient options that are both widely accessible and sustainable. More information about Bright Day™ can be found at brightdayfeed.com. *References available on request.

More information: Dan Northrup, PhD Director, Special Projects Benson Hill, USA E: dnorthrup@bensonhill.com

Hannah Lucas Manager, Business Development Benson Hill, USA E: hlucas@bensonhill.com

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Soy in aquaculture Lukas Manomaitis, Zhou (Joe) En Hua, U.S. Soybean Export Council (USSEC)

Photo credit: US Soybean Export Council.

Introduction In the early days of feed-based aquaculture, farmers felt that they had an easy way to gauge the quality of their feeds. If it was dark in color and smelled like fish, then the feed was good. However, as we know now, the days of feed heavy on fishmeal and fish oil (and generally aquatic animal proteins and oils) are over. Fortunately, alternative and effective formulation approaches exist, and the industry is seeing more ingredients for aquaculture become available seemingly every day. Having said that, one ingredient has already taken a central role in aquaculture diets, and that ingredient is soy. The idea of soy in aquaculture diets is something that the United States soy industry has taken seriously for over 35 years, and through their international marketing arm, the U.S. Soybean

Export Council (USSEC, www.ussec.org), they have conducted research, demonstrations and transferred the technical knowledge to make soy a central part of aquaculture today.

Soy, not just soybean meal While soy is not the only ingredient needed to create aquaculture feeds, the unique properties of soy have made it a critical part of formulations today. Soy has a good amino acid profile, one of the highest protein densities for a row crop, and is available internationally year-round as it is grown in many locations. While many are familiar with soybean meal, soy also provides high protein substitutes (soybean protein concentrate and soy isolates), a lipid source (soy oil), a phospholipid source (soy lecithin), as well as other unique products

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

such as fermented soy products (which may provide functional properties). In combination with other ingredients, cost-effective and efficient feeds can be created that reduce or eliminate the need for ingredients like those coming from aquatic animals. Those aquatic animal-derived ingredients can then be better targeted to key life stages or species that really require them.

Sustainability is critical The global need for more sustainable approaches is well understood, and the soy industry has already been in the lead of this effort. For example, the U.S. food and agriculture industry overall has a long-term view towards sustainable production, and specifically, U.S. soy farmers involved in this critical crop have already worked to make US soy one of the most sustainable ingredients currently available. Through the U.S. Soy Sustainability Assurance Protocol (SSAP, www.usses.org), the users of U.S. soy can obtain certified sustainable ingredients from U.S. soy with a certification program recognized by international aquaculture certification bodies like the Best Aquaculture Practices (BAP) program. BAP has already committed to requiring certified sustainable ingredients to be used in aquafeeds, with at least 50% of soy needing to be sourced from sustainable sources by July 1, 2022. Supporting the aquaculture industry From the beginnings of U.S. soy’s involvement in aquaculture, and particularly the work done together with the Chinese Extension service in the 1990’s to covert that nation’s aquaculture industry to a feedbased one, the U.S. soy industry has taken an industry approach to aquaculture. There has been a clear sense that improving the feed-based aquaculture production chain overall would yield better opportunities for soy. As a result, USSEC’s programs have worked to improve many parts of the aquaculture industry, some of which may not always seem directly linked to soy. These efforts have been diverse, and effective. For example, with the production technology packages. Approaches such as “80:20” pond production, “Low Volume, High Density” (LVHD) cage culture, the “Offshore Cage Aquaculture

Soybeans ready for harvest. Photo credit: United Soybean Board.

Technology” (OCAT) all have helped producers improve their yields, profitability, and sustainability (see more at www.soyaqua.org). The most recent technology package, the “In Pond Raceway System” (IPRS) has become something that the global aquaculture industry has taken note of, enabling farmers to increase yield by 2-3 times from the same water volume, while maintaining water in ponds without needing to drain it between harvests, and provides significantly more operational control. There are now more than 7,000 IPRS raceway cells operating in China and hundreds more in operation in Latin America, the Middle East and other parts of Asia. Another example is the International Aquaculture Feed Formulation Database (IAFFD, www.iaffd.com). USSEC has collaborated with other aquaculture stakeholders to provide the first publicly available, commercially oriented, comprehensive aquaculture feed formulation database to industry. Containing over 500 ingredient standards and target nutrient specifications for over 30 important aquaculture species (including at different age classes for every species), this database is strongly supported by USSEC and is improving every year. This resource enables feed mills to benchmark their own commercial databases with another database, as well as providing in-house and general training opportunities for aquaculture feed formulators. Commercial feed

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formulation program providers such as Adifo’s Bestmix® and A-System’s Allix3 provide their customers with the IAFFD as a reference database.

Using soy in aquafeeds As discussed, there are already many different types of soy ingredients that provide key nutrients for aquaculture formulations and soy ingredients are widely available and can be sourced from certified sustainable producers, such as through the U.S. Soy Sustainability Assurance Protocol (SSAP). But how does one determine how to use soy effectively and safely in aquaculture diets? Once again, the U.S. soy industry has been active in this space, doing the foundational work and research (internally, and determining the maximum amounts of various soy products that can be effectively and safely incorporated into feeds for different species at various life stages). With this information, formulators can have more confidence about soy inclusion levels, as when they know the maximum inclusion before there is a performance impact it allows them to quickly see if

the amounts suggested in a feed formulation program are below those levels, and therefore safe and effective.

Continuous improvement USSEC and the U.S. soy industry are always trying to be proactive as well. Aquaculture technology is always improving and feeds should also be improving in lockstep with industry. Feeds are not just a source of nutrients, but a production management tool. US soy has worked closely with industry “to promote a profitable and sustainable approach to aquaculture” through a better understanding of how soy can be a part of the solution and how feeds, in particular, can help. More information: Lukas Manomaitis Aquaculture Program Technical Contractor USSEC, Thailand E: LManomaitis@ct.ussec.org

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

Fermented sweet potato biomass: a sustainable ingredient for aquafeeds Rex Ferdinand Traifalgar, University of the Philippines Visayas, Maria Theresa Valdez, Tarlac Agricultural University, Leodevico L. Ilag, Plentex Limited The contribution of aquaculture to global food production is expected to increase, therefore the need for sustainable and cost-effective feed ingredients will also rise. It has been projected that aquaculture production will reach 105 Mt in the year 2029. However, this projected growth is dependent on feed inputs, which account for about 50-60% of the total production cost. Feed protein supply requirements for the expansion of aquaculture are considered the limiting factor that significantly affects the sustainability and economic viability of the industry. Currently, feed proteins for aquaculture are sourced from fish and soybean meals. However, utilization of these meals as fish feed has been viewed unfavorably by the public as an inefficient way of

resource utilization. Fish should be consumed directly as human food and should not be used as feed for other fish. Furthermore, the food and the livestock industries are competing with aquaculture for soybean meal leading to an increase in prices and erratic supply of this feed material. Moreover, there is also growing concern and trends against the use of soybean because of its association with nonsustainable production processes.

ProEnK: The future of sustainable feed production for aquaculture To address the global concern regarding the supply of feed ingredients required to sustain the growth of aquaculture, Plentex Philippines lnc., and experts

Figure 1. Weight gain of Pacific white shrimp fed diets containing increasing replacement levels of soybean meal with ProEnk.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

Figure 2. Total gut Vibrio load of the shrimp, P. vannamei fed with diets containing fermented sweet potato and the control, with soybean meal as the main protein ingredient.

from the Institute of Aquaculture of the University of Philippines Visayas and Tarlac Agricultural University have teamed up to develop ProEnK, a high protein feed ingredient produced from the fermentation of sweet potato biomass, a byproduct of the sweet potato processing industry. It has been estimated that up to 20% of sweet potato biomass ends up as waste from processing and is not used for human consumption. The group has developed an innovative process of using a cocktail of proprietary microbes that are able to convert agricultural biomass rich in carbohydrate into

high-protein biomass that is comparable with soybean meal in protein content but with the absence of omega-6 fatty acids. The developed process requires less water, bioconversion completed within 21 days and transforms sweet potato byproduct biomass with 3% protein into a feed ingredient containing 35-45% protein. This process is cheaper than intensive soybean agronomy that requires a significant amount of water, fetilizers, pesticides and time. ProEnK has the potential to reduce the utilization of fishmeal for aquaculture feed use.

Figure 3. Feed conversion efficiency of milkfish fed diets containing increasing levels of ProEnk.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

Figure 4. Omega-3/omega-6 fatty acid ratio of tilapia fed diets containing increasing levels of ProEnk.

This ingredient was found to be 70-90% digestible to milkfish, shrimp and tilapia. Analysis of the fermented biomass indicated an ideal content of carbohydrates, low fat and lower fiber contents. The fatty acid contents are dominated by medium-chain and short-chain fatty acids, which are ideal energy sources and immune activator molecules in aquatic animals. A complete set of essential amino acids required for most aquatic animals were also found to be present in ProEnK.

Trials supporting ProEnK as an aquaculture feed ingredient To confirm the nutritional and feed value of the ProEnk to aquatic animals, several feeding trials were conducted on Pacific white shrimp, milkfish and tilapia. In the trial with Pacific white shrimp, diets were formulated with increasing substitution of soybean meal (0-100%) in the diets of the treated aquatic animals. The diets were formulated to be isonitrogenous and isocaloric. In the trial with milkfish and tilapia, the diets were formulated containing increasing levels of ProEnK with a corresponding reduction of fish and soybean meals. This was done to determine the maximum inclusion level of ProEnK that could be included in the cultured finfish diet. Shrimp results Results from the pacific white shrimp trial indicated that ProEnK could completely replace soybean meal in the shrimp’s diet without affecting the overall growth

performance. Moreover, the results indicated that at 50% soybean meal replacement, the growth and feed conversion ratio (FCR) of shrimp is enhanced (Fig. 1). In addition, the fatty acid profiles of shrimp in the best treatment group showed a higher content of omega-3 fatty acids in their tissues. This significant enhancement of growth and a better fatty acid tissue profile was also associated with a low total Vibrio content in the treatments replacing 50% of soybean meal with ProEnK. The results suggested the activation of gut immunity by this dietary ingredient. ProEnk contains a good amount of short and medium-chain fatty acids that are known to activate gut-associated immune responses and are also known as potent antibacterial agents.

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Milkfish results The feeding trial with milkfish also demonstrated a similar pattern. Better growth response and Feed Conversion Efficiency in treatment with the highest inclusion level of dietary ProEnk was observed (Fig. 3). This indicated that ProEnK could be incorporated in up to 50% of the diet and still obtain better growth. It is also worth noting that dietary inclusion of ProEnK led to the improvement of the fatty acid profiles of milkfish. Diet treatments with ProEnk as sole protein source led to a two-fold improvement in the ratio of tissue omega-3 to mega-6 (omega-3/omega-6) fatty acids. Tilapia results The milkfish and tilapia feeding trials showed similar results whereby growth indices were similar in diets with the highest inclusion of ProEnK as compared to the control. Prominent improvement in the fatty acid profile of tilapia feed diets with ProEnK could be observed. Almost a three-fold improvement in omega-3/omega6 fatty acid ratio was observed in diets containing the highest inclusion of ProEnK (Fig. 4). The results suggest that utilization of this feed ingredient in fish feed may promote better fatty acid profiles in cultured tilapia.

Conclusions Collectively, the present data demonstrated the huge potential of ProEnk as a feed ingredient for the aquaculture of tilapia, shrimp and milkfish. The findings also suggest that among these species, dietary inclusion levels of ProEnk appear to influence the omega 3/ omega 6 ratio, an important health biomarker especially for inflammation, which is believed to be central to most chronic diseases. Animals that received the ProEnk diet have a better ratio of these fatty acids indicating less omega 6 fatty acid synthesis. Tilapia has been notoriously associated as unhealthy because of the high levels of omega-6 fatty acids in some studies. The inclusion of ProEnK has the potential to improve the fatty acid profile of tilapia and its image as part of a healthy diet. The high levels of omega 3 fatty acids in the treatment groups receiving the ProEnk diets suggest that this diet ingredient may have promoted the biosynthesis of omega 3 fatty acids rather than the omega 6 type of fatty acids. Both tilapia and milkfish are known to biosynthesize the long-chain polyunsaturated

fatty acids, EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid) from 18-carbon fatty acid precursors. Currently, the mechanism and conditions that can potentially induce the biosynthesis of EPA and DHA in tilapia and milkfish are unknown. However, the results from our studies suggest that dietary inclusion of 50% ProEnk in the diet could promote the biosynthesis of EPA and DHA fatty acids in the flesh of tilapia and milkfish. It is also tempting to speculate that the antibacterial property of ProEnk may limit the degree of bacterial infection and inflammation of the gut. Natural infection and inflammation are known factors to activate the biosynthesis of arachidonic acid, an omega 6 fatty acid. The inhibitory action of ProEnk against gut bacteria could also be a factor in the observed enhancement in performance and better fatty acid profile of these cultured aquatic animals. ProEnk could be included in the diets of tilapia, shrimp, and milkfish to about 50% by weight without affecting the overall biological performance and feed efficiency of the aquatic animals, with the added bonus of improving fatty acid profiles. This ingredient is sustainable, economical and could reduce the use of fish and soybean meals in aquaculture diets. This feed ingredient is envisioned to play a vital role in the development of sustainable global aquaculture. References available on request. More information: Rex Ferdinand Traifalgar Faculty, Institute of Aquaculture College of Fisheries and Ocean Sciences, University of the Philippines Visayas E: vilag@plentex.com.au

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Activated zinc oxide: An innovative nutritional approach to reduce WSSV-induced mortality in shrimp Benedikt Hein, Torben Liermann, Provita Supplements, Martin Rimbach, ISF GmbH

One of the most important fatal diseases in shrimp production is the White Spot Syndrome Virus (WSSV). WSSV is a virus causing high mortality rates, up to 100.0 % within 5 to 7 days and was first reported during the early 90s. Infected shrimp can be recognized by a loose cuticle with white spots (Lightner, 1996; Lin et al., 2011). Even though WSSV has been known for numerous years, shrimp producers still face enormous economic risks from high mortality rates due to the virus, especially during the cooler seasons. Treatment options against WSSV are still poorly developed and ineffective, mainly based on biomolecules like DNA or RNA vaccines, protein-based antigens and antibodies as well as herbal products (Feng et al., 2017). Furthermore, lower stocking densities have proven to reduce WSSV caused mortalities (Raja et al., 2015). While the above-

mentioned methods may be effective, they are neither very practical nor viable economically for shrimp producers. For this reason, we investigated the effect of feeding diets including different levels of our activated zinc oxide (MAXACTIVAT/Zn) on the susceptibility of WSSV-induced mortality in shrimp (Penaeus vannamei). Previous R&D work has already shown the positive influence of MAXACTIVAT/Zn against bacterial pathogens such as E. coli in calves and piglets. MAXACTIVAT/Zn (aZnO) has also shown a comparable greater impact on reducing bacterial growth in vitro compared with conventional Zn oxide (Hoffmann et al., 2019). The above-mentioned effects are often concluded to be related to an improvement of the barrier function of the gut mucosa (Li et al., 2001; Sales, 2013). This in turn allows the assumption that this also

Figure 1. Total mortality over the experimental period of the different dietary groups, IMAQUA (2020). *significant at p < 0.05

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

Figure 2. Cumulative mortality over the experimental period of the different dietary groups, IMAQUA (2020).

has a positive influence on the resilience against viral infections as those often occur in the gastrointestinal system (Katsuma et al., 2012). Moreover, a general positive influence of zinc oxides (ZnO) on health parameters has been observed when supplemented with dietary feeds (Janczyk et al., 2013; Liu et al., 2014).

Material and methods A challenge trial was conducted with three different groups. The differences between the groups arose from the different levels of MAXACTIVAT/Zn (0 ppm, 135 ppm and 405 ppm) added to the different diets. The zinc oxide has been activated through patented eccentric vibrating mill technology that creates higher reactivity when ingested by animals. The described process modifies the functional parameter, particle size and surface area of the aZnO (Hüttenrauch et al., 1985). The concentration of zinc in MAXACTIVAT/Zn is 74.5 %. Shrimp were infected via oral route with the WSSV Thai-1 strain (Escobedo-Bonilla et al., 2005) and monitored for clinical signs of disease, with observations for mortality performed twice a day. In order to control unknown influencing factors during the experiment, a negative control group (MOCK: mock inoculation) was added. Clinical outcome was evaluated by: Onset of mortality, cessation of mortality and cumulative mortality by day 7. Results Data received from the WSSV challenge trial can be seen as valid due to the fact that mortality within the

control (CTRL) group resulted to be within the expected range at 73.3 % while mortality within MOCK group was 0.0 %. Challenged shrimp fed with MAXACTIVAT/Zn at 405 ppm showed significantly lower mortality of 33.3 %, which was 40 % points less compared to CTRL (Fig. 1). Moreover, the mortality of the group that received aZnO at a concentration of 135 ppm was reduced by 20 % points (Fig. 1). With respect to cumulative mortality, a positive influence of the aZnO was observed. As illustrated in Figure 2, the onset of mortality within groups MA135 and MA405 was delayed by 18 and 30 hours, respectively, compared to CTRL. Moreover, next to a delayed onset of mortality, mortality levels remained constant once reached their peaks at 53.3 % after 108 hours and 33.3 % after 78 hours, respectively.

Discussion The high mortality rates related to WSSV, also recognizable within the control group of this study, highlight the great risks shrimp producers face. The above-described results indicate the high potential to decrease those risks. Both, the lower dosage (MA135), as well as the higher dosage (MA405) of MAXACTIVAT/ Zn, showed a positive influence on reducing mortality rates. Considering the onset of mortality it becomes clear that diets containing aZnO did not only delay fatality but also lead to constant low rates. Explanations of the mode of action of the tested aZnO can be described as follows. Previous studies

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investigating the effect of zinc oxide have shown a positive effect in the gastrointestinal tract of pigs. However, those effects have been observed by feeding high pharmacological dietary doses (Davin et al., 2012; Sales, 2013) which may not allow a direct relation to this study. Another possible explanation for the positive effect of ZnO may be the improvement of the intestinal barrier function (Li et al., 2001), the non-allowance of bacterial adhesion to the epithelium (Roselli et al., 2003), as well as the modification of different stress responding proteins (Sargeant et al., 2011). Similar results were also reported by Janczyk et al. (2013), who described a positive influence of ZnO on the immune response of piglets. Moreover, Liu et al. (2014) observed significant effects on various characteristics of the colonic tissue by feeding also a low concentration of ZnO to piglets. Those findings with respect to improved immune response and an improved gastrointestinal tract may also be valid for the results of the present study. Wang et al. (2018) already investigated the influence of WSSV on the microbiota of shrimp. They compared WSSV-infected with healthy shrimp and found significant differences between both groups. They concluded that WSSV could impact intestinal microbiota composition and, thus, function in L. vannamei. In addition, Huang et al. (2012) described possible adhesion mechanisms of WSSV. They identified the glucose transporter 1 (Glut1) surface protein in shrimp that may interact with VP53A, which is the envelope protein of WSSV. With regard to a possibly modified receptor system in the gut (through aZnO), the adhesion capacity of WSSV may be blocked. This mechanism in turn would be similar to observed

positive effects of aZnO against bacterial pathogens in calves and piglets. Thus, improving gut integrity through aZnO may be a comprehensible explanation for lowered mortality rates observed in the challenge trial. Feng et al. (2017) mentioned the negative aspects of vaccines creating non-practical applications. In contrast, supplementing aZnO to shrimp diets provides a nutritional as well as a practical approach. As WSSV pressure underlies seasonal changes (Peña et al., 2007), aZnO concentrations contained in diets may be adapted accordingly.

Conclusion The demand to clearly improve disease resistance in shrimp production is a considerable economic factor. This experiment consequently points out the significant efficacy of the novel activated trace mineral ingredient MAXACTIVAT/Zn on lowering WSSV induced mortality. This unique approach of a feed additive promotes interest for further research activities to investigate possible further antiviral effects of MAXACTIVAT/Zn. Moreover, the use of aZnO provides a nutritional path to reduce mortality in shrimp and has proven its high potential. References available on request. More information: Benedikt Hein Area Sales Manager Provita Supplements GmbH, Germany E: Benedikt.Hein@provita-supplements.de www.en.provita-supplements.com

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Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

Natural source of carotenoids for salmonids and shrimps Dominique Corlay, Aquaculture Natural Solutions

Color intensity for salmon and shrimp represent major quality criteria affecting consumer preferences (Parisenti et al., 2011) and giving a significant added value to the products. Carotenoids such as astaxanthin are also key nutrients in various physiological functions such as reproduction and can help to sustain the animal health status. On the other side, demands from customers lean towards more natural and sustainable products, free of any synthetic additives. Natural carotenoids such as Panaferd-AX are available on the aquafeed market and provide efficient nutritional solutions for both farmed salmonids and shrimps.

Sources of carotenoids In nature, wild salmon and shrimp get carotenoids from various primary producers such as microalgae, bacteria or yeast and then by live food. In aquaculture, dry feed is by far the only source. For shrimps, in some low-density production systems (<15 PL/m²) with good environmental conditions, farmed shrimp may find in the benthos sufficient quantities of carotenoids (Fig. 1). But in most intensive farming cases for shrimp, salmon and trout, the addition of carotenoids in the feed is fully needed. Various feed additives containing carotenoids such as astaxanthin are available from two major origins. Synthetic astaxanthin is chemically engineered through complex reactions from crude oil-based components. But this synthetic form only contains astaxanthin under various isomeric forms, not existing in nature. Considered as a “chemical dye” by some, it tends to be less used by premium farms in the salmon and trout industry and even ban by some food stores. Natural carotenoids can be extracted from various organisms and have the advantage to contain various carotenoids such as astaxanthin, adonirubin,

Credit: Kvarøy Artic, Norway

canthaxanthin and adonixanthin. These sources also have a nutritional value (protein content) when not synthetically formed. Three major sources of natural astaxanthin are available on the market: the microalgae Haematococcus pluviatilis, the yeast Phaffia rhodozyma and the bacterial micro-organism Paracoccus carotinifaciens, better known under the brand name Panaferd-AX®. Table 1 compares the main specifications between Panaferd-AX and synthetic astaxanthin.

Benefits for salmon: Color and health Natural carotenoid source is well known and used by part of the salmon industry for many years. In a study conducted in Scotland (ENEOS Corporation data), the efficacy of Panaferd pigment at different standard pigment rates (40 and 70 ppm) in Atlantic salmon (Salmo salar) was compared to synthetic astaxanthin. A stability study was also performed to evaluate the carotenoid stability of salmon fillets under freezing

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

conditions (-25°C) for 6 months. Atlantic salmon smolts from the same genetic strain, were sourced from a Scottish hatchery and raised to market size. Pigmented diets contained the same astaxanthin level: 40 or 70 ppm (PF1 and PF2, respectively) and either from Panaferd-AX or synthetic form (SA1 and S2). At the end of the trial (triplicates), no significant differences in growth, SGR and FCR were observed either during the growing period demonstrating there was no relationship between pigment source (synthetic versus Panaferd-AX) or carotenoid concentration. Regarding the results on the flesh quality, all Panaferd groups performed very well and after 17 months reached a color level on the Roche SalmoFan® in accordance with the market expectations (Fig. 2). The test stability results at freezing temperature (-25°C) confirm the excellent stability of carotenoids of Panaferd-AX salmon (Table 2) without any difference between natural and synthetic astaxanthin. In the smoking process, similar results were found by Lerfall et al. in 2016 and largely confirmed by the smoked Scottish salmons found in most food stores around

Figure 1. Total carotenoid content in shrimp P. vannamei according to their farming density (low density at 15 PL/m²; high density at 400 PL/m²). Adapted from Quintana-Lopez et al., 2019.

the world. Significant quantities of other carotenoids such as adonirubin in the salmon flesh are very typical of Panaferd®-AX use, where synthetic-based pigment contains only one form of carotenoid. Recent studies have proven the strong anti-oxidative, anti-tumorpromoting and anti-carcinogenic activities of adonirubin (Maoka et al., 2013). Panaferd®-AX carotenoids such

Table 1. Comparative table between PANAFERD-AX and synthetic astaxanthin.

Origin Source

Natural carotenoids

Synthetic astaxanthin

Bacterial micro-organism Paracoccus carofinifaciens

Synthetic product Crude oil derived origin

Product name/Brand name

Panaferd-AX™

Various suppliers: DSM, BASF…

Production method

Fermentation

Chemical reactions process

Product form

Powder

Free form Isomer 3S-3’S, same as wild salmon

Free form Mixture of enantiomers: 3S,3S' 3R,3R 3S3R; not found in nature

0.2 µ

N/A

2% mini

8 to 10%

Chemical form

Size of the organism

Astaxanthin concentration

Presence of other carotenoids

Adonirubin, canthaxanthin, adonixanthin …

No other carotenoids

Nutritional value (as is)

Crude protein: >50% Crude fat: 1.9%

No value

Color enhancing efficiency and stability

Good bioavailability for all species. Good stability

Good efficiency and stability

Authorized worldwide and by all food retailers Authorized in the EU organic standards

Authorized worldwide Fully ban by the organic standards Not authorized by some food retailers

Market and certifications

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

Table 2. Carotenoids in the salmon flesh (mg/kg) during the test stability at freezing temperature (-25°C).

At starting point (in mg/kg)

PF1

PF2

SA1

SA2

Canthaxanthin (coefficient 0.92)

0,57

0,42

0,10

0,07

Adonirubin

1,54

1,23

0,00

0,04

Astaxanthin

3,30

2,96

3,97

3,92

Total carotenoids

5,63

4,77

4,26

4,31

After 6 month at -25°C

PF1

PF2

SA1

SA2

Canthaxanthin (coefficient 0.92)

0,53

0,35

0,07

0,06

Adonirubin

1,43

1,06

0,05

Astaxanthin

2,97

2,54

3,57

3,43

Total carotenoids

5,12

4,06

3,69

3,55

Figure 2. Roche Color score after 17 month growing period.

Figure 3. Redness a* value on head and body in shrimp.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

as astaxanthin are present as free and the same isomeric forms (3S3’S) as in wild salmon. In synthetic astaxanthin, astaxanthin is a mixture of enantiomers, not existing in nature. This difference can be used to distinguish between farmed and wild salmon through an approved method by the FDA (Turujman, 1997).

Benefits for shrimp: Color and health Carotenoids can also be considered as essential nutrients for shrimp. Various studies have confirmed their implications in many physiological functions and health benefits such as survival, growth, reproduction, stress and disease resistance (Wade et al., 2015). In a study conducted at Songkhla University in Thailand (ENEOS data), juveniles (1.5 g) of P. vannamei were fed for 4 weeks with 3 diets: Control (no carotenoid addition), Panaferd-AX® at 0.5 kg and 1.25 kg/ton respectively. Color parameters such as redness (a* value) were obtained with a Hunterlab® ColorFlex on cooked shrimp (5 min boiling; Fig. 3). Shrimps fed with Panaferd-AX feature significant better red color compared to control. During a farm-scale evaluation in Panama (8 ponds 1.75 ha each; 4 control and 4 with Panaferd; same PL origin and stocking density), in the Panaferd ponds, shrimps were fed with natural carotenoids at 2.5 kg/ton, during 30 days before the final harvest. A color evaluation was conducted after cooking, using the color chart commonly used in Latin America (chart A1 to A4). Figure 4 details the significant difference in color variation between the

Figure 4. Color evaluation of shrimp at harvest after 30 days feeding with Panaferd-AX at 2.5 kg/ton feed and control (no carotenoids).

Figure 5. Antioxidant enzyme activities of P. vannamei fed with Panaferd for 4 weeks.

two diets and confirm the significant effect of natural carotenoids on color. The health benefits of carotenoids from Panaferd-AX® for shrimps have been also evaluated. After 30 days of feeding with the natural carotenoids, shrimps feature significant additional activities of the major antioxidant enzymes (SOD: SuperOxide Dismutase; and CAT: Catalase). Catalase is a major antioxidant enzyme, which is responsible for scavenging reactive oxygen species (ROS) and protecting tissue against damage. Shrimp fed with Panaferd-AX featured significantly higher CAT and SOD activities than the control (Fig. 5).

Conclusion Natural pigment Panaferd®- AX confirms it represents a reliable and stable pigment for salmon and shrimp.

Natural carotenoids also prove to be valuable nutriments enhancing the animal health status in intensive farming conditions. On the market side, consumer expectations for more food free of synthetic additives are now more prevalent around the world. As color is the symbol of salmon and shrimp, consumers expect clear statements on the pigment source.

More information: Dominique Corlay Consultant Aquaculture Natural Solutions, France E: ansaqua.dc@gmail.com E: panaferd.sales@jxanci.com

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

The gut microbiota drives the successful replacement of fishmeal by microbial and processed animal proteins in gilthead seabream Paula Solé-Jiménez, Fernando Naya-Català, M. Carla Piazzon, Itziar Estensoro, Josep À. Calduch-Giner, Ariadna Sitjà-Bobadilla, Danny Van Mullem, Jaume Pérez-Sánchez, LSAqua and IATS-CSIC

Figure 1. Facilities at Institut of Aquaculture Torre de la Sal (IATS-CSIC; left) and gilthead seabream (Sparus aurata; right).

The future of fish aquafeeds Traditionally, plant ingredients have been used for replacing marine feed ingredients in fish aquafeeds, achieving high levels of fishmeal (FM) and fish oil (FO) replacement in Atlantic salmon, European seabass or gilthead seabream. However, a shortage of FM ranging from 0.4 to 1.32 million metric tons may occur by 2050, significantly impairing the growth of the aquaculture industry. Therefore, there is an urgent demand for new raw materials to retain farmed fish performance and stabilize the supply of feed. Among others, insect proteins, processed animal proteins (PAP) or single-cell proteins (SCP) have the potential to play a major role in the development of new aquafeeds. PAPs (feather meal, blood meal,

meat or bone meal from non-ruminants) possess excellent palatability, competitive price, and high protein content with fewer carbohydrates than plants. SCP (yeast, bacteria, fungi and other microorganisms) are rich in nutrients, can rapidly grow in different carbon sources and taking up less land than other organisms. Therefore, both protein sources are very promising feed ingredients to reduce the FM content in marine aquafeeds, but the long-term suitability of new feeds formulations is often questioned. Thus, conventional methodologies, but also cutting-edge tools, are needed for unraveling the interactions between diets, host metabolism and gut microbiota, which has co-evolved with the host to develop a mutualistic relationship.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

granular cells and intraepithelial lymphocytes with no changes in the patterns of goblet cells. Pro-inflammatory effects are not an unexpected flaw, as it is a common metabolic derangement of high levels of FM/FO replacement by plant ingredients. This drawback effect can be partially or totally reversed by the use of feed additives that result in improved intestine integrity and function. How gut microbiota is involved in this process remains a matter of discussion, although the general thinking is that gut microbiota regulates feeding, digestive and metabolic processes as well as immune development and training, being dietary factors one of the most important drivers of intestinal microbial diversity. In fact, the core intestinal microbiota is clearly different among carnivorous, omnivorous, and herbivorous fish species.

Getting started LSAqua developed a protein source made by a combination of PAS and bacterial-SCP, LSAqua SusPro, taking into consideration the nutritional properties of FM as well as their substitutes, along with the nutrient requirements of the selected species. The suitability of this protein concentrate was previously assessed in terms of growth and digestibility parameters in shrimp, European seabass and trout. Such studies are now extended to gilthead seabream, focusing on growth performance and markers of gut health, including histopathological scoring of intestine sections, and in-depth analysis of autochthonous microbiota. The gilthead seabream study was conducted within the framework of a Transnational Access project (Breamreplacer, AE170009) in the European Project AQUAEXCEL 2020 with the support of Nutrigenomics and Fish Pathology groups of the Institute of Aquaculture Torre de la Sal (IATS-CSIC), Castellon, Spain (Fig. 1). The results of the study have been recently published in the open-access Journal Frontiers in Marine Science and highlighted that growth rates with the partial replacement of FM with LSAqua SusPro were indistinguishable from the control group, and only a slight impairment was found with the total replacement of FM. Nevertheless, a non-pathologic but prevailing proinflammatory effect was found on the intestine of fish fed LSAqua SusPro diets dominated by submucosal hyperplasia with intense infiltration of eosinophilic

Better microbiota for better feeds and fish As expected, the gut microbiota of gilthead seabream, as most marine fish, is constituted by high abundance (>90%) of Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes phyla. However, LSAqua SusPro diets had an impact on microbiota and changes in gut bacterial communities were already found at the phylum level, supporting the fact that intestinal microbiota is highly influenced by dietary nutrients. Besides, in contrast to the inflammatory histopathological scoring, microbiota pathway analysis from the inferred metagenome highlighted that proinflammatory signals were under-represented in fish fed LSAqua SusPro diets, whereas pathways pertaining to quorum sensing or anti-microbial production were over-represented (Fig. 2). Among discriminant bacteria in the different dietary groups, pro-inflammatory Gammaproteobacteria of the genus Psychrobacter, and Acinetobacter decreased with FM replacement, whereas the anti-inflammatory Paracoccus, Arthrobacter and Actinomycetales increased, with a remarkable presence of the Propioniciclava genus in LSAqua SusPro fed fish. This microbiota architecture might serve to trigger a counter-regulatory anti-inflammatory response. In other words, the gut microbiome structure is shifting to deal with the alternative fish feed formulations, becoming intestinal microbial communities a main target to mitigate the drawback effects of most alternative fish fed formulations.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

Figure 2. Simplified dotplot map depicting some of the bacteria with more than 1% of proportion in at least one dietary group. The size of the dots represents the normalized counts in percentage, of each bacterium within each group. CTRL and 50/100LSAqua refer to control and combined LSAqua-based diets, respectively. Bacteria above the dotted blue line showed a higher abundance in 50/100LSAqua group; bacteria below the blue dotted line showed a higher abundance in CTRL group.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

Concluding remarks The partial and total FM replacement by LSAqua SusPro is highly promising in terms of growth performance in gilthead seabream. Some detrimental effects were found in gut health markers, but supplementation with pro- or prebiotics emerges as a key strategy to enhance the counterregulatory responses driven by the gut microbiota. This opens new research opportunities for fish nutritionists, physiologists and breeders to promote a more ethical and sustainable farmed fish production according to the principles of the circular economy. This is in line with the LSAqua pursuit of innovative and sustainable solutions with a clear commitment to social responsibility and safety. References Solé-Jiménez P, Naya-Català F, Piazzon MC, Estensoro I, Calduch-Giner JÀ, Sitjà-Bobadilla A, Van Mullem D and Pérez-Sánchez J (2021) Reshaping of Gut Microbiota

in Gilthead Sea Bream Fed Microbial and Processed Animal Proteins as the Main Dietary Protein Source. Front. Mar. Sci. 8:705041

More information: Danny Van Mullem CEO LSAqua, Belgium E: hello@lsaqua.be

Jaume Pérez-Sánchez Head scientist Institute of Aquaculture Torre de la Sal (IATS-CSIC), Castellón, Spain E: jaime.perez.sanchez@csic.es

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Aquafeed evaluation under specific farming conditions: A RAS application Ana Nobre, Filipe Soares, Tomé Silva, Jorge Dias, Luis Conceição, Sparos, Paulo Rema, UTAD

Salmonid aquafeeds have evolved over the last halfcentury to meet the increased industrialization of this sector with the formulations representing a balance between nutritional requirements, cost, ingredient availability and sustainability standards. Currently, there is a wide variety of commercial aquafeeds designed for different generic farming conditions and fish growth stages. A precise evaluation of the suitability of aquafeeds for the particular conditions of the farm is required to optimize fish feeding and economic feed conversion. This is highly relevant for optimal fish growth and performance while ensuring optimal water quality, in RAS farms. Optimizing RAS production implies a balance between fish growth, feed efficiency and

water quality. Monitoring feeding efficiency indicators is very important not only for the economics of feed conversion but also for planning and managing the biofilter. For instance, decreasing nutrient and solid loads per kg of fish produced enables increasing the fish stocking capacity of the system. In this context, decision-supporting tools for monitoring and forecasting fish nutrient waste, including virtual environments based on mathematical models of fish physiological and metabolic processes, are critical for RAS operation planning. FEEDNETICSTM is a web application developed by SPAROS that includes a mechanistic nutrient-based model to predict fish growth and composition over time, using information on temperature, feed intake

Figure 1. FEEDNETICSTM validation charts for body weight: measured vs predicted. Based on a wide range of datasets regarding water temperature (8.5°C to 17.0°C) and feed composition: gross energy 17.5 - 24.5 MJ/kg, crude protein 37.5 - 53.5 %, crude lipids 5.8 - 27.9 %, ratio DP/DE 17.4 - 26.6.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

Table 1. Rainbow trout aquafeeds evaluated, based on commercial specifications.

Trout feeds

High energy

RAS High energy

Pellet (mm)

3 4.5 6

Price (% variation)

reference

+6%

Proximal composition (as fed basis)

Digestible protein (%)

38.6 37.0 33.9

41.6 40.5 35.2

Digestible lipids (%)

23.5 25.4 27.1

26.9 28.7 29.7

Ash (%)

7.0 6.0 6.0

5.2 5.4 4.3

Fiber (%)

2.1 2.1 2.1

2.0 2.0 2.0

Digestible P (%)

0.8 0.7 0.6

0.6 0.6 0.5

Digestible energy (MJ/kg)

21.1 21.4 21.9

22.4 22.6 22.8

DP/DE (g/MJ)

18.3 17.3 15.5

18.5 17.9 15.4

To focus on evaluating the different feed concepts, the amino and fatty acid profiles were the same for the 2 feeds, and comply with trout requirements.

and feed properties. The model has been calibrated with highly variable data and is currently available for gilthead seabream, European seabass, rainbow trout and Nile tilapia. Other species will follow and further calibration for a specific strain or other species not available can be provided upon request and pending on data availability. The validation charts shown in Figure 1 illustrate the model robustness for this use case. Herein, we illustrate a FEEDNETICSTM application to compare two high-energy feeds, including one that

is designed for RAS. Similar aquafeed performance evaluation can be carried out directly in the web application by fish farmers or aquafeed customer support teams.

Farming conditions and feeding regimes We considered a trout harvest size of 1 kg and the following typical trout farming conditions: initial stock of 15,000 trout of 50 g, a mortality rate of 1% per month, and two scenarios of water temperature, a reference profile ranging between 13°C and

Figure 2. FEEDNETICSTM model results for the comparison of two high-energy feeds under a temperature profile around 15°C (ranging between 13°C and 18°C).

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

Figure 3. FEEDNETICSTM model results for the comparison of two high-energy feeds under two temperature profiles around 15°C (ranging between 13°C and 18°C) and around 13°C (ranging between 11°C and 16°C).

18°C with an average around 15°C, and a lower temperature profile (2°C lower than the reference). The feed characteristics are presented in Table 1. Both feeds have a high energy content. However, the RAS feed is denser in nutrients, with higher digestible protein and lipid levels, targeting a reduced FCR thus allowing an increase in the farm fish production capacity maintaining the same waste discharges. In terms of DP/DE ratios, the feeds are similar. The feed improvements come at a higher cost (+ 6%). The feeding table considered is consistent with the commercial advised feeding rates for a high-energy feed. In a real application, we recommend that feeding tables are generated per feed and growth curve of the farm or production unit using FiT feeding tablesTM.

Evaluation of two high-energy aquafeeds The objective of this application is to compare the performance of the two high-energy trout feeds (Table 1). With FEEDNETICSTM, we can quantify the performance indicators for an overall production cycle, such as days in production to reach the target harvest weight, FCR, growth rate, total feed consumed, economic conversion, protein efficiency ratio and nitrogen (N) and phosphorus (P) wastes (total, metabolic and fecal). As expected, the FEEDNETICSTM simulations (Fig. 2) indicate that the high-energy RAS feed leads to a better performance: shorter (11%) production cycle, improved FCR by 0.1 units, and a decrease in total N and P wastes of about -12% and -25%, respectively. While there may be deviations between the simulation results and real measurements (due to, for instance,

Figure 4. FEEDNETICSTM model results for days in production and FCR, for the comparison of two high-energy feeds under two temperature profiles around 15°C (ranging between 13°C and 18°C) and around 13°C (ranging between 11°C and 16°C).

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

different fish origins, different stress factors, variations in the batch of the feed used, among many other), a mechanistic tool such as FEEDNETICSTM can be particularly useful to evaluate overall trends and carry out a cost-benefit analysis. For the particular simulated conditions, the RAS feed leads to a better economic conversion, lower N and P discharges, and with saving estimated around less €48 of feed per ton of fish produced, despite its higher unit cost.

Evaluation of two high-energy aquafeeds under two temperature profiles Temperature is an important parameter that affects fish intake, growth and metabolism and has a high impact on production performance. As such, the precise evaluation of aquafeeds will also depend on the particular temperature profiles under which fish are reared (i.e., not only the average temperature, but also its variation along time). To illustrate this, the objective of this second use case is to compare the performance of the two high-energy trout feeds detailed in Table 1, considering a second temperature profile with a lower average temperature compared to the reference used in the previous use case (Fig. 2). According to the FEEDNETICSTM results, the decrease in the average water temperature affects proportionally more the production performance with the non-RAS feed (Fig. 3). In particular, the results show a higher metabolic P waste for the non-RAS high-energy feed (0.7 kg P/ton produced at 15°C and 1.4 kg P/ton produced at 13°C) compared with the RAS feed (0.4 kg P/ton produced at both 15°C and 13°C). For the nonRAS feed, the Total P waste increases about 29% when decreasing the temperature by 2°C, which is due to a decrease of the P retention.

With the high-energy RAS feed, the FCR at the lower temperature is still within the sector standard (around 1), while the FCR increases to 1.1 with the non-RAS feed (Fig. 3, 4). With the high-energy feed, the production cycle (to grow fish from 50 g to 1 kg) increases by 35 days under the low temperature profile (Fig. 3, 4). At 13°C, the total production time considering the two feeds lags by about 1 month, with the RAS feed representing savings on feed of around €82 per ton of fish produced when compared with the non-RAS feed.

Conclusion The FEEDNETICSTM mechanistic nutrient-based model includes the fish physiological and metabolic processes that are required to predict the effects of feed composition and temperature on fish growth, feed conversion and wastes, among other variables. As illustrated in this use case, this type of tools can be used by the aquaculture industry for precision aquafeed evaluation under specific farm conditions. Besides enabling aquafeed evaluation, the FEEDNETICSTM results can help the design and planning of the RAS facilities concerning, for example, biofilter capacity and the cost-benefit assessment of active temperature control, and planning feed changes. FEEDNETICSTM is available to be used by fish farmers and the aquafeed sector as a web app.

More information: Ana Nobre Product Manager Sparos, Portugal E: ananobre@sparos.pt

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Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

COLUMN

Nutritional fish & shrimp pathology - III Albert G. Tacon, Ph.D. Dr. Albert Tacon is a Technical Editor at Aquafeed.com and an independent aquaculture feed consultant. E: agjtacon@aquahana.com

Disorders in mineral nutrition This column is the third of a six-part series extracted from a forthcoming publication by the author dealing with the major reported nutritional disorders in farmed fish and shrimp, and represents an update to a previous review published by FAO in 1992 (Tacon, 1992). In common with most farm animal species, fish and shrimp have a physiological requirement for over 20 minerals, including, 1) the macro-minerals: Calcium (Ca), Phosphorus (P), Potassium (K), Sodium (Na), Sulphur (S), Magnesium (Mg), Chlorine (Cl); 2) the trace minerals Iron (Fe), Zinc (Zn), Copper (Cu), Manganese (Mn), Cobalt (Co), Iodine (I), Selenium (Se), Chromium (Cr), Molybdenum (Mo); and 3) possibly the newer trace elements Aluminium (Al), Arsenic (As), Boron (B), Bromine (Br), Fluorine (F),

Germanium (Ge), Lead (Pb), Lithium (Li), Nickel (Ni), Rubidium (Rb), Silicon (Si), Tin (Sn), and Vanadium (V). In marked contrast to terrestrial farm animals, fish and shrimp live immersed in an aquatic environment, and as such, have the ability (depending upon the species and culture environment) to satisfy all and/ or part of their dietary mineral requirement through mineral absorption across the gills, body surface and/or through water ingestion. The following tables show the major reported deficiency signs and health impacts of fish and shrimp fed diets deficient in macro-minerals and trace minerals, including the macro-minerals calcium, phosphorus, potassium, and magnesium, and the trace minerals iron, zinc, copper, manganese, selenium, chromium, iodine and cobalt.

Table 1. Reported mineral/element deficiency signs and health impacts on fish and shrimp.

CALCIUM (Ca)

Reported deficiency signs & health impacts

Atlantic salmon (Salmo salar)

Reduced bone mineralization when dietary phosphorus was below requirement level Reduced growth & bone mineralization in low-calcium water, reduced growth & feed efficiency, reduced growth, feed efficiency & bone mineralization Reduced growth & feed efficiency in calcium-free water

Blue tilapia (Oreochromis aureus) Channel catfish (Ictalurus punctatus) Grass carp (Ctenopharyngodon Idella) Tiger puffer (Takifugu rubripes) Hybrid tilapia (Oreochromis niloticus x O. aureus) Indian major carp (Labeo rohita) Japanese flounder (Paralichthys olivaceus) Red lip mullet (Liza haematocheila) Nile tilapia (Oreochromis niloticus) Grouper (Epinephelus coioides) Rainbow trout (Oncorynchus mykiss) Red seabream (Pagrus major)

Increased body deformity (short body) - closed recirculating aquaculture systems Reduced feed efficiency & scale mineralization Delayed ossification of bone endochondral structures Reduced bone K and Mn content

White shrimp (Litopenaeus vannamei)

Reduced growth, feed efficiency & survival

Reduced growth, feed efficiency & bone mineralization Reduced growth & bone/scale mineralization Reduced growth, feed efficiency, protein efficiency ratio, and whole-body, vertebrae & scale Ca content Reduced growth

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

47 Table 1. Continued.

PHOSPHORUS (P)

Reported deficiency signs & health impacts

Atlantic salmon (Salmo salar)

Reduced growth, feed efficiency & bone mineralization, reduced whole-body P content, bone deformities and low Ca and P content of vertebrae

Black seabream (Sparus macrocephalus) Chinese sucker (Myxocyprinus asiaticus) Grass carp (Ctenopharyngodon Idella) Blue tilapia (Oreochromis aureus) Crucian carp (Carassius auratus) Red lip mullet (Liza haematocheila) Catla (Catla catla) Common carp (Cyprinus carpio) Channel catfish (Ictalurus punctatus) Chum salmon (Oncorhynchus keta) Japanese seabass (Lateolabrax japonicus) Milkfish (Chanos chanos) Mozambique tilapia (Oreochromis mossambicus) Siberian sturgeon (Acipenser baerii) Yellow catfish (Pelteobagrus fulvidraco) European seabass (Dicentrarchus labrax) Gibel carp (Carassius auratus gibelio var. CASIII) Gilthead seabream (Sparus aurata) Haddock (Melanogrammus aeglefinus) in body lipid content Indian major carp (Labeo rohita) Mrigal (Cirrhinus mrigala) Nile tilapia (Oreochromis niloticus) White shrimp (Litopenaeus vannamei) Olive flounder (Paralichfhys olivaceus) Rainbow trout (Oncorynchus mykiss)

Reduced growth, feed efficiency & protein efficiency ratio Reduced growth & body P content, reduced growth, feed efficiency, bone ash, and whole-body ash contents, reduced growth & bone mineralization

Reduced growth, feed efficiency, protein efficiency ratio & reduced bone, skin & scale mineralization Reduced growth, feed efficiency & bone mineralization Reduced growth & body P content Reduced growth, feed efficiency, bone mineralization, body ash and P, Ca & Mg content

Reduced growth & bone mineralization

Reduced growth, feed efficiency, protein efficiency ratio & increased mortality Reduced growth, feed efficiency, ash & fish P content Reduced growth Poor growth, loss of appetite, poor bone mineralization, deformed vertebrae and an increase Reduced growth, feed efficiency, protein efficiency ratio, alkaline phosphatase activity & P utilization efficiency Reduced growth & feed efficiency Reduced growth, feed efficiency & survival

Stinging catfish (Heteropneustes fossilis)

Reduced growth, feed efficiency, hematology (hemoglobin, hematocrit) & antioxidant status

Striped bass (Morone saxatilis)

Reduced growth, feed efficiency & reduced scale, vertebral and dorsal fin mineralization, scoliosis

Reduced growth & bone mineralization, tetany Reduced bone mineralization and hematology Reduced growth, protein efficiency ratio, body and vertebrae P content Reduced growth, bone mineralization & body P content

Sunshine bass (Morone chrysops) Tambaqui (Colossoma macropomum) Walking catfish (Clarias leather) Yellow croaker (Pseudosciaena crocea)

POTASSIUM (K)

Reported deficiency signs & health impacts

Chinook salmon (Oncorhynchus tschawytscha)

Anorexia, convulsions, tetany & death Reduced growth Reduced growth Reduced growth & antioxidant enzyme level Reduced growth, feed efficiency, protein efficiency ratio & antioxidant enzyme levels Reduced growth & protein efficiency ratio

Fleshy prawn (Penaeus chinensis) Giant tiger prawn (Penaeus monodon) Grass carp (Ctenopharyngodon Idella) Indian major carp (Labeo rohita) Kuruma prawn (Penaeus japonicus)

MAGNESIUM (Mg)

Reported deficiency signs & health impacts

Atlantic salmon (Salmo salar)

Reduced whole body, serum and vertebrae Mg content Loss of appetite, poor growth, high mortality, sluggishness & convulsions, reduced vertebrae Mg content Reduced growth, feed efficiency & decreased whole body, vertebrae, scales and plasma Mg content, reduced growth Reduced growth Reduced growth Reduced growth & electrolyte balance, reduced growth, appetite & movement, vertebral curvature & histological alterations of muscle, pyrolic caeca, and gill filaments. Reduced growth

Common carp (Cyprinus carpio) Grass carp (Ctenopharyngodon Idella) Hybrid tilapia (Oreochromis niloticus × O. aureus) Kuruma prawn (Penaeus japonicus) Rainbow trout (Oncorhynchus mykiss) Whiteleg shrimp (Litopenaeus vannamei)

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

Table 1. Continued.

IRON (Fe)

Reported deficiency signs & health impacts

Atlantic salmon (Salmo salar) Channel catfish (Ictalurus punctatus) Red sea bream (Chrysophrys major)

Reduced hematological values, liver and whole body Fe content Reduced growth, hemoglobin, hematocrit, plasma iron, transferrin saturation and erythrocyte count values Reduced mean corpuscular constants of blood and Fe content

ZINC (Zn)

Reported deficiency signs & health impacts

Channel catfish (Ictalurus punctatus) Common carp (Cyprinus carpio) Grass carp (Ctenopharyngodon Idella) Grass shrimp (Penaeus monodon) Malabar grouper (Epinephelus malaricus) Nile tilapia (Oreochromis niloticus) Orange-spotted grouper (Epinephelus coioides) Rainbow trout (Oncorynchus mykiss) Red drum (Sciaenops ocellatus) Siberian sturgeon (Acipenser baerii) Yellow catfish (Pelteobagrus fulvidraco)

Reduced serum and bone Zn content, reduced growth Reduced appetite, growth & increased mortality, skin & fin erosion Reduced growth & whole body, scale, vertebrae and plasma Zn content Reduced growth, whole-body Zn content & non-specific immune response Reduced growth & serum, muscle, vertebrae & scale Zn content Reduced growth, feed efficiency & bone Zn content, reduced growth & decreased liver, muscle, bone, and scale Zn content, reduced serum antioxidant enzymes, and altered liver histological signs (considerable nuclear migration) Reduced growth, feed efficiency, antioxidant enzyme activity and reduced whole body and vertebrae Zn content Reduced growth, high mortality, cataract, fin & skin erosion, reduced body Zn content Reduced bone & scale Zn content Reduced growth, feed efficiency, erythrocyte & leucocyte count, hemoglobin & hematocrit value Reduced growth & feed efficiency, reduced whole body Zn content, reduced lipase & antioxidant (SOD) enzyme level

COPPER (Cu)

Reported deficiency signs & health impacts

Channel catfish (Ictalurus punctatus) Cobia (Rachycentron canadum) Common carp (Cyprinus carpio) Indian catfish (Heteropneustes fossilis) Grass shrimp (Penaeus monodon) Malabar grouper (Epinephelus malaricus) Orange-spotted grouper (Epinephelus coioides) Russian sturgeon (Acipenser gueldenstaedtii) Snakehead (Channa punctatus) White shrimp (Penaeus indicus) Whiteleg shrimp (Litopenaeus vannamei) Yellow catfish (Pelteobagrus fulvidraco) Yellow croaker (Larimichthys croceus)

Reduced liver antioxidant enzyme level Reduced growth, feed efficiency, survival & whole-body and vertebrae Cu content Reduced growth in carp& tissue Cu content Reduced growth, feed efficiency & liver antioxidant enzyme activity Reduced growth, feed efficiency, protein efficiency, whole-body Cu content & non-specific immune response Reduced growth & liver antioxidant enzyme activity Reduced growth, feed efficiency & non-specific enzymes immunity Reduced growth, feed efficiency & liver antioxidant activity Reduced growth, feed efficiency, protein efficiency ratio, hemoglobin, hematocrit, RCBs & whole body Cu content Reduced growth, survival & whole-body Cu content Reduced growth, enlarged hearts & reduced hemolymph, carapace, and hepatopancreas Cu content Reduced growth & whole-body and liver Cu content Reduced growth, total antioxidant activity & whole-body and vertebrae Cu content

MANGANESE (Mn)

Reported deficiency signs & health impacts

Atlantic salmon (Salmo salar)

Reduced whole-body Mn content & liver oxidation enzyme activity Cobia (Rachycentron canadum) Decreased growth, feed efficiency, survival, whole-body, vertebrae & liver Mn content & decreased liver antioxidant enzyme activity Common carp (Cyprinus carpio) Reduced growth Grass carp (Ctenopharyngodon Idella) Reduced growth, feed efficiency & whole body, vertebrae and skin Mn content Hybrid grouper Reduced growth, feed efficiency, protein efficiency ratio, whole-body, liver and vertebrae Mn content, (Epinephelus lanceolatus × E. fuscoguttatus) reduced antioxidant enzyme activity, and increased oxidative stress Hybrid tilapia (Oreochromis niloticus × O. aureus) Reduced liver, bone and muscle Mn content & reduced liver antioxidant enzyme activity Rainbow trout (Oncorynchus mykiss) Reduced growth, abnormal tail growth, short body dwarfism Whiteleg shrimp (Litopenaeus vannamei) Reduced growth Yellow catfish (Pelteobagrus fulvidraco) Reduced growth & feed efficiency, decreased whole-body Mn content & decreased liver antioxidant activity Yellow croaker (Larimichthys croceus) Reduced growth, feed efficiency, whole-body, liver and vertebrae Mn content, reduced antioxidant enzyme activity, and increased oxidative stress

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

Table 1. Continued.

SELENIUM (Se)

Reported deficiency signs & health impacts

Atlantic salmon (Salmo salar)

Reduced plasma antioxidant (Glutathione Peroxidase - GPx) enzyme activity Reduced growth & liver and plasma antioxidant enzyme activity Reduced growth, feed efficiency, survivak & whole-body and vertebrae Se content, and reduced

Channel catfish (Ictalurus punctatus) Cobia (Rachycentron canadum) antioxidant enzyme activity Gibel carp (Carassius auratus gibelio var. CASIII) Japanese seabass (Lateolabrax japonicus) Rainbow trout (Salmo gairdneri) Malabar grouper (Epinephelus malabaricus) Nile tilapia (Oreochromis niloticus) Yellow croaker (Larimichthys croceus)

Reduced growth Reduced growth, reduced growth, feed efficiency & liver antioxidant enzyme activity Reduced growth & antioxidant capability, reduced growth, feed efficiency, survival & antioxidant enzyme activity Reduced growth & whole-body and vertebrae Se content, reduced total antioxidant activity

CHROMIUM (Cr)

Reported deficiency signs & health impacts

Common carp (Cyprinus carpio)

Reduced growth & carbohydrate utilization Reduced growth, feed efficiency, protein efficiency ratio, protein retention, and altered carbohydrate & lipid metabolism Reduced growth, feed efficiency, protein efficiency ratio & apparent net protein utilization Reduced growth Reduced immune response, reduced growth & feed efficiency

Grass carp (Ctenopharyngodon) Indian major carp (Labeo rohita) Nile tilapia (Oreochromis niloticus) Rainbow trout (Oncorhynchus mykiss)

Reduced growth & antioxidant capability

Due to space limitations Cobalt and Iodine were removed from the Table.

Notwithstanding the above-reported mineral deficiency signs, it should be stated that the majority of these studies were performed with animals fed semi-purified experimental diets (so as to elicit a deficiency response), and as such may not be applicable to animals fed practical diets. Animal and marine feed ingredient sources usually being richer sources of essential minerals and trace elements, compared with plant feed ingredient sources where many minerals and trace elements are usually less biologically available. This difference between animal and plant feed ingredient sources is particularly important for marine protein (ie. fishmeal) dependent cultured species, and the necessity to reduce their dependency on these finite feed resources with alternative terrestrial land animal and plant-based feed ingredient sources. As a general rule, animals reared under nutrient-poor freshwater environments are more likely to require dietary mineral and trace element supplementation than animals reared under marine culture conditions, and as such feeds should be formulated using a dietary mineral and trace element premix. Moreover, in view of the pro-oxidant properties of many inorganic mineral salts, it would be advisable to use minerals

chelated to organic molecules (such as specific amino acids or microorganisms) so as to maximize biological availability, and at the same time, limit unwanted biological interactions and nutrient leaching in the case of soluble inorganic mineral salts. As with other dietary essential nutrients, many minerals and trace elements have important functional properties, and as such, these minerals may require higher dietary fortification for maximum health and immune response, and in particular the immunestimulating trace elements zinc, copper, manganese, selenium and chromium. However, it is also important to mention here that most minerals and trace minerals may exert a negative effect on fish/shrimp growth and health if supplied in excess of dietary requirement, either due to direct toxicological effects or due to negative interactions with other minerals. For example, an excess of dietary calcium (calcium carbonate or limestone often being used as an inexpensive filler in aquafeeds) has been reported to exert a negative effect on the bone mineralization and the biological availability of other essential minerals, including P, Mg, K, Zn, Mn, and Fe. As a general guideline, the dietary Ca:P ratio should be kept at about <2:1.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

How to help the anti-infective immunity of European carp Mara Thomas, Justyna Andrysiak, Proteon Pharma

The excessive use of antibiotics has triggered the development of resistance in bacteria rendering most antibiotics partially or completely ineffective. The aquaculture industry is greatly responsible especially due to the overuse of antibiotics. In 2016, Chile used 382.5 tons of antibiotics for the salmon industry alone (Miranda et al. 2018). Antimicrobial resistance (AMR) also has a substantial economic impact. In Chile, the AMR crisis of 2009, dropped the value of their production by $2 billion until 2011 (Asche et al. 2009). This has revealed a necessity for innovative alternatives to help tackle the AMR threat. Bacteriophages, simply called phages, are viruses that attack only bacteria. They help eliminate excessively multiplying bacteria that could lead to pathological conditions without

disturbing the microbiome, as antibiotics do. As they are naturally occurring in the environment, they pose little to no threat of developing resistance. BAFADOR® is a bacteriophage cocktail that targets Aeromonas and Pseudomonas bacteria and helps strengthen the fish’s immune system. This study aims to assess its efficacy as a feed additive used prophylactically in European carp.

Methodology This study used 180 healthy carp (Cyprinus carpio). Upon their arrival, they were randomly split into three groups, each with 3 repetitions and they all underwent an acclimatization period of 12 days. They were kept in 180L tanks at a temperature of 18,6°C and were fed Ofeedo Extracarp 25 commercial diet manually.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

Figure 1. Mortality after infection with mix of A. hydrophila and P. fluorescens.

The average weight of fish was 80,15 g. The negative control groups were neither infected nor treated. The positive control groups were infected by intraperitoneal injection with an Aeromonas hydrophila and Pseudomonas fluorescens mix but were not given the prophylactic feed additive. The experimental groups were also infected with the same mix but were additionally given BAFADOR® mixed with the feed, in the amount of 0.02ml/fish. BAFADOR® was given 5 days before the experimental infection and daily after, at the same dose, for the remaining time of the experiment. The mortality in each group was recorded for 21 days in order to determine if BAFADOR® was effective in helping the immune system of carps and preventing bacterial infection. The study lasted a total of 40 days.

Results As can be seen in Figure 1, the experimental groups (3a, 3b and 3c) which were given BAFADOR® did not record any mortality during the experiment. In comparison, the positive control groups (2a, 2b and 2c) which were infected but did not receive the bacteriophage cocktail had an average mortality of 15%. BAFADOR® helped prepare the carp’s immune system and was effective in preventing infection by A. hydrophila and P. fluorescens. In addition, the water analysis showed that BAFADOR® was successfully absorbed by the feed, confirming this application method was the most suitable.

Conclusion The results of the experiment show that BAFADOR® is an effective solution for the prevention of A. hydrophila and P. fluorescens infections. The low mortality rates in the groups where BAFADOR® were administered compared to the infected control show the positive contribution of BAFADOR® to the fish’s immune system and general wellbeing. Improving the general health status of fish as well as lowering mortality rates among farmed fish can contribute to a better quality of production and financial savings to the farmer. References Asche, F., Hansen, H., Tveteras, R. and Tveterås, S., 2009. The salmon disease crisis in Chile. Marine Resource Economics, 24(4), pp.405-411. Miranda, C.D., Godoy, F.A. and Lee, M.R., 2018. Current status of the use of antibiotics and the antimicrobial resistance in the Chilean salmon farms. Frontiers in microbiology, 9, p.1284.

More information: Justyna Andrysiak Chief Product Developments Officer Proteon Pharma, Poland E: proteon@proteonpharma.com

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

How we used biosynthesis to produce the world’s strongest antioxidant Maxim Mikheev, BIOMEDICAN A

Figure 1. Summary of product development, from the beginning to production-ready strain. A) Initial red colonies. B) Improving carotenoids production. C) Scaling up production. D) Crystals inside cells contain 70% astaxanthin, 17% beta-carotene and 10% of canthaxanthin. D) Final biomass.

Biomedican entered into the astaxanthin market to diversify its products and show that its biosynthesis platform can target any high-value compound that has a significant market. There was a need to do something new so that we weren't overly concentrated on just the cannabinoid market. After several days of intense research, we found a strain producing beta-carotene with a very high yield. After a not-too-long analysis, we found that

by adding just a few genes, we could convert betacarotene into astaxanthin. Now, we’re able to produce a biosynthesized, non-GMO, organic astaxanthin molecule that is identical to what is found in nature. Astaxanthin is a high-value compound. It is the strongest natural antioxidant in the world. It is 6,000 times stronger than vitamin C and 550 times stronger than vitamin E. Astaxanthin’s estimated market was $1 billion in 2019.

Aquafeed: Advances in Processing & Formulation Vol 13 Issue 3 2021

The astaxanthin market consists of fish feed, animal feed and human consumption. Astaxanthin is heavily used for salmon farming. Wild salmon has a pink color due to the presence of astaxanthin inside. Astaxanthin is naturally produced by some alga, yeasts and bacteria. Then zooplankton, krill and shrimp consume the initial astaxanthin producers. Later, salmon eat krill and shrimp, accumulate astaxanthin, and get their pink color in the natural environment. Astaxanthin is necessary to maintain the natural color of farmed salmon. In order for farmed salmon to have the same pink color as salmon found in the wild, they must be fed astaxanthin. This compound also serves as a chicken food supplement, adding a deep yellow color to egg yolks. Salmon farms are currently using chemically synthetic astaxanthin. Synthetic astaxanthin is not identical to natural astaxanthin. Synthetic astaxanthin has a different molecular shape (stereoisomers) than its natural counterpart. The natural astaxanthin contains the only all-trans form. There are three main forms (3S, 3’S), (3R, 3’R), (3R, 3’S), with the most dominant being (3S, 3’S). Synthetic astaxanthin contains more than 500 stereoisomers. The current regulations have forbidden direct consumption of synthetic astaxanthin by humans, but it is permitted to use as animal/fish feed. It was shown that synthetic astaxanthin has no health benefits and significantly weaker antioxidant properties.

nature. The process works through proprietary yeasts that undergo fermentation to consistently produce large quantities of high-quality biosynthetic astaxanthin, identical to its natural counterpart, at the same economical price as chemical synthesis. We made an analysis of genes and some standard genetic designs last March. We ordered a gene synthesis by Twist Bioscience. One month later, we received a set of constructed genes optimal for biosynthesized astaxanthin. In summary, from the beginning (Apr 2020) to a production-ready strain (Sep 2020) took six months (Fig. 1).

Conclusion BIOMEDICAN can produce a biosynthesized astaxanthin product that is non-GMO, organic, and identical to what is found in nature. We can do this at 90% less cost than the current chemically synthetic product. The chemically synthetic product is less than ideal because it produces byproducts, toxins, and contaminants. Thus, there is great interest in biosynthesized astaxanthin. The largest buyers of astaxanthin are looking to change from chemically synthetic products to non-GMO and organic products that are significantly more bioavailable for humans and animal feed.

More information:

Developing a natural form of astaxanthin We decided to produce a natural form of astaxanthin using biosynthesis. Biosynthesis of astaxanthin eliminates the production of undesirable stereoisomers, allowing for the specific creation of astaxanthin found in

Maxim Mikheev CEO and Founder BIOMEDICAN E: max@biomedican.com

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AQUAFEED VOL 13 ISSUE 3 2021

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