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The effect of juice and jam processing of Physalis peruviana L. on β-carotene and ascorbic acid content

Student: Frederike Wiggers Registration: 930227 954 110 Course code: YFS-80824 European Credit Transfer and Accumulation System (ECTS): 24 Starting date: 2nd of September 2013 Planned completion date: 21st of January Examiner: Vincenzo Fogliano Supervisors: Mary Luz Olivares Tenorio, Jenneke Heising, Ruud Verkerk, Matthijs Dekker


Acknowledgements First of all I would like to thank some people that helped me during the process of execution and writing of my thesis. First, I would like to thank all the technicians at the chair group of Food Quality and Design, Frans Lettink, Xandra Bakker-de Haan and Charlotte van Twisk for helping me with the apparatus and familiarizing with the lab protocols. Also a special thanks to Geert Meijer, because he was always there to patiently help me when the HPLCs got their own will. Then I would like to say thanks to my supervisors Ruud Verkerk and Matthijs Dekker for their role in maintaining the main goal and focus of the project. Of course I would also like to thank Jenneke Heising, who was first not directly involved in my project but from who I got a lot of support when Mary Luz was not here anymore. Also a special thanks to Radhika Bongoni because she showed me some very important features for my investigation and a very big smile when I saw her. I would also like to thank Mary Luz Olivares Tenorio for guiding me through the first steps of lab work and outlining of the goals of this project. She was there to give me confidence that I could work on my own during the time that she was not here anymore. Last but not least I want to thank Ning Wang, because she helped me through everything in the lab and she was there for me when Mary Luz was gone to help me smile during the long days in the lab.

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Abstract The effect of jam and juice processing on the levels of ascorbic acid and β-carotene was investigated. One of the steps in the processing of jam is a heat treatment; usually jam is heated at 90° Celsius until a sugar content of 65° Brix is reached. The length of the heating time is dependent on the sugar content that must be reached. When lower temperatures are used, water will evaporate less quickly and the preferred final sugar content will be reached later. For jam three different temperatures (80,90 and 100° Celsius) were investigated in combination with two different final sugar contents (60 and 65° Brix) and three different ratios of fruit and sugar. The results show degradation for L-ascorbic acid between 88.95 and 99.61% compared to the initial value of 35.96 mg/100g. For total ascorbic acid the initial value is 41.84 mg/100g, with degradation in a range of 86.68 until 98.24%. For β-carotene, the initial value of 1.145 mg/100g had a maximal degradation of 99.03% and a minimal degradation of 62.45%(100° C). For both ascorbic acid and β-carotene the concentration of the compounds was degraded, but the concentration was higher when the cooking time increased. Analysis of the texture of the jam with the texture analyser showed that the strength of the gel depends on the fruit content of the jam. The more fruit is present in the mixture, the stronger the gel. Four different temperatures (60,70,80,90° Celsius) were used to pasteurise the juices for one minutes, except for 60° C which was pasteurised for four minutes. Samples were taken at t=0, t=heating up time and t=heat treatment to see the difference during the whole experiment. The concentration of L-ascorbic acid and β-carotene decreased when the temperature of the pasteurisation treatment increased. The concentration of L-ascorbic acid after blending was 37.15±1.34 mg/100g, which decreased to a final value of 12.38mg/100g after one minute of pasteurisation at 90° Celsius. For β-carotene the lowest concentration obtained was 0.79mg/100g after pasteurisation for one minute at 90° Celsius, which is a decline of 16.84% from the value of fresh juice, which is 0.95mg/100g.

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Table of Contents Acknowledgements ........................................................................................................... 2 Abstract ............................................................................................................................ 3 1.

Introduction ............................................................................................................... 5

2.

Background information ........................................................................................... 7 2.1 Ascorbic acid............................................................................................................ 7 2.2 Carotenoids ............................................................................................................. 8 2.3 Jam and Juice ......................................................................................................... 9

3.

Problem definition ....................................................................................................10

4.

Aim of research ........................................................................................................10

5.

Materials and Methods ............................................................................................11

6.

Results and discussion .............................................................................................16 6.1 Initial values ..........................................................................................................16 6.2 Jam .........................................................................................................................16 6.3 Juice .......................................................................................................................23 6.4 Texture analysis of jams ........................................................................................26

7.

Conclusion ................................................................................................................28

8.

Recommendations ....................................................................................................29

9.

References.................................................................................................................30

10. Appendix ....................................................................................................................33 10.1 Overview of jam samples .....................................................................................33 10.2 Texture analysis of jam ........................................................................................34 10.3 Raw data of HPLC analysis ...............................................................................36

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1. Introduction

The Cape gooseberry is a berry grown from the Physalis peruvia plant, originally grown in the higher areas of South America (Puente, Pinto-Muñoz et al. 2011). The fruit got its name because it was cultivated by settlers near Cape of Good Hope, in South Africa in the 1800s(Morton 1987). Colombia is the largest producer of Cape Gooseberries with 11,500 tonnes of Cape Gooseberries per year(Puente, Pinto-Muñoz et al. 2011). Other countries that grow Cape Gooseberries are South Africa, England, China, India and Australia(Morton 1987). The Physalis peruviana is family of the Solonaceae. This is one of the broadest plant families you can find worldwide and has its origin in the Andean and Amazonian areas in South America. The Solanaceae family encompasses tomato, eggplant, Cape gooseberry, peppers and potatoes, but also ornamental flowers, edible leaves, medicinal and seeds like coffee(Bombarely, Menda et al. 2011). The Solonaceae are able to live in very different climates, from deserts to high altitudes to rainforests. The Cape gooseberry has a smooth orange skin and contains a lot of small kernels in the center of the fruit, like tomatoes. Because the fruit is protected by a calyx, another common name is the husk cherry. Other names that are used for Physalis peruviana are Peruvian Ground cherry, goldenberry and Poha berry. The fruit itself grows on the softwooded, hairy branches of the bushes of the Physalis peruviana, of which physa means bladder and peruviana Peru. The berries can grow at a wide range of temperatures and heights, but the preferred circumstances are 18°C with lots of sunlight, acid ground with a pH range of 5-7, not too much wind and reduced rain when flowering. Multiple variables have influence on the content of β-carotene and ascorbic acid. It is shown by Singh et al. (Singh, Pal et al. 2012) that the highest levels of these compounds can be found after eight weeks of anthesis. This implies that pre or postharvest conditions may influence the content of compounds in the fruit. The amount of sun hours in combination with the altitude where the fruit is grown may also influence the β-carotene, ascorbic acid and sugar content of the fruit (Fischer 2000).

Figure 1 Physalis peruviana Linnaeus (Vincente)

The Cape gooseberry, together with the calyx and the leaves are traditionally used for medical treatments for e.g. cancer but also anti-inflammatory (Zavala, Quispe et al. 2006) (Wu, Tsai et al. 2006). The health promoting characteristics of this fruit are related to L-ascorbic acid (vitamin c), carotenoids and phenolic compounds, which are all present in Cape Gooseberries (Ramadan 2011). Ascorbic acid is found to be present for 43 milligrams per 100 grams of gooseberry pulp(Ramadan and Morsel 2004), βcarotene is present in 235μg per 100 grams of fruit weight (Fischer 2000), but the values differ a lot in literature, which is shown in Table 1 and Table 2. The total level of phenolic compounds was estimated to be 6.3 milligrams per 100 grams of gooseberry juice (Ramadan and Moersel 2007). These compounds are together partly responsible for the total antioxidant capacity of the fruit. Other compounds that are responsible for the 5


total antioxidant activity are phytosterols and Vitamin E, including α-,β- and γtocopherols(Ramadan and Moersel 2007). Table 1 and Table 2 show the range of values found in literature for ascorbic acid and β-carotene present in Cape gooseberries. Table 1 Values of ascorbic acid in cape gooseberry found in literature

Concentration ascorbic acid (mg/100g) 32.2 10.17 43 33.385 33.1 43.3

Source (Fischer 2000) (Singh, Pal et al. 2012) (Ramadan 2011) (Baijer 2013) (Valente, Albuquerque et al. 2011) (Repo de Carrasco and ENCINA ZELADA 2008)

Table 2 Values of β-carotene found in literature

Concentration β-carotene (mg/100g) 0.236 1.617 1.600 2.103 2.640

Source (Fischer 2000) (Singh, Pal et al. 2012) (Ramadan 2011) (Baijer 2013) (Repo de Carrasco and ENCINA ZELADA 2008)

Because of the health promoting compounds, the food industry is willing to use Cape Gooseberries in products more often(Ramadan and Moersel 2007). Therefore it is important to study the effect of heating during the process of making juice and jam of the fruit on the health promoting compounds. Vitamin C is known to be heat labile (Khraisheh, McMinn et al. 2004) which is also known for β-carotene (Cinquanta, Albanese et al. 2010) and flavonoids (Lee, Durst et al. 2002). Therefore it is important to study the influence of processing on the concentration of health promoting compounds.

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2. Background information 2.1 Ascorbic acid L-Ascorbic acid, which is also known as vitamin C, is a water soluble vitamin. The chemical name is 2-oxo-L-threo-hexono-1,4-lactone-2,3-enediol (Naidu 2003). The molecule structure can be found in Figure 2. This vitamin is present in lots of fruits and vegetables. The ascorbic acid level in cape gooseberry (46 mg/100 g) is higher than in most fruits, such as pear (4 mg/100 g), apple (6 mg/100 g), and peach (7 mg/100 g), but is quite similar to orange (50 mg/100 g) and strawberry (60 mg/100 g)(Ramadan 2011). Because humans lack the enzyme L-gulonolactone oxidase, they cannot synthesize ascorbic acid, and therefore ascorbic acid has to be included in the human diet. If the amount of consumed ascorbic acid is not sufficient, a deficient may occur which leads to scurvy. That is how the original hexuronic acid got its new name; ascorbic acid, which in Latin means “anti-scurvy”.

Figure 2 Molecule structure of L-ascorbic acid (Diederen 2012)

Vitamin C also has an important function as antioxidant. In a study of Gardner (2000), it was found that Vitamin C contributed to “65–100% of the antioxidant potential of beverages derived from citrus fruit but less than 5% of apple and pineapple juice.”(Gardner, White et al. 2000) Vitamin C is proven to contain antioxidant activity which decreases the free radicals and prevents damage on lipids and DNA (Halliwell and Gutteridge 1999). Other well-known characteristics of ascorbic acid are its sensibility for light, elevated temperature, oxygen, alkaline pH, oxidizing enzymes and divalent cations like copper and iron (Russell 2012). Most of these characteristics are less impetuous when ascorbic acid is not dissolved, which may imply an effect on the stability of the compound from the matrix where the compound is present. The role of vitamin C in the prevention and curing of several diseases, like cancer but also a common cold, has been widely spread, but the evidence for this has been conflicting. Ascorbic acid is known to be very heat labile. Vitamin C was measured in juice made from Physalis peruviana L., and the average was 46 milligrams per 100 grams of juice(Ramadan and Moersel 2007). When the juice was pasteurized for 10 minutes at 80° Celsius, this value decreased to 39 milligrams per 100 grams of juice. The degradation of Vitamin C was investigated, and it was found that it follows a first order reaction, with an activation energy of 58kJ/mol and a degradation rate constant for 100° C of 5.0*10-3 per minute(Belibel 2013). Compared with other research, also an activation energy of 58 kJ/mol was found and a kd100°C of 5.011*10^-3 per minute(Baijer 2013). Research done by Plaza et al. (Plaza, Sánchez-Moreno et al. 2006) on orange juice showed that the degradation of ascorbic acid was approximately 8 percent for juice treated with pulsed electric field, low pasteurization and high-pressure. Therefore all these treatments have a low impact on the degradation of ascorbic acid.

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2.2 Carotenoids Carotenoids are fat soluble compounds that can only be metabolised by plants and animals. Therefore it is necessary for humans to include them in their diet. The major carotenoids found in Physalis peruviana are all-trans β carotenes, responsible for 76,8% of the total carotenoids found in the samples (de Rosso and Mercadante 2007). Also 9-cis-β-carotene and all-trans-α-cryptoxanthin are found, responsible for 3.6 and 3.4% of the total carotenoids, respectively. The amount of β-carotene is Cape gooseberry pulp is found to be 1.613 milligrams per 100 grams. The values of β-carotene present in Cape Gooseberry vary a lot (see Table 2). The lowest value found was 235 micrograms per 100 grams of whole fruit(Fischer, Ebert et al. 1999) and the highest level of βcarotene found in literature was 2.64 milligrams per 100 grams(Repo de Carrasco and ENCINA ZELADA 2008). The difference in concentration can be caused by different factors like sun hours, harvest time, climate, transport, storage and more. Β-carotene is a carotenoid which is often present in fruits and vegetables. The structure of the molecule can be seen in Figure 3 (Rodriguez-Amaya 2001). The conjugated system - alternating single and double bonds- is responsible for the absorbance of wave lengths of 378 till 502λ (Rodriguez-Amaya 2001). Because of the absorption of these wavelengths, opposite wavelengths are reflected, which gives the molecules colours, from yellow to red. A minimum of seven alternating bonds is needed to get a perceptible colour. Therefore, β-carotene is responsible for the colour of the fruits. Carotenoids are in greater extent found in the peel of the fruit than in the pulp. When the fruit ripens, the chlorophyll transform to chromoplasts, which gives the transformation of colour from green to yellow/orange. Carotenoids are sensible for oxidation and isomerization by means of oxygen, metals, enzymes, heat, light, pro- and antioxidants. Bicyclic β-carotene is a carotenoid that is a provitamin A form. Provitamin A is a precursor for vitamin A, which is an important vitamin in the human body. Vitamin A plays a major role in the regulation of the immune system. When Vitamin A deficiency occurs, this can lead to night blindness and xerophthalmia, which is a deficiency in the production of tears, through which no cleaning of the eyes can take place, and these will cause infections.

Figure 3 Molecule structure of β-carotene (Rodriguez-Amaya 2001)

The behaviour of β-carotene in juices was investigated for different fruits and vegetables. Chen et al. demonstrated that the concentration of β-carotene in carrot juice decreases the most when heated for 30 minutes at 121° Celsius with a pH of 6.1 (Chen, Peng et al. 1995). The decrease was less for juice that was treated for 30 minutes at 120° Celsius and even less at 110° Celsius. When a high pressure treatment (500/800MPa, room temperature) is executed on a juice with oranges, lemon and carrot, the stability of carotenoids is minor influenced (Oey, Van der Plancken et al. 2008). When these juices are stored at 4° Celsius, the carotenoid levels stayed constant for 21 days (Fernández García, Butz et al. 2001). When the high pressure treatment was executed at elevated temperatures, the impact on the carotenoid levels was minimal(de Ancos, Sgroppo et al. 2002). These investigations show that higher temperatures have a greater influence on the degradation of β-carotene than lower temperatures and that high pressure treatments are even better in conservation of β-carotene. The degradation of β-carotene was investigated previously and an activation energy was found of 92.8 kJ/mol and a k value for 100° C of 5.863*10-4 per minute(Baijer 2013). 8


2.3 Jam and Juice In Kenya, where the Cape Gooseberry is very popular, it is already used in jams(1989). Jam making is a process of preservation fruit by lowering the water activity by means of sugar and vacuum packaging in jars that are heated when the jar is filled with jam. When heating, the water will evaporate and the sugar concentration will increase. Then the pectin is added, which will “set” the jam and gives it a good consistency. Pectin does not dissolve at room temperature, so higher temperatures are needed to dissolve the pectin and because water needs to evaporate in order to reach the preferred sugar content, temperatures are used in the range of 80 to 100 ° C. The mixture is heated until the preferred sugar content of 65° Brix is obtained. The Codex standard for jams, jellies and marmalade defines jam as a product “brought to a suitable consistency, made from the whole fruit, pieces of fruit, the diluted and/or concentrated fruit pulp or fruit puree, of one or more kinds of fruit, which is mixed with foodstuffs with sweetening properties as defined in Section 2.2, with or without the addition of water”(Alimentarius 2009). It is also stated that the minimal amount of fruit should be 35 percent. The average sugar content should be above 65 °Brix. It was found that Cape Gooseberries contain 4.9 grams of sugars per 100 gram of juice (Ramadan 2011). Compared with other fruits, this value is quite low. Orange and strawberry, for example, have a sugar content of 7.0 and 5.7 grams per 100 gram of juice, respectively. Therefore it is necessary to add 45 percent of fruit and 55 percent of sugar. The high amount of sugar is intended to increase the water activity and to reduce the risk of spoilage by microorganisms. For the pH, it is needed to have a pH below 3.5, because otherwise the pectin will not stabilise and the gel will not be formed(Zhao 2012). Although cape gooseberries are known for their high amounts of pectin (Zhao 2012), it might be necessary to add some pectin for proper gel formation. The amount of pectin that needs to be added will be tested during the trial phase. Pectin(Poly-D-galacturonic acid methyl ester) is a polysaccharide found in plant cell walls. The backbone of the molecule is build up from α 1-4 linked polygalacturonic acid molecules alternated with rhamnose(Ni, Yates et al. 2010). To this backbone different side chains are connected. The degree of methylation represents the percentage of carbonyl groups that are esterified with methanol. When this percentage exceeds 50, the molecule is called high-methoxy pectin (HM), otherwise it is an low-methoxy pectin(LM). Low-methoxy pectin, which was used for these experiments, forms a gel when Ca2+ is present to form ionic linkages via calcium bridges(Thakur, Singh et al. 1997). By means of these interactions junction zones are formed. The junction zones from High-methoxy pectin are stabilized by means of hydrogen bonds and hydrophobic interactions. For Cape Gooseberry juice the standards are different. The Codex standard for pulpy nectars of certain small fruits preserved exclusively by physical means, states that the minimum content of fruit should be 30 percent (Alimentarius 1981). The maximum soluble solids should not exceed 20 percent m/m, and it should be measured in °Brix at 20° C. To lengthen the shelf life the juice should be pasteurised or sterilized, depending on the preferred shelf life. The pasteurisation or sterilization time is depending on the pathogen you would like to reduce with 5 log (e.g. Salmonella,Listeria monocytogenes, E.coli O157:H7). For this product the E. coli O157:H7 is the dependent factor, because E. coli O157:H7 is known to be heat stable. The U.S. Food and Drug Administration stated that for apple juice a recommended pasteurisation time should be used of 0.3 seconds for a temperature of 82.2° C (180° Fahrenheit). This time is based on a 5-log reduction of oocysts of Cryptosporidium parvum, which is more heat labile than E.coli O157:H7.

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3. Problem definition

Cape gooseberries contain ascorbic acid (33.385±4.15 milligrams per 100 grams), βcarotene (2.103±0.23 milligrams per 100 grams) (Baijer 2013) and phenolic compounds (6.3 milligrams, per 100 grams of fruit juice) including flavonoids (Ramadan 2011). Ascorbic acid, carotenoids and phenolic compounds are all known for their health promoting characteristics, but ascorbic acid and carotenoids are also known for their sensibility during heat processing (Cinquanta, Albanese et al. 2010). To maintain a maximal content of health promoting compounds per 100g of product in both juice and jam, it is important to study in which extent these compounds degrade during the processing of the products. To study this degradation, the content of ascorbic acid and carotenoids will be measured before and after the processing steps, where temperature, fruit content and final sugar content are varied. By varying these three variables, it will be possible to measure in which extent β-carotene and ascorbic acid are sensible to the processing methods of juice and jam.

4. Aim of research

The aim of this investigation is to establish a process for jam and juice processing from Cape gooseberries (Physalis peruviana, L.) and to compare the content of β-carotenoid and ascorbic acid (Vitamin C)before and after the processes of juice and jam making.

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5. Materials and Methods Sample preparation The Cape gooseberries were peeled to remove the calyx and then checked for moulds, black spots and size. Some berries were rejected based on these characteristics, for example not fully grown berries. After the control the berries were frozen in a whole with liquid nitrogen and stored at -20° C. The gooseberries used for these experiments were prepared in April 2013 and they are stored in plastic trays that did not prevent light or oxygen from getting in. Jam making First the right amount of frozen fruit (see Table 4) is immersed in the same amount of hot water to extract the natural present pectin of the fruit, after this, the fruit and water are grinded in a blender (Waring commercial blender model HGB2WT83). Thereafter, one third of the weighed sugar is mixed with the pectin (Pectin from apples, BioChemika, Sigma Aldrich Co.) to divide it better over the fruit mixture. The other two third of the sugar is added first to the fruit. After this sugar is dissolved, the remaining sugar and pectin are added. Then the mixture is heated to the desired temperature on an induction cooking plate (ATAG induction cooking plate Type HI050B5U). During the cooking a sample is taken from the mixture and cooled. After cooling, the sugar content is measured by means of a Brix meter (Bellingham+Stanley eclipse serie number 039408). When the sugar content reaches 60 or 65° Brix, respectively, the jam is cooled by means of a bucket of ice and the pH is measured (WTW 720). To preserve the content of ascorbic acid and β-carotene, an antioxidant (BHT) is added. The vitamin C extraction is done immediately after the jams are prepared. The β-carotene samples are weighed and kept in the freezer at -20° Celsius for storage after addition of 5 ml of water, 10 ml of hexane and 0.4 ml of BHT . An overview of the different samples that will be measured is shown in Table 3, the exact ratio of ingredients can found in Table 4. Table 3 Experimental design for Physalis peruviana L. jam processing by heating. For the samples at 80, 90 and 100° C, water will evaporate and the sugar content will increase till 60 and 65° Brix, respectively.

Final sugar content (°Brix)

Initial sugar content (%)

Heating temperature (° Celsius)

60

Jam I

80 90 100 80 90 100 80 90 100 80 90 100 80 90 100 80 90 100

Jam II

Jam III

65

Jam I

Jam II

Jam III

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Table 4 Ingredients in grams per 100 g jam used for different types of jam

Ingredients

Jam I

Jam II

Jam III

Sugar (g) Fruit(g) Water(g) Pectin (g)

29.22 35.06 35.06 0.65

38.2 30.55 30.55 0.694

48.51 25.37 25.37 0.746

Juice making For the juice, the fruit is defrozen in the refrigerator overnight and then blended. After the blending the kernels and skins are removed from the mixture by sieving. After this, the different juices will receive different heat treatments by means of a heating block (Liebisch Type 53186301). Because the time needed to get a 5-log reduction in pathogens is very short (0.03 seconds at 80° C), it has been decided to take a fixed time (one minute) with the variable temperatures of 70, 80 and 90° C. For 60° C this would not be enough to get a 5-log reduction of E. coli O157:H7(FDA 2010), so for 60° C a heat treatment is applied for four minutes. These values are based on a 5-log reduction of Cryptosporidium parvum, which is believed to be more heat resistant than E. coli O157:H7. Because time is needed to reach the preferred temperature through the whole sample, samples were taken at different times. The first sample was taken when no heat is applied yet. The second sample when the temperature has reached 60,70,80 or 90 °C , respectively, and the third sample was taken when the actual heat treatment has been applied. All samples are immediately cooled by means of a bucket of ice to keep the level of present compounds as stable as possible. Ascorbic acid extraction was done immediately after cooling. The juices meant for β-carotene extraction were stored in the freezer at -20° C for one night after the addition of 0.4 ml of BHT,10 ml of hexane and 15 ml of Milli-Q water . An overview of these treatments can be found in Table 5. Table 5 Heat treatments applied to Cape Gooseberry juice

Treatment No heat treatment 60°C- 4minutes

Sample taken at t=0 t=sample heated to 60°C t=4 minutes at 60°C

70°C- 1 minute

t=sample heated to 70°C t=1 minute at 70°C

80°C- 1 minute

t=sample heated to 80°C t=1 minute at 80°C

90°C- 1 minute

t=sample heated to 90°C t=1 minute at 90°C

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Determination of L-ascorbic acid and dehydroascorbic acid For the determination of L-ascorbic acid and Dehydroascorbic acid, protocol 35, “Determination of L- Ascorbic Acid(AA) and L-dehydroascorbic Acid(DHA) using HPLC” by Charlotte van Twisk and Frans Lettink was used. During the extraction, the samples were treated in subdued (red) light and on ice to prevent the degradation of ascorbic acid as good as possible. For jam, approximately 0.25 grams of sample was weighed and put in a 15 ml Greinder tube. To this sample, 0.4 ml of BHT (Sigma Aldrich Co.) and 3.5 millilitre of 3% MPA(Merck), and 1.0 ml of 1 M THBQ(Merck) in Milli-Q water solution were added and this mixture was homogenized by means of an Ultra Turrax (IKA T25 ) with a small Ultra Turrax tube at the highest speed for one minute. After the homogenization, the samples are centrifuged (ThermoFischer Scientific Hereaus Multifuge X3R, serie number 41064061), at 3000 rpm for 5 minutes at 4° C. After the centrifugation the supernatant is collected in a pre-weighed tube, and 3.5 ml of MPA THBQ solution is added to the pellet. Then the previous steps are repeated until the supernatant is collected for the third time. Then the weight of the supernatant is obtained and the supernatant is divided over four Eppendorf tubes of two ml. These tubes are centrifuged for 10 minutes at 10,500 rpm at 4° C. After this, the supernatants are filtered using an 0.2μm CA filters(Sartorius Stedim Biotech). Two of the four tubes are for the determination of L-ascorbic acid (AA) and can be put into the amber vials for measurements. The other two tubes are reserved for total ascorbic acid determination (TAA). The amber vials(Grace 1.5ml Tread 8-425) are filled with 1.485 ml of extract and 15 microliter of TCEP solution(Sigma Aldrich Co.). TCEP reduces the L-dehydroascorbic acid to L-ascorbic acid, so the amount of oxidation can be determined For the juice samples, there are two methods used to determine the content of L-ascorbic acid; from the juice and from the pellet. For the juice, 10 ml is pipetted into a 15 ml Greinder tube and centrifuged for 10 minutes at 3000 rpm at 4° C. Then the juice is deposited in four Eppendorf tubes of each two ml and centrifuged for 10 minutes at 10500 rpm at 4° C. The content of the tubes are further processed like the jam samples. For the pellets of the juice, again 10 ml of juice is pipetted in a 15 ml Greinder tube and centrifuged for seven minutes at 3500 rpm at 4° C. The supernatant is collected and the pellet is threated three times with 3.5 ml of MPA THBQ solutions as described for the jam samples. When the samples are ready, a calibration curve has to be made. This will be done with a stock solution of 0,1 grams of L-ascorbic acid(Sigma Aldrich Co.) and 10 ml MPA THQ solution. This stock solution will be diluted, after filtration through a 0.2μm CA filter(Sartorius stedim biotech), 50, 100, 200,400, 800, 1600, 3200, 6400, 12800, 25600 and 51200 times with accompanying concentrations of 0.2, 0.1, 0.05, 0.025, 0.0125, 0.00625, 0.003125, 0.00156, 0.00078, 0.00039 and 0.000195 mg/ml, respectively. After the dilutions are made, they are pipetted in amber vials for analysis by the High Performance Liquid Chromatography, HPLC(Thermo Separation Products). For the HPLC analysis, an 0.2% Orthophosphoric acid solution (Merck) was used as eluent and 10% aceton nitrile(Biosolve) was used to rinse the system. For each measurement, a 20μl sample was taken an read at a wavelength of 245 nm for 5.5 minutes with a flow of 1 ml/min. The column used for this investigation was the Polaris 5C18a.

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Determination of β-carotene For the determination of β-carotene the protocol by Mary Luz Olivares Tenorio was used, adjusted from protocols for watermelon, mango and carrots. The extraction of β-carotenes was done under subdued light and on ice to prevent the compound from degradation. For jam, approximately 2 grams of jam is put in a 50 ml Greinder tube and five ml of Milli-Q water, 0.4 ml of BHT(Sigma Aldrich Co.) and 10 ml of hexane(Biosolve) are added and stored in the freezer at -20° C. This mixture is homogenized with Ultra Turrax(IKA T25) for three minutes and flushed with nitrogen before centrifuged for ten minutes at 2800 rpm at 4° C(ThermoFischer Scientific Hereaus Multifuge X3R, serie number 41064061). After this, the supernatant is collected in a preweighted 50 ml Greinder tube and 10 ml of THF(Biosolve) is added to the pellet. This mixture is again homogenized by the Ultra Turrax for 3 minutes and flushed with nitrogen and then centrifuged. When all the supernatant is collected, it is frozen by means of liquid nitrogen to freeze the water that is present in the sample and the solvent is poured into round bottom flasks. The solvent is evaporated by means of a rotary vacuum evaporator(Büchi Switserland) at 40° C at 270 millibar vaccuum with nitrogen to prevent the compounds from oxidation. After the solvent is evaporated, the carotenoids remain. These are obtained by addition of 5 ml of THF and 5 ml of eluent(92.4% Methanol (Biosolve), 7.5% THF, 0.1% TEA (Sigma Aldrich Co.))to dissolve the carotenoids again. This solvent is collected in 15 ml Greinder tube and weighed. After this, the samples are filtered by 0.2 μm RC filters(Sartorius Stedim Biotech) and put in amber vials(Grace 1.5ml Tread 8-425). For juice, the procedure is exactly the same, with the exception of the amount of extractions . The extraction should be repeated until the pellet is colourless. The calibration curve used for the analysis of β-carotene is made with a 20mg β-carotene (Sigma Aldrich Co.)per 20ml THF stock solution. This stock solution is diluted until 800x with hexane after filtration with 0.2 μm RC filters. Dilution 800x, 400x and 200x are measured for their absorbance by means of a spectrophotometer (Varian Cary 50 Bio). With these values, the purity of the standard can be determined. After this measurement, new dilutions are made with 1mg/ml stock solution and 50/50 solution of THF and eluent in a range from 5 until 5120 times diluted, which stands for a range of 0.2 until 0.000195mg/ml. This has to be adjusted for the measured purity of the standard. After the dilutions are made, they can be put into amber vials for HPLC analysis(Ultimate 3000). For the HPLC analysis the Vydac reverse phase column C18 201TP is used with a flow of 1 ml/min and one measurement takes 25 minutes. The areas of the compounds were read at 453 nm. The eluent used for this measurement is a 92.4% methanol, 7.5% THF and 0.1% TEA. Using the areas obtained from the HPLC, the content of β-carotene can be calculated.

14


Texture analysing To analyse the difference between the jams the Texture Analyser (Stable Micro Systems) was used. A project from the Application guide(Marmalade), provided by the program, was used for the determination of the jams. A cylindrical probe was used with a diameter of 12 millimeter in combination with a table to lift the samples to a stable level, which can be seen in Figure 4. The samples were put in plastic cups to prevent the samples from spreading out during the force that is applied to the sample during measurement. The samples were put in the measuring room for one hour to adapt to the conditions of the room. The temperature was 20.8째 C. First a calibration was executed for the height of the arm. After this calibration some test samples were taken from commercial jams to see how the Marmalade project worked and if the settings could be used. When the settings were set, the real measurements were executed. Because of the difference in the samples, the T.A. settings had to be adjusted for every sample separately. To prevent overload of the sample, the Trigger Force has to be adjusted to minimum value.

Figure 4 Test setting for Texture Analyser

15


6. Results and discussion 6.1 Initial values The levels of ascorbic acid and β-carotene can fluctuate from berry to berry. There is a great variety in levels because of seasons, sun hours, transport, harvest and many more other factors. Therefore it cannot be said that the values found during this investigation apply to all berries from Physalis peruviana, or even all berries from the used batch. The values presented here are taken from four samples and the standard deviations are calculated to show the varieties between the samples. The initial values of ascorbic acid and β-carotene in the whole frozen Cape gooseberries are presented in Table 6. Table 6 Initial values of L-ascorbic acid, total ascorbic acid and β-carotene in whole frozen Cape Gooseberries.

Concentration L-ascorbic acid(mg/100g) 35.96±1.72

Concentration total ascorbic acid(mg/100g) 41.84±1.88

Concentration β-carotene (mg/100g) 1.145±0.28

6.2 Jam

An overview of all the samples taken can be found in Appendix number 1. Because the jams vary in temperature and final sugar content, the results will be presented with a separation between these variables. The values from the extraction of L-ascorbic acid will be presented first and after that the results from the extraction of β-carotene. Because the weight of water and fruit was the same for every jam, the assumption was made that the calculated concentration of L-ascorbic acid and β-carotene was two times diluted. The real dilution is not known, since there has been evaporation during the process of jam making. The concentration found was also corrected for the unequal distribution of fruit in the different jam samples (see Table 4). L-ascorbic acid First, a division is made between the three different temperatures at which the jams were made. These are 80, 90 and 100° C. In Figure 5, Figure 6 and Figure 7 the concentrations of L-ascorbic acid (AA) and Total Ascorbic Acid(TAA) are shown for the samples with a final sugar content of 60 and 65° Brix. These graphs show that the Total Ascorbic Acid level is higher than the level of L-Ascorbic acid for all samples. This is as expected, because it is quite possible that L-ascorbic acid oxidises to L-dehydroascorbic acid, since L-ascorbic acid is a quite unstable compound, which has been experienced during the trial phase. Figure 5 shows that the concentration of L-ascorbic acid remains quite stable, but there can be a slight trend of formation noticed. The degradation of L-ascorbic acid compared to the initial values is quite the same for all six samples. This can be due to the fact that the level of L-ascorbic acid was already degraded in such an extent that there is no difference perceptible between the times of 420 and 2880 seconds of heat treatment at 80° C. The biggest part of the degradation could be influenced by another step of the jam processing, like the blending where the cell structure was ruptured and oxygen could enter the cell matrix or the immersing step with hot water, where the first heat shock was applied. For the samples that have a final sugar content of 65° Brix, the levels of Total ascorbic acid fluctuate a lot, but the shape is comparable with the levels of L-ascorbic acid. There

16


is no real trend visible because the values fluctuate too much. When a trend line would be drawn, it would be a horizontal line.

Concentration (mg/100g)

Concentration AA and TAA for samples prepared at 80° C 4.5 3.5

60Brix samples

2.5

65Brix samples

1.5

TAA 60Brix samples

0.5 -0.5 0

1000

2000 Time (s)

3000

4000

TAA 65Brix samples

Figure 5 Concentration L-ascorbic acid(AA) and Total Ascorbic acid (TAA) for jams prepared at 80° Celsius.

Figure 6 shows the levels of L-ascorbic acid and total ascorbic acid for the jams that were prepared at 90° C. When you take only the values in account from L-ascorbic acid, you can see a slight trend that shows an increase in concentration when the cooking time is longer. It was expected that the longer the jam was exposed to heat, the more the compound would degrade. Therefore the values that were found are not as expected. It can also been seen that the difference between the level of L-ascorbic acid and total ascorbic acid is almost the same for all samples.

Concentration (mg/100g)

Concentration AA and TAA for samples prepared at 90° C 4.5 3.5 60Brix samples

2.5

65Brix samples

1.5

TAA 60Brix samples

0.5 -0.5 0

TAA 65 Brix samples 500

1000 Time(s)

1500

2000

Figure 6 Concentration L-ascorbic acid(AA) and Total Ascorbic acid (TAA) for jams prepared at 90° Celsius.

In Figure 7, the concentrations of L-ascorbic acid and total ascorbic acid are presented for the jams that were prepared at 100° C. Compared to the values of the samples prepared at and 90° C, the obtained values of the concentrations of L-ascorbic acid in the 100°C samples are higher. This can be explained by the difference in time. Because the samples that were heated until 100° C were only heated for a short time, the treatment could be of less impact. This can be related to the fact that high temperature-short time(HTST) treatments may retain higher levels of L-ascorbic acid than longer 17


treatments at lower temperatures, which has been proven for black currant jam (Ó BEIRNE, Egan et al. 1987). It has been proven that after HTST treatments no microbial growth was detected during storage(Almela, Nieto-Sandoval et al. 2002) and that it is a good method to retain carotenoids, including β-carotene(Lin and Chen 2005). But this theory cannot be proven in this research because the times used here are not comparable to times used during High Temperature Short Time-treatments, which are quite shorter.

Concentration (mg/100g)

Concentration AA and TAA for samples prepared at 100° C 4.5 3.5

60Brix samples

2.5

65 Brix samples

1.5

TAA 60Brix samples

0.5 -0.5 0

200

400 Time(s)

600

800

TAA 65Brix samples

Figure 7 Concentration L-ascorbic acid(AA) and Total Ascorbic acid (TAA) for jams prepared at 100° Celsius.

In Figure 8 and Figure 9 a division is made between the samples that had a final sugar content of 60 (Figure 8) or 65°Brix (Figure 9). From these graphs you can see the difference between the different temperatures more clearly. For the jams that were made at 80° C, the time needed to reach the sugar content of 60° Brix was longer than the times of the jams that were treated with 90 or 100° C. It is shown that the concentration of L-ascorbic acid and total ascorbic acid are higher for the samples that were prepared at 100° C. It was expected that higher temperatures would have a greater impact on the degradation of ascorbic acid, so these differences between the temperatures are not as expected. Also, the measured values do not show a great variation, since the standard deviation of some of the samples is even too low to see the error bar in the graph.

Concentration(mg/100g)

Concentration AA and TAA for samples at 60°Brix 4

80C

3

90C

2

100C

1

TAA 80C

0 0

1000

2000

3000

Time(s)

4000

TAA 90C TAA 100C

Figure 8 Concentration of L-ascorbic acid(AA) and Total Ascorbic Acid (TAA) for all samples with a final sugar content of 60° Brix.

18


Concentration(mg/100g)

Concentration AA and TAA for samples at 65°Brix 6 5 80C

4

90C

3

100C

2

TAA 80C

1

TAA 90C

0 -1

0

500

1000 Time(s)

1500

2000

TAA 100C

Figure 9 Concentration of L-ascorbic acid(AA) and Total Ascorbic Acid (TAA) for all samples with a final sugar content of 65° Brix.

Figure 10 shows the differences in concentration of L-ascorbic acid between the jams with a final sugar content of 60 and 65° Brix. With one exception for a sample with final sugar content of 65° Brix, prepared at 100° C, it is clear that the concentration of Lascorbic acid is lower for the jams that had a final sugar content of 65° Brix. This might be due to the fact that the cooking time was longer for jams that had to reach a higher sugar content, because more water needed to evaporate before the preferred sugar content was reached.

Concentration(mg/100g)

Concentration L-ascorbic acid for samples at 60 and 65°Brix 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0

80C, 60Brix 90C, 60Brix 100C, 60Brix 80C, 65Brix 90C, 65Brix 100C, 65Brix 0

1000

2000

3000

4000

Time(s)

Figure 10 Concentration of L-ascorbic acid for all temperatures and all Brix levels.

All these values do not show a clear trend. This might be due to the fact that the values are already that low, that no clear conclusion can be drawn. It might be possible that some steps during the process of jam making are more important than others. Previous research has been done on jam making from guava(Jawaheer, Goburdhun et al. 2003), 19


and this showed that every step (peeling, blending, exhausting) all have influence on the degradation of ascorbic acid. Research on orange juice showed that deaeration causes a smaller degradation of ascorbic acid (Roig, Rivera et al. 1995). The difference in ascorbic acid between these temperatures and times in Figure 10 are not clear to draw conclusions. So the degradation process of ascorbic acid must be investigated in different processing steps to see which steps are crucial. Β-carotene In Figure 11,Figure 12 and Figure 13, the results are shown from the extraction of βcarotene from the different jams. The results are split up per temperature. In Figure 11 a trend is visible that shows an increase in concentration of β-carotene when the cooking time is longer. This can be due to the fact that during the cooking (different times and temperatures) the cell walls are broken down and more β-carotene is released (Bernhardt and Schlich 2006). Blending and cooking can soften the cell walls and destruct the matrix where the β-carotene is attached to. This can make the β-carotene more (bio)available to extraction and digestion. Heat treatments can provide degradation by means of oxidation or retention of β-carotene(Dietz, Kantha et al. 1988). It seems that for the process of jam making β-carotene is released and retained.

concentration β-carotene (mg/100g)

Concentration β-carotene for samples at 80°C 0.3 0.25 0.2 0.15 0.1 0.05 0

60Brix 65Brix 0

1000

2000

3000

4000

time (s) Figure 11 Concentration beta-carotene in jam, cooked at 80° Celsius, for two different final sugar concentrations.

Figure 12 shows the values of β-carotene for the samples that were prepared at 90° C. Although one point is slightly off, there is also for these values a trend that the concentration increases with an accompanying increasing cooking time. This can also be explained by the theory that more β-carotene can be released when the cooking time in longer, because more cell walls can be broken down. For these samples the theory discussed previously about the increasing concentration of the jam samples during longer heating and thus more evaporation of water can also be applied. When more water is evaporated, the concentration of β-carotene might be increased, but because the precise dilution is not known, all samples are corrected with the same dilution factor.

20


concentration β- carotene (mg/100g)

Concentration β-carotene for samples at 90°C 0.5 0.4 0.3 0.2

60Brix

0.1

65Brix

0 0

500

1000

1500

2000

time (s) Figure 12 Concentration beta-carotene in jam, cooked at 90° Celsius, for two different final sugar concentrations.

Figure 13 shows an increase in concentration when the time is longer for the jams that had a final sugar content of 65° Brix and the opposite applies to the samples with a final sugar content of 60° Brix. It is also remarkable that although the time of the cooking process was shorter for the 60° Brix-samples, the concentration of β-carotene is still lower. The samples with a final sugar content of 65° Brix show a trend that has also been seen for the samples that were prepared at 80 and 90° C.

concentration β- carotene (mg/100g)

Concentration β-carotene for samples at 100°C 0.5 0.4 0.3 0.2

60Brix

0.1

65Brix

0 -0.1 0

200

400 time (s)

600

800

Figure 13 Concentration beta-carotene in jam, cooked at 100° Celsius, for two different final sugar concentrations.

In Figure 14 and Figure 15 the results are shown per temperature for 60 (Figure 14) and 65 (Figure 15)° Brix. Figure 14 shows a trend for the temperatures 80 and 90 °C of increasing concentration of β-carotene while the cooking time increases. This is not the case for the samples that have been prepared at 100° C. For the samples that have a final sugar content of 65° Brix, this increasing trend is also visible with the exception of one point of the 80 degrees samples.

21


Concentration β-carotene for jams with final sugar content of 60° Brix Concentration β-carotene(mg/100g)

0.5 0.4 0.3

80C

0.2

90C

0.1

100C

0 0

1000

2000

3000

4000

Time(s) Figure 14 Concentration beta-carotene is jams with final sugar content of 60° Brix prepared at 80,90 and 100° Celsius.

Concentration β-carotene for jams with final sugar content of 65° Brix Concentration β-carotene(mg/100g)

0.5 0.4 0.3

80C

0.2

90C

0.1

100C

0 0

500

1000

1500

2000

Time(s) Figure 15 Concentration beta-carotene is jams with final sugar content of 65° Brix prepared at 80,90 and 100° Celsius.

Because the weight of water and fruit was the same for every jam, the assumption was made that the calculated concentration of L-ascorbic acid and β-carotene was two times diluted. The real dilution is not known, since there has been evaporation during the process of jam making. The samples that have higher temperatures or longer cooking times might have lower concentrations of water present in the jam. Since it is not known what the exact dilution is for each sample, the obtained values were corrected with a factor 2, although this is not right for each sample since water had to evaporate to get the preferred final sugar content. The samples that were cooked for a longer time or at a higher temperature might contain less water and therefore have higher measured concentrations of ascorbic acid and β-carotene. This might be the reason that the graphs show an increase in concentration when the time of cooking increases.

22


6.3 Juice L-ascorbic acid For the extraction of L-ascorbic acid, the sample was divided between a supernatant and a pellet. These were separately investigated and later these values were combined to get an indication what the concentration of L-ascorbic acid was in de different juices. In Figure 16, the concentration of L-ascorbic acid for the whole juice samples(supernatant and pellet) are presented for all the temperatures that were used. It was expected that when the temperatures increased and the time of the treatment was not changed, the concentration of L-ascorbic acid would decrease, because of the heat sensibility of the compound. This theory can be supported by the values found in Figure 16. This figure shows that with increasing temperatures, the concentration of L-ascorbic acid decreases.

Concentration (mg/100g)

Concentration L-ascorbic acid for all pasteurisation temperatures 50 40

fresh juice

30

60C

20

70C

10

80C

0 0

10 20 30 40 50 60 70 80 90 100

90C

Temperature (°C) Figure 16 Concentration L-ascorbic acid after pasteurisation at different temperatures

The value of L-ascorbic acid of the fresh juice is approximately 37.15 mg/100g. This is close to the initial values found in the frozen fruit, 35.96¹1.72. The value might be slightly higher because of the blending step. The blending could make the compound better available for extraction than for the (frozen) berry samples taken for the initial value determination. It could also be possible that the freezing step influenced the content of ascorbic acid present in the fruit. No large amounts of L-ascorbic acid are degraded during the defreezing and blending of the fruit. The figure also shows the standard deviations, which are very small. Table 7 shows the difference between the values obtained from the samples that were heated up to the desired temperatures of 60 to 90° Celsius and the samples that were pasteurised at these temperatures. It was expected that the concentrations would be lower after the pasteurisation step compared to the concentrations after the heating up time, but this is not the case for all the samples. This means that the heating up time has a great influence on the final concentration of L-ascorbic acid. This might be due to the time range it takes to heat the samples up to the desired temperatures, which varied between 4 minutes and 43 seconds to 6 minutes and 20 seconds. These times are much longer than the applied pasteurisation times.

23


Table 7 Difference in concentration of L-ascorbic acid between samples taken after heating until preferred temperature and after pasteurisation time. Tfinal (°C) 60

Concentration L-ascorbic acid after heating to Tfinal(mg/ml) 34.80902

Concentration L-ascorbic acid after pasteurisation (mg/ml) 33.31222

Difference between concentration after heating up and after pasteurisation 1.496802

60

34.30348

32.35556

1.947913

60

32.961

31.73919

1.221816

60

33.70202

33.33292

0.369109

70

12.42785

26.15009

-13.7222

70

22.21074

25.73766

-3.52692

70

22.25752

27.1463

-4.88878

80

15.23684

13.43193

1.804908

80

14.12299

13.41003

0.71296

80

13.52167

12.94215

0.579512

80

12.67257

13.36473

-0.69216

90

12.13918

12.18659

-0.04741

90

11.80056

11.89302

-0.09246

90

12.2012

13.24757

-1.04636

90

11.90125

12.20906

-0.30781

Β-carotene Figure 17 shows the concentration of β-carotene after the pasteurisation of the different juices for 60,70 80 and 90° Celsius. The concentration of the fresh juice is the highest. This is as expected, because no treatment has been executed on these samples. The samples show a slight degradation of concentration of β-carotene when the temperature of the pasteurisation treatment increases. This is as expected, because higher temperatures have a greater influence on the compounds when they are treated for the same time interval. Only the samples that were heated until 60° C were pasteurised for 4 minutes. But the figure shows that this treatment has less impact on the concentration of β-carotene then one minute at 70,80 or 90° Celsius. Compared to the degradation of Lascorbic acid, the degradation of β-carotene shows a less fast decrease. This might be due to the fact that L-ascorbic acid is less heat stable than β-carotene. This theory can be supported by the degradation constants found in previous investigation by Baijer (2013). It was found that the kd80°C for L-ascorbic acid was found to be 1.7228*10-3 min-1, whilst for β-carotene this value was 1.07*10-4 min-1.

24


Concentration β-carotene (mg/100g)

Concentration β-carotene for all pasteurisation temperatures 1.2 1 fresh juice

0.8 0.6

60C

0.4

70C

0.2

80C

0

90C 0

10 20 30 40 50 60 70 80 90 100 Temperature (°C)

Figure 17 Concentration β-carotene after pasteurisation at different temperatures

Table 8 shows the difference between the concentration of β-carotene after heating up to 60 to 90° Celsius and the concentration of β-carotene after pasteurisation treatment. It was expected that the concentration of β-carotene would be even lower after pasteurisation. But this is not the case for all samples. Therefore the heating up time, which differs from 5 minutes and 37 seconds to 6 minutes and 20 seconds, has a great impact on the degradation of β-carotene compared to the pasteurisation. Table 8 Difference in concentration of β-carotene between samples taken after heating until preferred temperature and after pasteurisation time. Tfinal (°C)

Concentration β-carotene after heating to Tfinal(mg/ml)

Concentration β-carotene after pasteurisation (mg/ml)

60

0.883245303

0.904686783

Difference between concentration after heating up and after pasteurisation -0.02144148

60

0.898787536

0.879314401

0.019473135

60

0.918193443

0.871012346

0.047181096

60

0.907453943

0.907569499

-0.000115556

70

0.905772671

0.87856297

0.027209701

70

0.919116044

0.885153443

0.033962601

70

0.833641087

0.73384737

0.099793717

80

0.843020431

0.725157454

0.117862977

80

0.767304924

0.828412995

-0.061108071

80

0.787545419

0.839427393

-0.051881974

80

0.880235044

0.832323707

0.047911337

90

0.884142336

0.842309555

0.041832781

90

0.762760958

0.793168731

-0.030407773

90

0.766098102

0.787457316

-0.021359213

90

0.807896333

0.811616707

-0.003720375

25


6.4 Texture analysis of jams During the jam making process different variables were adjusted; the final sugar content, amount of sugar and fruit and the temperature(for an overview see Appendix 10.1). The amount of pectin added was kept the same for all samples because some variables should stay the same. The difference between the jams were analysed by means of the Texture Analyser. Because the jams were very runny, the force needed to push the probe into the jams was very low. Therefore, no real “breaking point� was found. For the comparison, Figure 18 shows the graph for a commercial jam and Figure 19 shows an experimental jam that is representative for all samples. This is the graph from sample 17. The values on the y-axis differ a hundred times, which already shows the difference between the commercial and experimental jam. In Figure 18, you can clearly see a breaking point at approximately three seconds, which is not clear for the experimental jams . From the difference between these two figures, it can be concluded that the experimental jams are not as strong as the commercial jams.

Texture Analysis for Commercial Apricot Jam Force (N)

0.2 commercial apricot jam 1

0.1 0 0

-0.1 -0.2

2

4

6

commercial apricot jam 2 commercial apricot jam 3

Time (s)

Figure 18 Texture Analysis of Commercial Apricot Jam

Texture Analysis on Sample 17 0.008 0.006

Sample 17.1

Force (N)

0.004

Sample 17.2

0.002 0 -0.002 0

2

4

Sample 17.4

-0.004 -0.006

Sample 17.3

Time (s)

Figure 19 Texture Analysis on Sample 17

The measurements of all the jams can be found in Appendix 10.2. Here you can see that the force needed varied from -0.006 until 0.008 Newton, sample 4 and 10 excluded. Because the jams cannot be separated on breaking point, the average force at t=2 seconds was taken to see the difference between the jams. The values measured from t=1.98 until 2.02 seconds for all measurements were taken into account. The average of the measurements is displayed in Table 9. From this table you can see that sample 4 and 26


10 have the highest values. This can be explained by the composition of the jam. Jam 4 and 10 both have a fruit content of 35.1 percent, which is the highest amount of fruit used in these samples. In line with the extra pectin that is added to the sample by the fruit, the gel that is formed will be stronger (Carbonell, Costell et al. 1991). The other samples show some resemblance and depend on the point where the graph drops down. In appendix 10.2, samples 3, 6, 9 and 15 do not show a drop in the graphs, which means that the viscosity of the samples is too low for a proper texture analysis. Table 9 Composition of jams and average force used at t=2 seconds.

Sample

Fruit content (g)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Fruit content (% w/w) 54 44 34 54 44 34 54 44 34 54 44 34 54 44 34 54 44 34

Pectin (g)

35.1 30.6 25.4 35.1 30.6 25.4 35.1 30.6 25.4 35.1 30.6 25.4 35.1 30.6 25.4 35.1 30.6 25.4

Pectin (%w/w) 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Force at “t=2s� (*10-3N) 0.65 0.69 0.75 0.65 0.69 0.75 0.65 0.69 0.75 0.65 0.69 0.75 0.65 0.69 0.75 0.65 0.69 0.75

0.048009 0.781481 0.668635 6.222222 2.844444 -0.37373 -30.8644 0.955556 1.355556 12.65 -6.25185 -0.73333 -2.06389 1.381481 1.233333 -0.00833 -1.74444 0.788889

Figure 20 shows graphically that it depends on the amount of fruit in the jam what force is needed to invert the samples. For the lowest percentage of fruit (25.4%), the strength of the gel is so minimal that the force does not vary a lot and stays quite close to zero. For the highest fruit content (35.1%), the values vary a lot. From the graphs in Appendix 10.2 it is shown that sample 4 and 10 show the greatest resistance against the force applied to the sample. Figure 20 Force measured at "t=2s" for different fruit content percentages

Force at "t=2s"(*10-3N)

Force vs. Fruit content(%) 20 10

60 Brix, 80C

0

60Brix, 90C

-10 20

25

30

35

40

60Brix, 100C

-20

65Brix, 80C

-30

65Brix, 90C

-40

Fruit content(%)

27

65Brix, 100C


7. Conclusion For ascorbic acid, it is demonstrated that the compound is very sensitive to the process of jam making. The initial values were 35.96±1.72 mg/100g for L-ascorbic acid (AA) and 41.84±1.88mg/100g Total Ascorbic Acid (TAA). After the jam making process L-ascorbic acid was decreased to values between 0.14 and 3.97mg/100g, representing a degradation of 88.95 - 99.61%. For total ascorbic acid, the degradation was found to be in a range of 86.68 - 98.24%. There was not a clear effect seen from higher processing temperatures or longer cooking times. The initial value of β-carotene in de frozen fruit was 1.145±0.28mg/100g. The lowest concentration that was encountered was 0.011mg/100g, which means a degradation of 99.03%, but the concentration of β-carotene also increased when the cooking time increased. This may be due to the fact that during the process the cell walls are ruptured and the β-carotene is released which lead to an improved availability for extraction. This is also shown by the fact that the highest concentration β-carotene obtained was 0.43mg/100g, which gives a decrease of 62.45%. This value was found for the sample that was thought to have the treatment with the most impact: 100° Celsius and highest percentage of water and final sugar content of 65° Brix. The strength of the gel of the jams is dependent on the fruit content. The only two jams that gave a reasonable resistance were two jams with 35.1% of fruit. Less fruit means less ascorbic acid and β-carotene and also less pectin, which contributes to the strength of the gel. Therefore the fruit content is responsible for the concentration of ascorbic acid, β-carotene and strength of the gel. For juice, ascorbic acid and β-carotene show a decrease in concentration when the temperature of the pasteurisation treatment is increased. For L-ascorbic acid, the lowest value was found to be 12.38 mg/100g after one minute pasteurisation at 90° Celsius, which is a decrease of 65.57% compared to the initial value found in Cape gooseberry. The content of Β-carotene in juice shows a less steep decrease during heating compared to L-ascorbic acid, but with the lowest obtained value of 0.79mg/100g, this is a decrease of 31% compared to the initial value of 1.145mg/100g. This shows that L-ascorbic acid is a more heat sensitive compound compared to β-carotene. The heating up step has more influence on the degradation of the compounds in the juice than the actual pasteurisation. Because of this, it might be better to use another way to apply the heat to the samples, for example with high pressure.

28


8. Recommendations During the execution of the experiments, some methods were found to be questionable for the results. For example, during the process of jam making, oxygen, heat and UVlight had access to the jam samples. Because some of the results are not as expected, it cannot be sure which of these factors had the most influence on the degradation of the compounds. For further investigation the jam could be made in a room with red light or by means of a vacuum cooker to prevent light or oxygen from getting in, respectively. With a vaccuum cooker you can use lower temperatures than in an open system, so this will retain the compounds even better. For further investigation one could take a deeper look in the different steps during the jam making, and see which step is the key step for degradation of L-ascorbic acid or βcarotene. Maybe the blanching step or the blending step have more influence on the degradation of the compounds because they provide the first heat shock and oxygen, respectively. During the process of jam making, one could also look at the dilution due to the added water, or if there is no dilution but concentration during the evaporation of the water during the cooking. Maybe this can elucidate why the concentration of Lascorbic acid rises when the cooking time is longer. In literature, a lot of information was found on the retention of health promoting compounds with other methods than the traditional heat treatments like pasteurisation. It would be interesting to see the difference between the degradation of ascorbic acid and β-carotene in Cape gooseberry juice during heat treatments and treatments with highpressure, thermo sonication, pulsed electric field or high temperature short time(HTST) treatments. For the texture analysis the Texture Analyser could be replaced by the viscosity meter the next time, because the jams were too runny to have a real breaking point during the analysis. This can also be changed by adjusting the amount and sort of pectin used in the jam. This was not done this time because some variables were decided to stay the same, but when the next investigation focusses on the improvement of jam making, this has to be taken into account. During the extraction of β-carotene, a homogenizing step is included with the Ultra Turrax for three minutes. It was experienced that the samples becomes very hot, even after one minute. Because β-carotene is known for its sensibility to heat, it would be recommended that the Ultra Turrax is only used for 45 seconds to one minute, to prevent the compound from degradation during extraction. The results showed that heat increased the concentration of β-carotene, but the purpose of extraction is to retain the compounds as good as possible for your product and to see what the process of making these products have on the concentration of the compound, not the influence the extraction itself has on the concentration.

29


9. References Picture at front page: Janet Wippell, December 7, 2012 (1989). Lost Crops of the Incas:Little-Known Plants of the Andes with Promise for Worldwide Cultivation, The National Academies Press. Codex Alimentarius (1981). "The Codex standard for pulpy nectars of certain small fruits preserved exclusively by physical means." Codex Alimentarius (2009). Codex Standard for Jams, Jellies and Marmalades. Almela, L., J. M. Nieto-Sandoval, et al. (2002). "Microbial Inactivation of Paprika by a HighTemperature Short-X Time Treatment. Influence on Color Properties." Journal of Agricultural and Food Chemistry 50(6): 1435-1440. Baijer, H. (2013). "The effect of thermal processing on carotenoids and ascorbic acid in Colombian Cape gooseberries.". Belibel, A. (2013). "Effect of thermal processing on flavonoids and vitamin C in Colombian Cape Gooseberries." Bernhardt, S. and E. Schlich (2006). "Impact of different cooking methods on food quality: Retention of lipophilic vitamins in fresh and frozen vegetables." Journal of Food Engineering 77(2): 327-333. Bombarely, A., N. Menda, et al. (2011). "The Sol Genomics Network (solgenomics. net): growing tomatoes using Perl." Nucleic acids research 39(suppl 1): D1149-D1155. Carbonell, E., E. Costell, et al. (1991). "Fruit content influence on gel strength of strawberry and peach jams." Journal of Food Science 56(5): 1384-1387. Chen, B., H. Peng, et al. (1995). "Changes of carotenoids, color, and vitamin A contents during processing of carrot juice." Journal of Agricultural and Food Chemistry 43(7): 1912-1918. Cinquanta, L., D. Albanese, et al. (2010). "Effect on Orange Juice of Batch Pasteurization in an Improved Pilot-Scale Microwave Oven." Journal of Food Science 75(1): E46-E50. de Ancos, B., S. Sgroppo, et al. (2002). "Possible nutritional and health-related value promotion in orange juice preserved by high-pressure treatment." Journal of the Science of Food and Agriculture 82(8): 790-796. de Rosso, V. V. and A. Z. Mercadante (2007). "Identification and Quantification of Carotenoids, By HPLC-PDA-MS/MS, from Amazonian Fruits." Journal of Agricultural and Food Chemistry 55(13): 5062-5072. Diederen, J. (2012). Carbohydrates. Food Chemistry FCH 20806. Dietz, J., S. Kantha, et al. (1988). "Reversed phase HPLC analysis of α- and β-carotene from selected raw and cooked vegetables." 38(4): 333-341. FDA (2010). "Recommended Pasteurization Time/Temperatures." Fernández García, A., P. Butz, et al. (2001). "Antioxidative capacity, nutrient content and sensory quality of orange juice and an orange-lemon-carrot juice product after high pressure treatment and storage in different packaging." 213(4-5): 290-296. Fischer, G., G. Ebert, et al. (1999). Provitamin A carotenoids, organic acids and ascorbic acid content of cape gooseberry (Physalis peruviana L.) ecotypes grown at two tropical altitudes. II ISHS Conference on Fruit Production in the Tropics and Subtropics 531.

30


Fischer, G., Ebert, G. and Lüdders P. (2000). "PROVITAMIN A CAROTENOIDS, ORGANIC ACIDS AND ASCORBIC ACID CONTENT OF CAPE GOOSEBERRY (PHYSALIS PERUVIANA L.) ECOTYPES GROWN AT TWO TROPICAL ALTITUDES.". Gardner, P. T., T. A. C. White, et al. (2000). "The relative contributions of vitamin C, carotenoids and phenolics to the antioxidant potential of fruit juices." Food Chemistry 68(4): 471-474. Halliwell, B. and J. M. Gutteridge (1999). Free radicals in biology and medicine, Oxford university press Oxford. Jawaheer, B., D. Goburdhun, et al. (2003). "Effect of processing and storage of guava into jam and juice on the ascorbic acid content." 58(3): 1-12. Khraisheh, M., W. McMinn, et al. (2004). "Quality and structural changes in starchy foods during microwave and convective drying." Food Research International 37(5): 497-503. Lee, J., R. W. Durst, et al. (2002). "Impact of Juice Processing on Blueberry Anthocyanins and Polyphenolics: Comparison of Two Pretreatments." Journal of Food Science 67(5): 1660-1667. Lin, C. H. and B. H. Chen (2005). "Stability of carotenoids in tomato juice during processing." 221(3-4): 274-280. Morton, J. F. (1987). Cape Gooseberry. Fruits of warm climates. Miami: 430–434. Naidu, K. A. (2003). "Vitamin C in human health and disease is still a mystery? An overview." Nutrition Journal 2(1): 7. Ni, Y., K. M. Yates, et al. (2010). High molecular weight, low methoxyl pectins, and their production and uses, Google Patents. Ó BEIRNE, D., S. Egan, et al. (1987). "Some effects of reduced boiling time on the quality of fruit preserves." Lebensmittel-Wissenschaft+ Technologie 20(5): 241-244. Oey, I., I. Van der Plancken, et al. (2008). "Does high pressure processing influence nutritional aspects of plant based food systems?" Trends in Food Science & Technology 19(6): 300-308. Plaza, L., C. Sánchez-Moreno, et al. (2006). "Effect of refrigerated storage on vitamin C and antioxidant activity of orange juice processed by high-pressure or pulsed electric fields with regard to low pasteurization." 223(4): 487-493. Puente, L. A., C. A. Pinto-Muñoz, et al. (2011). "< i> Physalis peruviana</i> Linnaeus, the multiple properties of a highly functional fruit: A review." Food Research International 44(7): 1733-1740. Ramadan, M. F. (2011). "Bioactive phytochemicals, nutritional value, and functional properties of cape gooseberry (< i> Physalis peruviana</i>): An overview." Food Research International 44(7): 1830-1836. Ramadan, M. F. and J. T. Moersel (2007). "Impact of enzymatic treatment on chemical composition, physicochemical properties and radical scavenging activity of goldenberry (Physalis peruviana L.) juice." Journal of the Science of Food and Agriculture 87(3): 452-460. Ramadan, M. F. and J.-T. Morsel (2004). "Goldenberry, a novel fruit source of fat-soluble bioactives-A minor fruit of the Andes is gaining international popularity." Inform-International News on Fats Oils and Related Materials 15(2): 130-131. Ramadan, M. F. H. (2011). "Physalis peruviana: a rich source of bioactive phytochemicals for functional foods and pharmaceuticals." Food Reviews International 27(3): 259-273.

31


Repo de Carrasco, R. and C. R. ENCINA ZELADA (2008). "Determinación de la capacidad antioxidante y compuestos bioactivos de frutas nativas peruanas." Rev. Soc. Quím. Perú 74(2): 108-124. Rodriguez-Amaya, D. B. (2001). A guide to carotenoid analysis in foods, ILSI press Washington^ eD. C DC. Roig, M., Z. Rivera, et al. (1995). "A model study on rate of degradation of L-ascorbic acid during processing using home-produced juice concentrates." International journal of food sciences and nutrition 46(2): 107-115. Russell, L. F. (2012). "Water-soluble vitamins." Food Analysis by HPLC 100: 325. Singh, D., A. Pal, et al. (2012). "Growth and developmental changes of cape gooseberry (Physalis peruviana L.) fruits." Asian Journal of Horticulture 7(2): 374-378. Thakur, B. R., R. K. Singh, et al. (1997). "Chemistry and uses of pectin — A review." Critical Reviews in Food Science and Nutrition 37(1): 47-73. Valente, A., T. G. Albuquerque, et al. (2011). "Ascorbic acid content in exotic fruits: A contribution to produce quality data for food composition databases." Food Research International 44(7): 22372242. Vincente, M. M. Wu, S., J. Tsai, et al. (2006). "Supercritical carbon dioxide extract exhibits enhanced antioxidant and anti-inflammatory activities of< i> Physalis peruviana</i>." Journal of ethnopharmacology 108(3): 407-413. Zavala, D., A. Quispe, et al. (2006). "Efecto citotóxico de Physalis peruviana (capulí) en cáncer de colon y leucemia mieloide crónica." An. Fac. med 67(4): 283-289. Zhao, Y. (2012). "Jams, Jellies, and Other Jelly Products." Specialty Foods: Processing Technology, Quality, and Safety: 135.

32


10. Appendix 10.1 Overview of jam samples Sample nr. 1a 1b 2a 2b 3a 3b 4a 4b 5a 5b 6a 6b 7a 7b 8a 8b 9a 9b 10a 10b 11a 11b 12a 12b 13a 13b 14a 14b 15a 15b 16a 16b 17a 17b 18a 18b

째Brix (end) 60 60 60 60 60 60 65 65 65 65 65 65 60 60 60 60 60 60 65 65 65 65 65 65 60 60 60 60 60 60 65 65 65 65 65 65

째Celsius

% sugar

80 80 80 80 80 80 80 80 80 80 80 80 90 90 90 90 90 90 90 90 90 90 90 90 100 100 100 100 100 100 100 100 100 100 100 100

29 29 38 38 48 48 29 29 38 38 48 48 29 29 38 38 48 48 29 29 38 38 48 48 29 29 38 38 48 48 29 29 38 38 48 48

% fruit 35 35 30 30 25 25 35 35 30 30 25 25 35 35 30 30 25 25 35 35 30 30 25 25 35 35 30 30 25 25 35 35 30 30 25 25

33

% pectin 0.65 0.65 0.69 0.69 0.75 0.75 0.65 0.65 0.69 0.69 0.75 0.75 0.65 0.65 0.69 0.69 0.75 0.75 0.65 0.65 0.69 0.69 0.75 0.75 0.65 0.65 0.69 0.69 0.75 0.75 0.65 0.65 0.69 0.69 0.75 0.75

pH

time (min:s)

3.97 3.97 3.70 3.70 3.72 3.72 3.65 3.65 3.46 3.46 3.68 3.68 3.88 3.88 3.67 3.67 3.57 3.57 3.24 3.24 3.09 3.09 3.67 3.67 3.60 3.60 3.4 3.4 3.42 3.42 3.33 3.33 3.43 3.43 3.35 3.35

48 48 29 29 7 7 30 30 18:04 18:04 8:23 8:23 18:3 18:3 11:7 11:7 2:48 2:48 24:16 24:16 14:5 14:5 8:32 8:32 9:44 9:44 7:2 7:2 2:25 2:25 9 9 6:50 6:50 4:25 4:25


10.2 Texture analysis of jam

Sample 1

Sample 2

0.01

0.006 0.004

Force (N)

Force(N)

0.005

Sample 1 0

0

2

4

sample 1.2

6

-0.005

Sample 2.1 0.002

sample 2.2

0

sample 1.3

Sample 2.3 0

-0.01

-0.002

time(s)

0.05

0.004

0 -0.05 0

Sample 3.1

0.002 0.001

Sample 3.2

0

Sample 3.3

-0.001 0

2

4

Force(N)

Force (N)

0.003

5

Sample 4.2

-0.15

Sample 4.3

-0.2

Sample 4.4

-0.25

Sample 4.5 Time(s)

Time(s)

Sample 5

Sample 6

0.01

0.006

0.006

Sample 5.1

0.004

Sample 5.2

0.002

Sample 5.3

0

Sample 5.4 2

4

6

8

0.004

Force (N)

Force (N)

0.008

Sample 6.1

0.002

Sample 6.2 0 -0.002

0

4

Sample 6.3

6

Sample 6.4

-0.004

Time (s)

Time (s)

Sample 8

0.006 0.006

0.004 0.002

0.004

Sample 7.1

0

2

4

-0.004

6

Force (N)

Force (N)

2

Sample 5.5

Sample 7

Sample 7.2 Sample 7.3

Sample 8.1 0.002

Sample 8.2

0

-0.006 -0.008

Sample 4.1

10

-0.1

-0.3

-0.002

-0.002 0

6

Sample 4

0.005

-0.004

4

Time (s)

Sample 3

-0.002 0

2

Sample 8.3 0

Time(s)

34

-0.002

2

4

Time(s)

6


Sample 9

Sample 10

0.005

1.2 1

0.004

0.8

Sample 9.1 0.002

Sample 9.2

0.001

Sample 9.3

0 -0.001

Force (N)

Force (N)

0.003

0

2

4

Sample 10.1

0.6 0.4

Sample 10.2

0.2

Sampl 10.3

0

Sample 9.4

Sample 10.4

-0.2 0

5

-0.6

Time (s)

0.005 0.004 0.003

Force (N)

Forc (N)

Sample 12

Sample 11.1 Sample 11.2 2

4

6

Sample 11.3

0.002

Sample 12.1

0.001

Sample 12.2

0 -0.001 0

Sample 12.3

Time (s)

Sample 14 0.005

0.004

0.004

0.002

Sample 13.1

0

Sample 13.2 2

4

6

Force (N)

Force (N)

6

Sample 12.4

-0.004

Time (s)

Sample 13.3 Sample 13.4

-0.004

0.003

Sample 14.1

0.002

Sample 14.2

0.001

Sample 14.3

0 -0.001 0

Time (s)

Sample 15

2 Time (S)

4

Sample 16 0.006 0.004

Sample 15.1

Force (N)

Force (N)

4

-0.003

0.006

0.005 0.004 0.003 0.002 0.001 0 -0.001 0 -0.002

2

-0.002

Sample 13

-0.006

Sampe 10.5

Time (s)

Sample 11

-0.002 0

15

-0.4

-0.002

0.006 0.005 0.004 0.003 0.002 0.001 0 -0.001 0 -0.002 -0.003 -0.004

10

Sample 15.2 Sample 15.3 2

4

-0.004

Time (s)

35

Sample 16.2

0 -0.002

Sample 15.4

Sanmple 16.1

0.002

0

2

4

6

Sample 16.3 Sample 16.4

Time (s)


Sample 17 0.008

0.005

0.006

0.004

0.004 0.002

Sample 17.2

0 -0.002 0

2

4

Sample 17.3

Sample 18.1

0.002

Sample 18.2

0.001

Sampl 18.3

0

Sample 17.4

-0.004 -0.006

0.003

Sample 17.1

Force (N)

Force (N)

Sample 18

-0.001 0 -0.002

Time (s)

2

4

Sample 18.4

6

Time(s)

10.3 Raw data of HPLC analysis Ascorbic acid and β-carotene analysis for jam samples Injection Injection Name Ret.Time Rel.Area Area Name Selected Selected Peak: min % mV*min Peak: [2.80..2.95] [2.80..2.95] [2.80..2.95] [2.80..2.95] [2.80..2.95] UV_VIS_1 UV_VIS_1 UV_VIS_1 Eluent n.a. n.a. n.a. 4a AA1 1a Ascorbic Acid (AA)AA1 2.862 3.01 0.6368 4a AA2 1a AA2 2.865 3.55 0.6516 4a TAA1 1a Total Ascorbic Acid (TAA) TAA1 2.863 10.83 1.8134 4a TAA2 1a TAA2 2.863 7.24 1.145 4b AA1 1b AA1 2.874 5.59 0.8078 4b AA2 1b AA2 2.874 5.9 0.8067 4b TAA1 1b TAA1 2.875 11.8 1.1167 4b TAA2 1b TAA2 2.874 8.38 1.1128 Eluent Eluent n.a. n.a. n.a. 5a AA1 2a AA1 2.871 6.38 0.841 5a AA2 2a AA2 2.874 4.58 0.6212 5a TAA1 2a TAA1 2.874 9.85 0.8817 5a TAA2 2a TAA2 2.875 10.15 0.8908 5b AA1 2b AA1 2.871 4.01 0.5279 5b AA2 2b AA2 2.872 4.08 0.5166 5b TAA1 2b TAA1 2.874 9.33 0.7058 5b TAA2 2b TAA2 2.874 9.56 0.7243 Eluent Eluent n.a. n.a. n.a. 6a AA1 3a AA1 2.878 3.33 0.3755 6a AA2 3a AA2 2.874 4.79 0.4076 6a TAA1 3a TAA1 2.876 12.67 1.0092 6a TAA2 3a TAA2 2.877 13.13 1.0121 6b AA1 3b AA1 2.878 10.77 0.8701 6b AA2 3b AA2 2.877 10.75 0.8088 6b TAA1 36

Ret.Time

Rel.Area

Area

min [2.80..2.95] UV_VIS_1 2.854

% [2.80..2.95] UV_VIS_1 8.03

mV*min [2.80..2.95] UV_VIS_1 1.0452

2.857 2.854

10.32 17.1

1.0522 1.7475

2.856 2.855 2.856 2.856 2.855

19.42 12.51 12.85 22.59 22.13

1.8757 1.1642 1.1646 2.0522 2.0357

n.a.

n.a. 2.857 2.86 2.862 2.865 2.863 2.863 2.867 2.869

n.a.

n.a. 7.71 9.76 18.47 18.84 12.75 13.25 22.19 22.11

n.a. 2.87 2.868 2.865 2.869 2.871 2.871 2.868

0.9451 0.9044 1.6404 1.6004 1.0765 1.0625 1.7792 1.768 n.a.

8.76 11.64 22.17 21.97 10.95 11.21 21.64

1.0105 1.0112 1.8542 1.7823 0.836 0.8201 1.596


3b TAA1 3b TAA2 Injection Name Selected Peak: [2.80..2.95] 7a AA1 7a AA2 7a TAA1 7a TAA2 7b AA1 7b AA2 7b TAA1 7b TAA2 Eluent 8a AA1 8a AA2 8a TAA1 8a TAA2 8b AA1 8b AA2 8b TAA1 8b TAA2 Eluent 9a AA1 9a AA2 9a TAA1 9a TAA2 9b AA1 9b AA2 9b TAA1 9b TAA2 Eluent 10a AA1 10a AA2 10a TAA1 10a TAA2 10b AA1 10b AA2 10b TAA1 10b TAA2 Eluent

2.875 2.877

23.34 23.27

1.8093 1.7953

Ret.Time

Rel.Area

Area

min [2.80..2.95] UV_VIS_1 2.856 2.859 2.858 2.859 2.86 2.86 2.862 2.859 n.a. 2.858 2.862 2.863 2.865 2.859 2.86 2.866 2.864 n.a. 2.866 n.a. 2.862 2.864 2.868 2.869 2.866 2.865 n.a. 2.861 2.861 2.86 2.859 2.87 2.883 2.878 2.873 n.a.

% [2.80..2.95] UV_VIS_1 4.78 7.42 12.1 13.34 9.63 9.07 13.58 14.29 n.a. 6.04 8.47 14.14 15.13 9.31 8.98 14.16 14.98 n.a. 4.91 n.a. 16.13 17.39 8.59 7.8 17.11 18.03 n.a. 3.96 4.43 9.33 9.27 7.68 10.11 10.07 9.5 n.a.

mV*min [2.80..2.95] UV_VIS_1 0.84 0.8129 1.2246 1.2324 0.9032 0.8885 1.2962 1.291 n.a. 0.8833 0.8995 1.3731 1.3678 0.8429 0.8525 1.3022 1.2977 n.a. 0.6854 0.7196 1.5578 1.5522 0.7424 0.7023 1.5789 1.5772 n.a. 1.1302 1.1341 2.0399 1.9826 1.2654 0.65 1.0567 1.0761 n.a.

37

6b TAA2 Eluent Injection Name Selected Peak: [2.80..2.95] 11a AA1 11a AA2 11a TAA1 11a TAA2 11b AA1 11b AA2 11b TAA1 11b TAA2 Eluent 12a AA1 12a AA2 12a TAA1 12a TAA2 12b AA1 12b AA2 12b TAA1 12b TAA2 13a AA1 13a AA2 13a TAA1 13a TAA2 13b AA1 13b AA2 13b TAA1 13b TAA2 Eluent 14a AA1 14a AA2 14a TAA1 14a TAA2 14b AA1 14b AA2 14b TAA1 14b TAA2 Eluent

2.868

21.45

1.5756

n.a.

n.a.

n.a.

Ret.Time

Rel.Area

Area

min [2.80..2.95] UV_VIS_1 2.881 2.884 2.878 2.884 2.877 2.887 2.881 2.88 n.a. 2.886 2.883 2.883 2.88 2.893 n.a. 2.89 2.883 2.842 2.845 2.844 2.841 2.843 2.843 2.843 2.843 n.a. 2.852 2.855 2.855 2.853 2.856 2.857 2.854 2.85 n.a.

% [2.80..2.95] UV_VIS_1 6.01 5.82 8.87 8.85 7.74 8.46 9.28 10.04 n.a. 3.09 2.34 5.28 5.62 2.58 n.a. 3.73 3.44 25.85 26.26 35.36 36.72 30.52 32.08 41.51 41.87 n.a. 23.1 23.48 40.7 40.56 23.47 29.56 40.71 41.11 n.a.

mV*min [2.80..2.95] UV_VIS_1 0.5647 0.6229 0.9175 1.0259 0.7263 0.6455 1.2025 1.2042 n.a. 0.2595 0.2989 0.7171 0.5698 0.2942 n.a. 0.3144 0.4855 1.5233 1.554 2.0585 2.0735 1.6171 1.6226 2.1542 2.1508 n.a. 1.3877 1.4212 2.0688 2.2119 1.2231 1.2151 1.9965 2.0101 n.a.


Injection Name Selected Peak: [2.80..2.95]

Ret.Time

Rel.Area

Area

15a AA1 15a AA2 15a TAA1 15a TAA2 15b AA1 15b AA2 15b TAA1 15b TAA2 Eluent 16a AA1 16a AA2 16a TAA1 16a TAA2 16b AA1 16b AA2 16b TAA1 16b TAA2 17a AA1 17a AA2 17a TAA1 17a TAA2 17b AA1 17b AA2 17b TAA1 17b TAA2 Eluent 18a AA1 18a AA2 18a TAA1

min [2.80..2.95] UV_VIS_1 2.859 2.856 2.862 2.863 2.859 2.86 2.866 2.862 n.a. 2.863 2.878 2.864 2.864 2.867 2.861 2.865 2.863 2.851 2.856 2.856 2.856 2.853 2.851 2.858 2.86 n.a. 2.86 2.856 2.856

% [2.80..2.95] UV_VIS_1 21.7 24.18 46.01 46.69 20.87 21.04 40.86 41.22 n.a. 39.82 49.59 58.24 59.01 37.69 30.73 48 47.02 29.28 30.62 41.34 42.89 33.78 36.32 44.31 44.82 n.a. 19.66 20.71 35.93

mV*min [2.80..2.95] UV_VIS_1 1.1648 1.2584 2.1569 2.1484 0.8708 0.8729 1.7195 1.8252 n.a. 2.7679 1.7793 3.6319 3.8378 1.5032 1.6388 2.0405 2.1263 1.5089 1.5087 1.9973 2.0704 1.5584 1.6448 2.0454 2.0462 n.a. 0.9323 0.9325 1.4315

18a TAA2

2.859

36.88

1.4293

18b AA1 18b AA2 18b TAA1 18b TAA2

2.856 2.858 2.857 2.858

27.59 28.67 39.54 39.95

1.0083 1.0329 1.5183 1.5122

Injection Name Selected Peak: [2.80..2.95] 5a AA1 5a AA2 5a TAA1 5a TAA2 5b AA1 5b AA2 5b TAA1 5b TAA2 Eluent 6a AA1 6a AA2 6a TAA1 6a TAA2 6b AA1 6b AA2 6b TAA1 6b TAA2 Eluent 12a AA1 12a AA2 12a TAA1 12a TAA2 12b AA1 12b AA2 12b TAA1 12b TAA2

38

Ret.Time

Rel.Area

Area

min [2.80..2.95] UV_VIS_1 2.861 2.865 2.862 2.862 2.866 2.867 2.868 2.865 n.a. 2.863 2.865 2.866 2.865 2.863 2.863 2.867 2.867 n.a. 2.867 2.863 2.864 2.867 2.868 2.869 2.867 2.867

% [2.80..2.95] UV_VIS_1 16.06 16.41 29.07 29.39 19.66 21.06 34.3 34.32 n.a. 13.98 15.68 29.51 29.82 14.7 15.14 29.11 29.2 n.a. 16.97 18.16 31.38 32.25 19.29 19.05 32.89 33.12

mV*min [2.80..2.95] UV_VIS_1 0.9796 0.9569 1.7703 1.7643 1.0285 1.0306 1.7796 1.7734 n.a. 0.6856 0.7059 1.3714 1.367 0.585 0.5821 1.1737 1.1738 n.a. 0.7698 0.7755 1.4079 1.4139 0.7873 0.7468 1.4126 1.4174


200 150 100 50 0

y = 849.98x + 0.1753 R² = 0.9999 0

0.1

0.2

calibration curve L-ascorbic acid 14/11 Area (mV*min)

Area (mV*min)

calibration curve L-ascorbic acid 13/11 calibration curve Lascorbic acid 13/11

0.3

200 150 100 50 0

y = 945.67x + 0.3698 R² = 0.9998 0

concentration (mg/ml)

150 100 y = 843.38x + 0.4295 R² = 0.9999

0 0.2

0.3

Area (mV*min)

concentration (mg/ml)

Area (mV*min)

Calibration curve L-ascoric acid 15-11

0.1

Linear (Calibration curve L-ascoric acid 15-11)

200 150 100

y = 920.06x + 0.4403 R² = 0.9999

50 0 0

0.1

0.2

calibration curve Lascorbic acid 18/11

0.3

concentration (mg/ml)

Calibration curve L-ascorbic acid 19/11

Calibration curve L-ascorbic acid 20/11

200

200

100

y = 885.93x - 0.3118 R² = 0.9984

0 0 -100

0.1

0.2

0.3

Calibration curve Lascorbic acid 19/11

Concentration (mg/ml)

200 150 100 50 0

y = 888.14x + 0.4697 R² = 0.9997 0

0.1

0.2

concentration(mg/ml)

100

y = 845.3x + 0.8591 R² = 0.9993

0 0

0.1

0.2

0.3

Concentration (mg/ml)

calibration curve L-ascorbic acid 28/11 area (mV*min)

0.3

calibration curve L-ascorbic acid 18/11

Area (mV*min)

area (mV*min)

200

0

0.2

concentration (mg/ml)

Calibration curve L-ascoric acid 15-11

50

0.1

calibration curve Lascorbic acid 14/11

0.3

calibration curve Linear (calibration curve)

39

Calibration curve Lascorbic acid 20/11


Injection Name Selected Peak: alfa carotene water 1a 1 1a 2 1b 1 1b 2 2a 1 2a 2 2b 1 2b 2 water 3a 1 3a 2 3b 1 3b 2 4a 1 4a 2 4b 1 4b 2 h20 5a 1 5a 2 5b 1 5b 2 6a 1 6a 2 6b 1 6b 2 water 7a 1 7a 2 7b 1 7b 2 8a 1 8a 2 8b 1 8b 2

Ret.Time

Rel.Area

Area

min alfa carotene UV_VIS_2 n.a. 7.443 7.443 7.46 7.453 7.473 7.463 7.487 7.493 n.a. 7.503 7.507 7.513 7.52 7.527 7.527 7.543 7.547 n.a. 7.627 7.577 7.583 7.587 7.617 7.6 n.a. 7.617 n.a. 7.627 7.65 7.623 7.64 7.63 7.65 7.663 7.643

% alfa carotene UV_VIS_2 n.a. 39.6 43.68 69.82 100 44.78 100 46.23 100 n.a. 40.09 36.07 30.67 31.2 100 58.25 100 46.45 n.a. 71.28 50.6 53.54 52.65 35.94 40.67 n.a. 0.51 n.a. 55.75 52.99 59.59 59.43 76.11 53.79 51.66 61.67

mAU*min alfa carotene UV_VIS_2 n.a. 3.2992 4.6403 5.5654 5.6014 3.3584 3.5675 3.6334 3.4913 n.a. 2.6608 2.5828 1.9568 2.0178 4.4642 5.4149 4.7756 4.627 n.a. 1.6101 4.562 3.8722 3.8359 2.0545 2.2174 n.a. 0.0111 n.a. 5.3921 5.5737 6.1096 6.0397 4.2956 4.4843 4.1293 4.1431

Injection Name Selected Peak: alfa carotene water 9a 1 9a 2 9b 1 9b 2 10a 1 10a 2 10b 1 10b 2 water 11a 1 11a 2 11b 1 11b 2 12a 1 12a 2 12b 1 12b 2 water 13a 1 13a 2 13b 1 13b 2 14a 1 14a 2 14b 1 14b 2 15a 1 15a 2 15b 1 15b 2 16a 1 16a 2 16b 1 16b 2

40

Ret.Time

Rel.Area

Area

min alfa carotene UV_VIS_2 n.a. 7.673 7.68 7.64 7.66 7.683 7.667 7.69 7.68 n.a. 7.713 7.733 7.727 7.733 7.727 7.75 7.743 7.747 n.a. 7.763 7.753 7.757 7.75 7.76 7.77 7.797 7.783 8.077 8.057 8.027 8.057 8.04 8.057 8.043 8.053

% alfa carotene UV_VIS_2 n.a. 84.16 56.48 58.64 83.97 63.82 62.73 63.52 73.97 n.a. 78.67 77.68 77.55 78.43 100 100 80.97 100 n.a. 100 100 75.14 100 77.59 77.16 100 77.59 100 100 100 100 100 100 100 100

mAU*min alfa carotene UV_VIS_2 n.a. 2.4313 2.5494 2.7451 2.7485 4.6058 5.2241 5.7736 5.828 n.a. 4.5308 4.0559 3.7473 3.8356 1.9534 1.972 2.256 2.2432 n.a. 3.5831 3.6799 4.3256 4.1827 3.2957 3.2234 2.4082 2.3244 2.0234 2.1944 1.7093 1.7477 5.7936 5.8146 5.2822 5.4007


Injection Name Selected Peak: alfa carotene

water 17a 1 17a 2 17b 1 17b 2 18a 1 18a 2 18b 1 18b 2 water

Ret.Time

Rel.Area

min alfa carotene UV_VIS_ 2 n.a. 8.097 8.09 8.093 8.103 8.09 8.09 8.093 8.137 n.a.

% alfa carotene UV_VIS_ 2 n.a. 100 100 100 100 100 100 100 100 n.a.

Area mAU*mi n alfa carotene UV_VIS_ 2 n.a. 3.427 3.6147 3.762 3.6891 2.0126 1.9887 1.6665 1.5872 n.a.

Injection Name Selected Peak: alfa carotene

5a 1 5a 2 5b 1 5b 2 6a 1 6a 2 6b 1 6b 2 water 12a 1 12a 2 12b 1 12b 2

1000 y = 5655.1x + 1.3099 R² = 0.9998 500

0.1

Area

min alfa carotene UV_VIS_ 2 8.107 8.09 8.117 8.113 8.107 8.117 8.14 8.12 n.a. 8.103 8.143 8.143 8.133

% alfa carotene

mAU*min alfa carotene

UV_VIS_2 100 100 100 100 100 100 100 100 n.a. 100 100 100 100

UV_VIS_2 4.5265 4.3847 2.9461 2.7916 1.6286 1.654 1.9008 2.0056 n.a. 2.5207 2.6269 2.1331 2.1156

1000 calibration curve betacarotene 21/11

0 0

Rel.Area

calibration curve betacarotene 25/11 area (mV*min)

Area (mV*min)

calibration curve betacarotene 21/11

Ret.Time

0.2

500 0

0 -500

concentration (mg/ml)

calibration curve betacarotene 26/11

0 0

0.1

0.2

Concentration (mg/ml)

1000

1000 500

0.1

calibration curve betacarotene 25/11

calibration curve Area(mV*min)

area (mV*min)

calibration curve betacarotene 26/11 y = 4693x + 2.6186 R² = 0.9997

y = 4997.5x - 0.8906 R² = 0.9998

0.2

Concentration (mg/ml)

y = 5275.2x 1.1244 500 R² = 0.9997 0 0 -500

41

calibration curve

0.1

0.2

Concentration(mg/ml)

Linear (calibration curve)


Ascorbic acid and β-carotene analysis for juice samples Injection Name Selected Peak: [2.82..2.97] Eluent

Ret.Time

Rel.Area

Area

min

%

mV*min

Injection Name Selected Peak:

[2.82..2.97]

[2.82..2.97]

[2.82..2.97]

[2.82..2.97]

UV_VIS_1

UV_VIS_1

UV_VIS_1

n.a.

n.a.

n.a.

Eluent

Ret.Time

Rel.Area

Area

min

%

mV*min

[2.82..2.97]

[2.82..2.97]

[2.82..2.97]

UV_VIS_1

UV_VIS_1

UV_VIS_1

n.a.

n.a.

n.a.

pellet 1a AA1

2.838

77.67

23.6398

1a AA1

2.921

87.4

308.7171

Pellet 1a AA2 Pellet 1a TAA1 Pellet1a TAA2

2.842

79.21

23.5884

1a AA2

2.888

91.35

326.3245

2.839

85.8

26.5613

1b AA1

2.91

80.6

226.2411

2.84

86.69

26.8811

1b AA2

2.913

86.12

249.0731

pellet1b AA1

2.845

80.93

21.0519

2a AA1

2.93

83.45

286.2517

pellet1b AA2 pellet1b TAA1 pellet1b TAA2

2.85

81

21.0016

2a AA2

2.929

84.43

281.4068

2.848

85.48

26.2108

2b AA1

2.927

84.23

264.5295

25.9269

2b AA2

Eluent

2.84 n.a.

85.47 n.a.

n.a.

Eluent

2.922 n.a.

88.06 n.a.

270.6754 n.a.

pellet2a AA1

2.843

84.4

36.6276

3a AA1

2.916

86.88

268.2078

pellet2a AA2 pellet2a TAA1 pellet2a TAA2

2.852

85.81

36.6563

3a AA2

2.886

89.22

258.7833

2.853

89.46

44.011

3b AA1

2.904

83.4

244.2366

2.854

89.57

44.1559

3b AA2

2.902

83.35

258.5925

pellet2b AA1

2.853

85.7

34.8089

4a AA1

2.944

73.96

94.7101

pellet2b AA2 pellet2b TAA1 pellet2b TAA2

2.851

86.86

35.4173

4a AA2

2.856

90.15

42.5615

4b AA1

2.901

87.89

186.2935

2.856

90.17

41.9731

4b AA2

2.902

87.48

183.5652

Eluent

n.a.

n.a.

n.a.

Eluent

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

pellet3a AA1

2.861

84.52

34.018

5a AA1

2.912

86.96

221.973

pellet3a AA2 pellet3a TAA1 pellet3a TAA2

2.859

85.42

34.463

5a AA2

2.911

90.44

217.95

2.858

90.13

42.373

5b AA1

2.913

83.13

223.9346

2.862

90.08

42.1478

5b AA2

2.894

82.46

221.6637

pellet3b AA1

2.862

87.12

36.1824

6a AA1

2.868

77.88

122.4131

pellet3b AA2 pellet3b TAA1 pellet3b TAA2

2.863

87.45

36.2405

6a AA2

2.871

71.88

112.9231

2.863

93.99

42.1298

6b AA1

2.873

66.49

108.0088

2.866

90.89

43.5816

6b AA2

2.875

70.56

100.491

7a AA1

2.872

71.74

106.9735

Eluent

n.a.

n.a.

n.a.

pellet4a AA1

2.858

85.1

19.8846

7a AA2

2.869

71.25

106.6356

pellet4a AA2

2.859

86.5

20.0611

7b AA1

2.866

69.77

102.8179

pellet4a

2.865

91.07

26.9455

7b AA2

2.869

70.59

105.9467

42


TAA1 pellet4a TAA2

2.864

91.13

26.8528

eluent

2.839

11.76

3.0336

pellet4b AA1

2.868

88.03

21.3969

8a AA1

2.873

92.37

95.1216

pellet4b AA2 pellet4b TAA1 pellet4b TAA2

2.868

88.41

21.4088

8a AA2

2.873

71.88

92.5885

2.865

92.2

29.0151

8b AA1

2.872

76.77

95.8368

28.9919

8b AA2

2.87

76.53

93.2784

9a AA1

2.869

75.59

97.3957

Eluent

2.86 n.a.

92.14 n.a.

n.a.

pellet5a AA1

2.87

84.42

18.2893

9a AA2

2.873

73.96

95.179

pellet5a AA2 pellet5a TAA1 pellet5a TAA2

2.87

85.9

18.4544

9b AA1

2.868

64.8

105.212

2.871

86.54

18.4435

9b AA2

2.873

74.67

96.0195

2.872

86.72

18.4221

Eluent

pellet5b AA1

2.871

86.33

17.9949

pellet8a AA1

2.843

76.74

9.7346

pellet5b AA2 pellet5b TAA1 pellet5b TAA2

2.873

86.56

18.0116

2.845

77.45

9.334

2.872

86.71

18.0406

2.841

91.36

23.6335

2.872

86.72

18.1009

pellet8a AA2 pellet8a TAA1 pellet8a TAA2

2.839

91.28

22.7595

pellet6a AA1

2.847

54

6.635

pellet8b AA1

2.84

79.64

9.4043

pellet6a AA2 pellet6a TAA1 pellet6a TAA2

2.849

58.24

6.5783

2.846

80.5

9.3363

2.852

79.71

16.0218

2.845

92.09

23.2079

2.85

82.65

16.7216

pellet8b AA2 pellet8b TAA1 pellet8b TAA2

pellet6b AA1

2.849

62.62

5.8326

Eluent

pellet6b AA2 pellet6b TAA1 pellet6b TAA2

2.845

65.68

6.0905

pellet9a AA1

2.841

75.43

8.3705

2.843

84.69

15.4392

2.848

78.27

8.029

2.847

84.98

15.4274

pellet9a AA2 pellet9a TAA1 pellet9a TAA2

2.841

86.98

22.0754

2.846

91.17

21.4101

Eluent

n.a.

n.a.

n.a.

n.a.

n.a.

2.836 n.a.

n.a.

92.02 n.a.

23.1416 n.a.

pellet7a AA1

2.833

62.03

5.5883

pellet9b AA1

2.851

79.3

8.6429

pellet7a AA2 pellet7a TAA1 pellet7a TAA2

2.832

64.5

5.7335

2.85

80.34

8.791

2.839

84.36

14.3223

2.85

87.21

22.6958

2.839

85.17

14.6182

pellet9b AA2 pellet9b TAA1 pellet9b TAA2

2.845

91.74

20.8385

pellet7b AA1

2.838

66.31

5.4829

pellet7b AA2 pellet7b TAA1 pellet7b TAA2

2.832

66.76

5.8996

2.828

87.45

15.4479

2.832

87.35

15.5808

43

Eluent

n.a.

n.a.

n.a.


calibration curve ascorbic acid 18/12

200 100

y = 822.89x + 0.302 R² = 0.9998 0.2 0.3

0 0

0.1

Area(mV*min)

Areq (mV*min)

Calibration curve ascorbic acid 17/12 Calibration curve ascorbic acid 17/12

Concentration (mg/ml)

Injection Name Selected Peak: β-carotene

water

Ret.Time

Rel.Area

Area

min

%

mAU*min

β-carotene UV_VIS_ 1

β-carotene UV_VIS_ 1

β-carotene UV_VIS_ 1

n.a.

n.a.

n.a.

200 100 0 -100

Injection Name Selected Peak: βcarotene

0

0.1

y = 805.42x 0.3837 R² = 1 0.2 0.3

calibration curve ascorbic acid 18/12

Cocentration(mg/ml)

Ret.Time

Rel.Area

Area

min

%

mAU*min

β-carotene

β-carotene UV_VIS_ 1

β-carotene UV_VIS_ 1

UV_VIS_1 5b 1

7.793

100

20.5973

1a 1

7.677

98.48

34.6399

5b 2

7.79

100

20.3543

1a 2

7.72

97.82

23.5422

6a 1

7.807

100

19.323

1a 3

7.73

100

22.3396

6a 2

7.803

100

19.8307

1a 4

7.733

100

21.7212

6b 1

7.807

100

21.7934

2b1

7.74

95.44

22.2737

6b 2

2b 2

7.733

100

22.6643

water

2b 3

7.743

97.86

23.152

7a 1

7.81

100

21.6998

2b 4

7.743

97.75

22.8821

7a 2

7.787

100

21.9873

7b 1

7.79

100

20.6806

water

n.a.

n.a.

n.a.

7.807 n.a.

100 n.a.

21.8898 n.a.

3a 1

7.753

100

21.4096

7b 2

7.793

100

20.9278

3a 2

7.753

100

20.8113

8a 1

7.8

100

16.9675

3b 1

7.767

100

22.0321

8a 2

7.81

100

17.0414

3b 2

7.757

97.79

22.9536

8b 1

7.807

100

19.3253

4a 1

7.77

100

19.3209

8b 2

7.807

100

19.4676

4a 2

7.77

100

19.6044

water

4b 1

7.78

100

19.3457

9a 1

7.817

100

19.4077

4b 2

7.783

97.46

19.5625

9a 2

7.813

100

19.2685

9b 1

7.817

100

19.169

9b 2

7.817

100

17.8335

water

n.a.

n.a.

n.a.

5a 1

7.783

100

19.0328

5a 2

7.79

100

19.175

Area (mV*min)

calibratie curve β-carotene for juice samples 1000

y = 5722.4x - 0.9356 R² = 0.9997 500

calibratie curve slecht

0 -500

0

0.05

0.1

0.15

Concentration (mg/ml)

0.2

Linear (calibratie curve slecht)

44

n.a.

n.a.

n.a.

Bsc. Thesis report on Physalis peruviana L.