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EDITOR-IN-CHIEF Prof. dr hab. Henryk Zieliński, Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Olsztyn, Poland ASSOCIATE EDITORS Food Technology Section Prof. Alberto Schiraldi, DISTAM, sez. Chimica, Università di Milano, Milano, Italy Assoc. Prof. Dr. Özlem Tokuşoğlu, Celal Bayar University, Manisa, Turkey Dr hab. inż. Marek Adamczak, prof UWM, University of Warmia and Mazury, Olsztyn, Poland Food Chemistry Section Dr. Maria Dolores del Castillo, Institute of Food Science Research (CSIC-UAM), Madrid, Spain Dr hab. Magdalena Karamać, Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Olsztyn, Poland Dr. Agnieszka Kosińska-Cagnazzo, University of Applied Sciences and Arts Western Switzerland, HES-SO Valais, Sion, Switzerland Food Quality and Functionality Section Dr. Maria Juana Frias Arevalillo, Institute of Food Science, Technology and Nutrition (ICTAN), Madrid, Spain Dr. Zuzana Ciesarova, NPPC National Agricultural and Food Centre, VUP Food Research Institute, Bratislava, Slovak Republic Prof. dr hab. Ryszard Amarowicz, Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Olsztyn, Poland Nutritional Research Section Prof. André Mazur, Human Nutrition Unit, INRA, Clermont, France Prof. dr hab. Anna Brzozowska, University of Life Sciences, Warsaw, Poland Prof. dr hab. Jerzy Juśkiewicz, Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Olsztyn, Poland Language Editor Prof. Ron Pegg, University of Georgia, Athens, USA Statistical Editor Dr Tomasz Jeliński, Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Olsztyn, Poland Executive Editor Joanna Molga,“News” Section, Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Olsztyn, Poland

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ISSN (1230–0322) 2 0 1 6, Vol. 66, No. 3

Published by the Division of Food Sciences, Institute of Animal Reproduction and Food Research of Polish Academy of Sciences, Olsztyn

Pol. J. Food Nutr. Sci.

2016, Vol. 66, No. 3

Advisory Board of PJFNS 2015–2018

Huda Al-Kateb Coventry University, Coventry, UK Jennifer M. Ames University of the West of England, Bristol, UK Wilfried Andlauer University of Applied Sciences, Sion, Switzerland Sa’eed Halilu Bawa University of the West Indies, St. Augustine, The Republic of Trinidad and Tobago Bhaskar C. Behera Agharkar Research Institute, India Vural Gökmen Hacettepe University, Ankara, Turkey Adriano Gomes Da Cruz UNICAMP, Sao Paulo, Brazil Liwei Gu University of Florida, Gainesville, USA Henryk Jeleń Poznań University of Life Sciences, Poland Georgios Koutsidis Northumbria University, Newcastle-upon-Tyne, UK Theodore P. Labuza Department of Food Science and Nutrition, University of Minnesota, USA Andrzej Lenart Warsaw University of Life Sciences, Poland Johns Lodge Northumbria University, Newcastle-upon-Tyne, UK Claudine Manach INRA, Centre de Recherche de Clermont-Ferrand, Theix, France Adolfo J. Martinez-Rodriguez CSIC, Madrid, Spain Brian McKenna National University of Ireland, Dublin, Ireland

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Pol. J. Food Nutr. Sci.

2016, Vol. 66, No. 3

POLISH JOURNAL OF FOOD AND NUTRITION SCIENCES COVERED BY CURRENT CONTENTS/AGRICULTURE, BIOLOGY & ENVIRONMENTAL SCIENCES, JOURNAL CITATION REPORTS AND SCIENCE CITATION INDEX EXPANDED, BIOSIS, SCOPUS, FSTA (FORMERLY FOOD SCIENCE AND TECHNOLOGY ABSTRACTS) AND CAS ABSTRACTS, AND AGRO-LIBREX, FROSTI, PSJC, AGRIS AND INDEX COPERNICUS DATA BASES 2016, VOL. 66, NO. 3 REVIEW Use of Whey and Whey Preparations in the Food Industry – a Review. J.B. Królczyk, T. Dawidziuk, E. Janiszewska-Turak, B. Sołowiej ..................................................................................................... 157

ORIGINAL PAPERS Antioxidant Capacity of a Turkish Traditional Alcoholic Drink, Raki. G. Yalcin ......................................................................................................................................................................................... 167

Total Oil Content and Fatty Acid Profile of Some Almond (Amygdalus communis L.) Cultivars.

A.N. Yildirim, F. Akinci-Yildirim, B. Şan, Y. Sesli ........................................................................................................................... 173 Biological Activities and Nutraceutical Potentials of Water Extracts from Different Parts of Cynomorium coccineum L. (Maltese Mushroom). P. Zucca, A. Argiolas, M. Nieddu, M. Pintus, A. Rosa, F. Sanna, F. Sollai, D. Steri, A. Rescigno ................................................... 179

Effects of Encapsulated Fish Oil by Polymerized Whey Protein on the Textural and Sensory Characteristics of Low-Fat Yogurts.

D. Liu, T. Zhang, N. Jiang, Ch. Xi, Ch. Sun, J. Zheng, M. Guo ..................................................................................................... 189 Kinetics of Texture and Colour Changes in Chicken Sausage During Superheated Steam Cooking. A.A. Abdulhameed, T.A. Yang, A.A. Abdulkarim ............................................................................................................................ 199 Nutritional Value and Consumer Acceptance of New Cereal Bars Offered to Children. M. Białek, J. Rutkowska, J. Radomska ........................................................................................................................................... 211 Longer Breastfeeding in Infancy Decreases Systolic Hypertension Risk in Young Adults. K. Rak, D. Kornafel, M. Bronkowska ............................................................................................................................................. 221

SHORT REPORT Effect of Microbial Transglutaminase on Ice Cream Heat Resistance Properties – a Short Report. I. Kasprzyk, J. Markowska, E. Polak .............................................................................................................................................. 227 Conference Announcements ..................................................................................................................................................... 233 Instruction for Authors ............................................................................................................................................................. 237

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Pol. J. Food Nutr. Sci., 2016, Vol. 66, No. 3, pp. 157–165 DOI: 10.1515/pjfns-2015-0052 http://journal.pan.olsztyn.pl Review article Section: Food Technology

Use of Whey and Whey Preparations in the Food Industry – a Review Jolanta B. Królczyk1*, Tomasz Dawidziuk1, Emilia Janiszewska-Turak2, Bartosz Sołowiej3 Opole University of Technology, Prószkowska 76 St., 45–758 Opole, Poland Department of Food Engineering and Process Management, Faculty of Food Sciences, Warsaw University of Life Sciences, Nowoursynowska 159c St., 02–787 Warsaw, Poland 3 Department of Milk Technology and Hydrocolloids, Faculty of Food Sciences and Biotechnology, University of Life Sciences in Lublin, Skromna 8 St., 20–704 Lublin, Poland 1

2

Key words: whey, whey preparations, sweet whey powder, deminaralised whey powder, WPCs, WPI The interest in whey and whey preparations has considerably increased in recent years. Whey and whey preparations are the so-called “forgotten treasure” and, because of their unique properties, they have been “rediscovered” and have been increasingly frequently and successfully used by various production plants in the food industry. They have also been eagerly purchased by consumers who are aware of the role of whey preparations in adequate human nutrition. For many years, there has been a tendency in the food processing industry to use substitutes of ingredients in recipes of many products. This situation can be observed in the case of foods with reduced fat and sugar, or products for lacto-ovo-vegetarians. Whey – and more specifically, its preparations – can also be used as a substitute. According to many literature sources, its use can have a positive impact not only on the consumers’ health but also on the finances of many companies, by reducing the costs of raw materials, and thus production costs. This review paper presents selected uses of whey and whey preparations in the food industry. The uses of whey discussed include: meat and meat products, reduced-fat products, yoghurts and ice creams, cheeses, bakery products, confectionery and pastry products, infant formulas, and whey drinks.

INTRODUCTION Only a few decades ago, whey was a serious problem for dairy plants. It was not recycled to the extent it currently is. Whey was removed along with sewage, which posed a threat to the ecosystem due to the organic compounds it contained [Wesołowska-Trojanowska & Targoński, 2014]. In  the  case of cheese production, ten parts of milk give nine parts of whey and  one part of  cheese [Bylund, 2003]. The  current use of whey, as well as its preparations, is made possible thanks to numerous studies in this area. Whey derived from cow’s milk, as well as sheep [Salvatore et al., 2014], goat [Philippopoulos & Papadakis, 2001] or camel milk [Laleye et  al., 2008] can be  processed. Production of  whey powder has recently increased in the European Union from 1,950,000 t in 2011 to 2,200,000 t in 2014 [EWPA, 2015]. A  tendency to use substitutes of  ingredients in  recipes of  many products has been observed for several years in  the  food processing industry. It  pertains to foods with reduced fat and  sugar, or food products for vegetarians and  people with lactose intolerance [Bolumar et  al., 2015; Garcia-Serna et  al., 2014]. Whey and  its preparations may serve as substitutes. According to many sources, their use can have a positive impact not only on the consumers’ health, but *  Corresponding Author: E-mail: [email protected]

also on the finances of many companies by reducing the costs of raw materials, and thus lowering production costs [Božanić et al., 2014; Keaton, 1999; Singh & Singh, 2012]. Cost reduction is achieved by the use of whey preparations as partial or complete replacements of milk powder [De Wit, 2001], eggs [Stoliar, 2009], fat [Prabhu, 2006; Stoliar, 2009], sucrose [Pernot-Barry, 2008], or even other proteins [Keaton, 1999]. There are two different types of whey: sweet whey and acid whey. Sweet whey is a by-product of ripened cheese production (pH 5.8–6.6) whereas acid whey is obtained from cottage cheeses (pH 3.6–5.1) [Anand et al., 2013]. Regulations about the use of whey are based on Codex Alimentarius concerning milk and  milk products launched by  the  World Health Organization Food and Agriculture Organization of the United Nations [WHO, 2011]. The  so-called whey proteins, which include β-lactoglobulin, α-lactalbumin, lactoperoxidase, and  lactoferrin, are the  main source of  whey health-promoting properties [Kumar et  al., 2008]. The  following peptides were identified in the β-lactoglobulin sequences: β-lactorfin which influences the  smooth muscles, β-lactotensin which exhibits hypocholesterolemic and  anti-stress activities, and  in  the  α-lactalbumin sequences – α-lactorfin which displays effects similar to that of morphine, namely blood pressure reduction [Chatterton et  al., 2006]. In  turn, lactoferrin is  a  bioactive milk protein with a  comprehensive activity. Although the  mechanisms of  its action are not

© Copyright by Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences ©2  016 Author(s). This is an open access article licensed under the Creative Commons Attribution-NonCommercial-NoDerivs License (http://creativecommons.org/licenses/by-nc-nd/3.0/).

158 fully known, a  broad spectrum of  its properties has been confirmed by  scientific research. It  shows many physiological functions, i.e. antifungal, antiviral, antibacterial, antitumour, and  anti-inflammatory. Also, lactoferrin has a positive effect on the nervous system and is able to bind iron [Darewicz et al., 2014]. Amino acids, minerals, and vitamins should also be taken into account [De Wit, 2001]. This review paper presents selected uses of  whey and  whey preparations in  the  food industry. The  uses of  whey discussed include: meat and  meat products, reduced-fat products, yoghurts and ice creams, cheeses, bakery products, confectionery and pastry products, infant formulas, and whey drinks. MEAT AND MEAT PRODUCTS Whey processing products used in the meat industry are as follows [Keaton, 1999; Prabhu, 2006]: sweet whey powder, whey protein concentrates (WPCs) (34–80% protein content), whey protein isolate (WPI) (> 90% protein), whey with reduced lactose content, demineralised whey, and lactose. They are used especially in  the  production of  comminuted products, such as: frankfurters, sausages, mortadellas, luncheon meat, or surimi [De Wit, 2001]. Whey protein may partially replace meat protein, as well as partially or completely substitute for soy protein and other binding agents, fillers, modified starch and hydrocolloids [Keaton, 1999; Prabhu, 2006; Youssef & Barbut, 2011]. Whey proteins with improved flavour and increased functionality are obtained with new technologies,. While choosing a particular whey product, it is essential to match its function to the characteristics we want to achieve. For example, highprotein concentrates or isolates are used to modify fat content [Prabhu, 2006]. A slight increase in sweetness occurs, especially with the addition of sweet whey (which enables reduced addition of sweeteners) [Keaton, 1999]. According to Keaton [1999], the  properties of  whey proteins used in  the  processing of  meat products, poultry, and fish are as follows: water binding capacity that prevents the depletion of mass during thermal processing and  storage of  the  product, increases juiciness of  the  final product and  facilitates cutting cold meat products into slices; viscosity which improves the consumers’ palatable impressions during consumption of the product (biting and chewing); which is directly related to the ability to bind water; high solubility – in the range of pH from 2 to 10 (ideal for use in injected products), while sodium caseinate is soluble at above pH 5.0, and soy protein isolate only at pH above 5.5; the  formation of  stable emulsions, which is  particularly important in the production of finely comminuted meat products, especially when the raw material is of poor quality; here, whey proteins may partially or completely replace other emulsifiers. Furthermore, the addition of whey proteins affects the taste and improves the gelation. They can be used in the production of edible sausage casings. They are also used in the finishing of  semi-products, as their addition has a  positive effect on the adhesion of batter to portions of meat, poultry, or

Whey and Whey Preparations in the Food Industry

fish. They may also exhibit antioxidant activity (this refers to the oxidation of fat in pork meat, in salmon meat, or in products rich in lipids) [Prabhu, 2006]. Keaton [1999] in  his paper presented an example for a recipe of mortadella using WPC34 (whey protein concentrate with protein content of  approx. 34%) and  WPI (90% protein), and revealed an increase in the effectiveness of this product (Tables 1–3). TABLE 1. Contents of fat, protein, and moisture in meat and whey products used in the production of Frankfurter/Bologna (adopted from Keaton [1999]). Specification

Fat (%)

Protein (%)

Moisture (%)

Lean beef

20.0

18.0

63.0

Pork trim

40.0

10.0

49.0

WPI (90% protein)

1.5

90.0

3.5

WPC34

4.0

34.0

3.5

TABLE 2. Composition of  raw materials in  production of  Frankfurter/ Bologna (adopted from Keaton [1999]).

Specification

Control sample

WPC34

WPI90

kg

%

kg

%

kg

%

Lean beef

21.50

15.96

12.0

7.76

20.0

11.56

Pork trim

78.50

58.25

88.0

56.89

90.0

52.02

WPI(90% protein)









3.00

1.73

WPC34





4.68

3.03





Salt

2.25

1.67

2.25

1.45

2.25

1.30

Corn syrup solids

2.00

1.48









1.00

0.74

1.00

0.65

1.00

0.58

0.50

0.37

0.50

0.32

0.50

0.29

0.45

0.33

0.45

0.29

0.45

0.26

0.50

0.37

0.50

0.32

0.50

0.29

Sodium erythorbate

0.06

0.04

0.06

0.04

0.06

0.03

Modern cure (nitrite/salt)

0.25

0.19

0.25

0.16

0.25

0.14

Ice

27.70

20.56

45.00

29.09

55.00

31.79

Total

134.71

100

154.69

100

173.01

100

Hydrolysed milk protein Hydrolysed beef stock Sodium tripolyphosphate Frank/bologna seasoning

TABLE 3. Increase in productivity in production of mortadella with use of WPC34 and WPI90 (adopted from Keaton [1999]). Specification The increase in productivity compared to a control sample (kg) The increase in productivity compared to a control sample after heat treatment (kg)

WPC34

WPI (90% protein)

19.98

38.30

17.98

34.47

159

J.B. Królczyk et al.

PRODUCTS WITH REDUCED FAT CONTENT High consumption of fats, particularly those of animal origin, may have a negative impact on human health. It contributes to many diseases that are nowadays classified as civilization diseases, e.g. atherosclerosis. This leads to the simple conclusion that it is advisable to limit the consumption of fat and replace fatty foods with the low-fat ones [Ellander et al., 2015; Sołowiej, 2012]. Fat is  an important component of  many food products. It affects the taste of the finished product, improves the texture, springiness, mouthfeel, juiciness and  stability during storage. As a  result of  its reduction, the  obtained product is  tasteless and unacceptable to consumers [Johnson, 2000; Prabhu, 2006]. Replacing part of the lipids with the so-called fat substitutes can help prevent negative phenomena. Whey proteins are widely used during the  production of  salad dressings, soups and sauces, mayonnaise, meat, yoghurts, and ice cream preparations [De Wit, 2001; Johnson, 2000; Yilsay et  al., 2006; Zhang et al., 2015]. According to Johnson [2000], these replacers can be divided into two groups: substitutes and mimetics. The WPCs are classified as fat mimetics because they have various functional properties similar to those that lipids have. WPC34 and WPC80 are commonly used. They can completely or partially replace egg yolk, hydrocolloids, soy protein, or modified starch. The  most important functions of  WPC in  low-fat products are: water binding, emulsification, high solubility, gelation, increasing the viscosity, and increasing the adhesive interactions [Johnson, 2000]. DeWit [2001] proposes that, to fully utilise the emulsifying properties of WPCs, emulsions could be prepared before the  addition of  components with low pH.  Johnson [2000], in turn, suggests a new approach in which WPC34 and carrageenan are combined in  order to obtain best quality in the production of low-fat sausages. WPCs with a high protein content have very little perceptible flavour. Low-protein WPCs such as WPC34 can, in turn, impart a slightly milky, sweet aftertaste to products to which they have been added. Therefore, when fat content is reduced and a WPC is added, the composition of the product should be corrected; addition of spices and flavourings may be required [Johnson, 2000]. BAKERY AND CONFECTIONERY PRODUCTS Whey may be  widely used in  the  baking, confectionery, and  pastry industries for the  production of  breads, cakes, cookies, biscuits, crackers, muffins, and  icing [Burrington, 1999; Ceglińska et al., 2007; De Wit, 2001; Stoliar, 2009]. The product, in which eggs play a key role are cakes. Eggs contribute to the  development of  their structure and  taste. Protein is  a  significant factor affecting the  structure, therefore it is recommended to replace it only partially. A hen’s egg weighs, on average, from 52 to 55 g, of which 76% is water, so when replacing whole fresh eggs, water needs to be added to the WPCs. The proposed proportions of WPCs and water, if added, in substitution of hen’s eggs are as follows: 100 g fresh whole eggs = 15 g WPC80 + 75 g water 100 g fresh whole eggs = 35 g WPC34 + 75 g water

100 g dried whole eggs = 57 g WPC80 Replacing eggs with whey proteins is  also an effective means of  reducing production costs (the  obtained product crumbles less during cutting and  packaging – which means a lack of additional costs) [Stoliar, 2009]. Furthermore, as a  result of  the  presence of  cholesterol in egg yolk, due to dietary reasons, there is a growing interest in the replacement of this component. An experiment was conducted in which one cake was prepared using a standard cake recipe and a second with the addition of WPCs. The appearance of both cakes was similar, but the volume of the sample with the addition of WPCs was increased. However, replacing eggs with WPCs resulted in a poorer taste and dry structure of the cake [De Wit, 2001]. Baked goods are products rich in  carbohydrates, but poor in  proteins, as a  result of  which they do not belong to dietary products. Therefore, whey processing products in  combination with sugar alcohols or artificial sweeteners contribute to a reduction in carbohydrate content of the discussed products. For example, WPC34 is suitable for products such as spice cookies or chocolate chip cookies as a  partial replacement for both egg and  fat. On the  other hand, WPC80 is a good substitute for eggs in products such as bread, cakes and  biscuits (both dry and  soft) and  muffins. Using WPCs, which are classified as fat mimetics, we can lower fat content by  up to 50%, and  thereby increase the moisture content of the finished product, such as cakes, cookies, muffins [Stoliar, 2009]. In baking, confectionery and pastry, lactose is often used as a substitute for sucrose as it enhances the Maillard reaction, improves emulsification and  crumb structure, and  enhances the flavour [Burrington, 1999; Stoliar, 2009]. In addition, whey proteins contain a high level of essential amino acids; they are also considered a source of high quality protein. In addition, they are characterised by a high content of calcium and other minerals, such as potassium and zinc. Thanks to these properties, whey protein is a valuable additive to bakery products [Burrington, 1999; Ceglińska et al., 2007; Munaza, 2012]. Ceglińska et  al. [2007] proposes replacing sodium chloride (table salt) in  the  production of  bread with minerals derived from ultrafiltration of whey. The results of  quality tests of  bread with various combinations of  contents of salt and of minerals from whey powder are presented in Table 4. The largest volume of bread and the smallest crumb hardness was obtained in sample 2. The use of minerals derived from whey as substitutes for table salt is therefore recommended in bread production in quantities not exceeding 3% [Ceglińska et al., 2007]. In turn, the addition of whey preparations to bakery and cake products proposed by Burrington [1999] is shown in Table 5. Whey products, including, demineralised whey powders, low-lactose whey powders, WPCs and isolates, and lactose have been used in  the  following confectionery: chocolates and  chocolate chips, candies, jellies and  chewing gums [Bouzas, 1999; Pernot-Barry, 2008]. Lactose – milk sugar – can serve as a  bulking agent. It  is  slightly sweet, less soluble than sucrose, and  has a  low hygroscopicity level; however, it  influences the  colour, the  taste, and  the  texture

160

Whey and Whey Preparations in the Food Industry

TABLE 4. Results of quality tests of bread in various combinations of content of salt and of minerals from whey powder (adopted from Ceglińska et al. [2007]). Hardness (N)

Baking loss (%)

Yield of bread (%)

Bread volume (cm3)

Absolute weight (g/cm3)

24 h

72 h

1

11.5

138.6

213.9

0.289

5.35

8.10

2

11.7

137.6

297.5

0.252

3.21

5.37

3

10.4

141.0

223.6

0.287

6.15

9.77

4

9.6

141.4

184.4

0.332

6.38

10.22

5

9.5

142.2

213.8

0.295

4.99

10.58

6

9.7

142.0

187.7

0.315

7.59

10.45

Additive

The legend for the applied additives 1–6: 1 – 2.0% of table salt, 2 – 2.0% of whey minerals, 3 – 3.0% of whey minerals, 4 – 4.0% of whey minerals, 5 – 1.5% of table salt + 1.5% of whey minerals, and 6 – 1.0% of table salt + 3.0% of whey minerals. TABLE 5. Recommended proportions of addition of whey preparations for selected bakery and cake products (adopted from Burrington [1999]). Sweet whey powder (%)

WPC34 to WPC50 (%)

WPC80 (%)

Demineralised whey powder (%)

White bread

1–5

1–4

1–3

2–6

Sweet rolls

2–5

1–4

1–3

2–6

Cakes and biscuits

1–5

1–5

1–4

2–5

Crackers

1–5

1–4

1–3

2–6

Pizza dough

1–5

1–4

1–3

2–6

Cakes

1–6

1–4

1–3

2–6

Type of product

of the finished product and takes part in the Maillard reaction [De Wit, 2001; Pernot-Barry, 2008]. For these reasons, use of lactose can be more or less reduced, which depends on many characteristics of  the  confectionery. For example, in the case of milk fudge, replacing sucrose by lactose causes the  formation of  caramel flavour, and  sugar content is  reduced. Products in  whose manufacture lactose was added include hard candy, fillings, chocolate bars, and toffee candy [Pernot-Barry, 2008]. Another use of  derivatives of  WPCs is  the  production of  the  so-called aerated confectionery and  chocolate. The  foaming properties of  concentrates are used in  this case. In addition, WPI and concentrates of high protein content (WPC80) can be  successfully used in  the  production of protein bars for athletes [Bouzas, 1999]. Another example

is meringues, in whose production great emphasis is put on foam stability [De Wit, 2001]. The  author proposes a  total replacement of egg protein with skimmed WPCs, as the shape and size of the product (meringue) containing WPCs is similar to that obtained on the basis of egg protein. On the other hand, Nastaj et  al. [2014] found that in  the  case of  foams obtained from WPI and  WPC80  prior to thermal fixation (in  the  production of  meringues), increased protein content and reduced sucrose addition led to an improvement of their rheological properties. The  obtained high-protein meringue with a  reduced sucrose level may be  a  new food product, attractive for physically-active people and  athletes with increased demand for complete protein. Both demineralised whey powder, as well as WPCs can be used as a source of milk solids in the production of chocolate-flavoured coatings (for ice cream and bars), and the kaymak mass. The additive also affects the costs of raw materials and the production costs. Fat is reduced as well [Bouzas, 1999]. Examples of  whey preparations additives in  selected confectionery indicated by  Bouzas [1999] are presented in Table 6. DAIRY PRODUCTS Yoghurts and ice creams A growth in the consumption of fermented milk beverages has been observed in recent years; the most important of these is  the  consumption of  yoghurt. For this reason, the  quality characteristics of  the  finished product are very important. These characteristics can be successfully modified using whey preparations [Kozioł et al., 2014; Liu et al., 2016].

TABLE 6. Typical whey preparations and lactose additive in the selected confectionery (adopted from Bouzas [1999]). Demineralised whey powder (%)

WPC34 (%)

WPC80 (%)

WPI90 (%)

Lactose (%)

Milk chocolate

0–5

0–5





3–7

Chocolate flavour topping

0–20

0–20





3–7

Kaymak mass

0–50

0–50











0–20

0–35



Product

Protein bars

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Hugunin & Lucey [2009] presented the  whey products used in the production of yoghurt. These include: 1. sweet whey powder, which may replace skimmed milk powder at the level 2–5.2%; 2. WPCs, which are most often used by  manufacturers of yoghurt. Addition of WPC34 at the level 0.7–2.0% or WPC80 at 0.5–0.8% is sufficient in the case of mixed yoghurt (a  greater amount of  the  additive may adversely affect some quality characteristics). Replacing skimmed milk powder with WPCs causes, among other effects, an increased gel strength in solid yoghurt, increasing the viscosity of mixed yoghurt, and reduces the risk of syneresis in both types of yoghurt; 3. WPI which, due to the low content of lactose and milk fat, is used in yoghurts with reduced lactose content; 4. demineralised whey powder, with reduced mineral content, which accelerates the  fermentation process. On the other hand, low mineral content weakens the structure of the gel, so it is necessary to add milk protein hydrolysates when this type of formulation is used. The consistency of the finished product has a significant influence on the choice of product by consumers. The texture of  yoghurt can be  improved through the  use of  the  abovementioned products by increasing the viscosity and stability and reducing the risk of syneresis. Furthermore, the addition of whey protein gives yoghurt a smooth and creamy texture; it also increases its nutritional value [De Wit, 2001; Hugunin, 2009; Hugunin & Lucey, 2009]. It  is  suspected that the  bioactive components present in  whey and  whey protein can stimulate the  growth of  probiotic bacterial cultures (both in  the  finished product, and in the human digestive tract). Research carried out by Hugunin [2009], Hugunin & Lucey [2009] indicate that the addition of Bifidobacterium bifidum to standard yoghurt cultures (Lactobacillus delbrueckii ssp. bulgaricus, Streptococcus thermophilus), increases the number of viable B. bifidum in  the  samples that contain the  sweet whey or whey proteins. Whey preparations used in the manufacture of ice-creams and sundaes include whey powder, demineralised whey powder, WPCs and WPI [Young, 2007]. Features and benefits that result from the use of whey products are presented in Table 7. In  addition to the  aforementioned properties of  whey preparation, their other important properties include: water binding capacity, ability to form foam, and  high nutritional value [Young, 2007; Jasińska et al., 2012]. On the  other hand, from the  point of  view of  ice-cream manufacturers, it is important to reduce the costs of production. Rationalisation of  these costs can be  achieved by  using cheaper substitutes of  certain ingredients of  the  recipes. The  relatively expensive components include milk powder and  egg yolk [Alfaifi & Stathopoulos, 2010; Jasińska et  al., 2012]. Jasińska et  al. [2012] suggested replacing milk with WPCs and  with protein-fat preparations. They compared the control sample without the addition of whey products or fat-protein preparations with ice mixtures with a  50% addition of  WPC65  and  WPC80, as well as with 50% addition of  whey-fat preparations containing vegetable fats (coconut oil, palm oil). The organoleptic properties, fat content and dry

TABLE 7. Selected functional properties of whey preparations and benefits that can be obtained with their use in the production of ice creams and sundaes (adopted from Young [2007]). Functions Viscosity

Gelation

Emulsification Taste and flavour

Impact Compaction Formation of gel during heat treatment and the improvement of viscosity The formation of a stable emulsion Mild, sweet, milky flavour

Benefits Stabilising air bubbles, obtaining creamy structure Increasing resistance to high temperatures; improvement of the structure The ability to partially replace casein Compatible with flavouring additives

matter content was evaluated, and the acidity and hardness of the blend and the ice cream, as well as fluffiness and meltability were measured. The  best result was obtained with the  addition of  WPC80 (a  high score in  the  organoleptic evaluation, low melting behaviour and the degree of aeration) [Jasińska et al., 2012]. Alfaifi & Stathoupolos [2012] analysed the impact of substituting powder from dried egg yolks with WPC80 in Gelato ice cream production. They replaced 0, 20, 50 80  and  100% of the egg yolks with WPC80 in two different parts of the samples. In the first part of the samples, the whole egg yolk was 4.5% of the whole sample; in the second 9%, of the whole sample. Based on the results, they noticed a pronounced effect on the physical properties of Gelato ice cream, such as viscosity of the mixture of ice, fluffiness (degree of aeration) and texture. Lower viscosity was achieved with 4.5% of  added egg yolk. Additionally, increasing the content of WPC80 led to an increase of  fluffiness [Alfaifi & Stathopoulos, 2010]. Along with an increase of  percentage of  protein concentrate, improvement in the structure of the finished product has been observed as well. Thus, WPC80  as a  replacer for egg yolk seems to be a rational solution in the case of Gelato ice creams for producers who want to reduce manufacturing costs without change desired characteristics of the finished product [Alfaifi & Stathopoulos, 2010]. However, according to Palka & Palich [2008], increased aeration can lead to a deterioration in the quality of ice creams during storage. Before developing a product’s recipe, it should be experimentally determined what amount of whey products should be  in  the  final product. More recipes are given by  Young [2007] who recommend the  addition of  different types of  whey powders to the  entire ice blend, e.g. whey powder (2–3%) or WPC34 (1.5–3%) or WPI (0.5–1) or WPC60 – WPC80 (0.5–2%). Cheese, processed cheese, and their analogues Whey preparations, such as sweet whey powder, powdered whey with reduced lactose content, WPCs and WPI can be  successfully used in  the  production of  processed cheese and processed cheese analogues [Young, 1999]. Liquid, sweet whey left after cheese making from cow and sheep milk, can be  used in  manufacturing whey cheeses [Philippopoulos & Papadakis, 2001; Salvatore et al., 2014; Wendorff, 2008].

162 For the production of processed cheese analogues, cheese is  replaced by  milk proteins (casein, whey proteins) or vegetable proteins [Sołowiej, 2007; Sołowiej et al., 2014, 2015]. Vegetable proteins are mainly used because of  their price. Production of  1  kg of  soy protein is  more than three times cheaper than the production of milk protein [Aljewicz et al., 2011]. A  key factor in  the  use of  whey preparations in  this product range is, therefore, their cost-effectiveness [Sołowiej, 2007; Young, 1999]. Their emulsifying properties are valuable, especially during heat treatment, packaging, and cooling. The  ability to bind water can disrupt the  technological process if it is carried out in an inappropriate way. This affects the  melting of  the  mixture and  spreadability of  the  finished product. The big advantage of whey preparations is their high nutritional value [Young, 1999]. Sołowiej [2007] and Sołowiej et al. [2008] showed that the addition of whey protein preparations to processed cheese analogues, as well as replacing casein with these proteins, increases the hardness of the final product which may be important for the preparation of products for slicing. In  turn, the  analogues supplemented with whey products exhibited lower meltability compared to cheese analogues prepared solely on the basis of acid casein in a pH range of  5.0–7.0 [Sołowiej et  al., 2008]. Moreover, the  use of  high-protein formulations such as WPI and  WPC80  allowed for the partial elimination of the flux emulsifying salts (i.e. a reduction in disodium phosphate content from 2.0% to 0.8%) [Sołowiej et al., 2014]. Young [1999] recommends control of  lactose content during the  production process to prevent its crystallisation and  browning of  the  product (therefore, it  seems reasonable to use products with a  low content of  milk sugar – WPCs, WPI). The recommended addition of whey preparations to cheese mixtures is as follows: sweet whey powder: 4–8%, whey with reduced content of  powder lactose: 5–8%, WPC34 and WPC80: 1–5%, WPI90: 0.5–1% [Young, 1999]. The most popular whey cheeses include myzithra, ricotta, mysost, gjetost, manouri, anthotyros and giza [Philipopoulos & Papadakis, 2001; Salvatore et al., 2014; Wendorff, 2008]. Wendorff [2008] presents two ways of producing cheese on the basis of whey: 1. exposure of  whey to heat treatment and  then acidifying it  to separate the  fat and  the  protein. Optionally, milk components can be  added. The  final step is  to separate the liquid from the curd, for example in ricotta. 2. slow evaporation of the whey in open containers until lactose contained in it causes browning, e.g. mysost, gjetost. Ricotta is produced from sweet whey left after the production of  such cheeses as Cheddar, Mozzarella, etc. It  is  recommended to add skimmed milk in  the  amount of  5–10% to increase dry milk mass and to improve the taste of the final product [Wendorff, 2008]. It  is  believed that production of ricotta cheese is one of the most convenient, least problematic ways to utilise whey [Salvatore et al., 2014]. This cheese has a soft, fine texture and a slightly caramel flavour [Wendorff, 2008]. Salvatore et al. [2014] showed that the use of whey concentrate in the production of ricotta cheese increases the productivity and the recovery rate of α-lactalbumin protein. Myzithra, a  traditional Greek cheese with a  fat content of  19–25%, is  used as a  table cheese and  as an ingredient

Whey and Whey Preparations in the Food Industry

of many dishes. It is produced in a partially dehydrated form as grated cheese, the so-called “dry myzithra” [Philippopoulos & Papadakis, 2001]. It is recommended to filter the whey before production to remove any residues [Wendorff, 2008]. In  the  case of  mysost and  gjetost cheeses, lactose plays a key role (Maillard reaction). Mysost is produced from sweet whey from cows’ milk, while gjetost is based on sweet sheep’s whey. The production process of the aforementioned cheeses is  very unusual, because the  whey is  subjected to a  process of  condensation; thus, a  brown concentrate is  obtained. It is then heated to a temperature of approx. 95°C, until it obtains the desired colour and intense caramel flavour. The final product contains approx. 33% of fat and 40% of lactose [Wendorff, 2008]. OTHER USES OF WHEY IN THE FOOD INDUSTRY Other uses of whey in the food industry include the production of infant formulas and whey drinks. Whey preparations, being a  source of  high quality protein and  of  active peptides, are widely used by  manufacturers of  baby foods [Chung & Yamini, 2012; Lloyd, 2002; Murphy et al., 2015]. It  is  a  standard procedure to establish an appropriate ratio of whey proteins to casein, which in the case of whey-based supplements should reach 60:40, i.e. the  same ratio that is found in breast milk (in cow’s milk the ratio is 20:80). WPCs and demineralised whey powder are mainly used in this case [De Wit, 2001; Lloyd, 2002]. Also important is the increased amount of amino acids in infant formulas (they are of particular importance in the nutrition of infants born prematurely). It  is  especially true about lysine, methionine and  threonine. The essential amino acids also include phenylalanine, the percentage of which is lower in supplements based on whey than in human milk. It is very important for infants suffering from phenylketonuria [De Wit, 2001]. Whey preparations are also used as media in the microencapsulation of sensitive food ingredients which are fragrances, dyes, or various types of probiotic bacteria (e.g. Bifidobacterium-BB-12). The  use of  whey protein supports protection of  the  above-mentioned active ingredients and  prevents the  loss of  their properties in  the  long-term. Additionally, after the  microencapsulation process, a  product is  obtained in the form of powder or granules, which allows for controlled release of  the  component and  new uses for food additives [López-Rubio & Lagaron 2012; Pinto et al., 2015]. Another protective use of  whey proteins is  as an edible coating for food [Galus & Kadzińska, 2016; Frenzel & Steffen-Heins, 2015; Pintado et  al., 2009]. According to many authors, coating based on whey preparations is  characterised by  good mechanical properties, a  good barrier against lipids, aromatics and, especially, oxygen. Fruit and  vegetables can be coated successfully with whey protein [Galus & Kadzińska, 2016; Seydim & Sarikus, 2006]. Production of alcoholic beverages based on whey seems to be an interesting application of whey. These products include: whey beer, wine, and sparkling wine called whey “champagne”. They are characterised by a low alcohol content (≤1.5%). They are manufactured mostly from whey permeate (characterised by a low content of protein) with the addition of yeast strains Kluyvero-

163

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myces fragilis or Saccharomyces lactis. Addition of sweeteners and flavours is optional [Jeličić et al., 2008; Wendorff, 2008]. According to Wendorff [2008], whey is the ideal raw material for beer production, mainly due to the high mineral content (as in the case of broth) and lactose content, which, as a result of the Maillard reaction, affects the colour and the flavour of the final product (as in the case of roasted malt). However, as Jeličić et al. [2008] declare, the fat content in whey might be problematic, as fat has a destructive effect on beer foam. In addition to alcoholic beverages, there are also soft whey drinks [Smithers, 2008; Baldissera et al., 2011]. Fruit concentrates based on citrus fruits, mangoes, bananas, strawberries and others are used in their production. It is possible to add cocoa, vanilla, honey (which can be a substitute for other sweeteners) or bran (which enriches the finished product with dietary fibre). Powdered whey drinks (instant), which may be enriched with vitamins and  minerals are a  large group [Jelčić et  al., 2008]. Other products popular among consumers include RTD (Ready To Drink) beverages, manufactured on the basis of whey protein isolates and concentrates [Rittmanic, 2006]. CONCLUSIONS In recent years there has been a growing interest in the use of whey and whey preparations in food production. The practical application of whey proteins is due to their high nutritional value, excellent functional properties and the absence of negative taste. Optimal utilisation of whey ingredients has a significant impact on reducing the production cost of many food products. Such a solution contributes in consequence to greater profitability in production and to lower environmental risk. Whey proteins, thanks to a better knowledge of their physicochemical and biological properties, are currently conceived as main nutrients in food products and physiologically-active substances in novel foods. Whey preparations which are used in  the  food industry are: sweet and acid whey powder, demineralised whey powder, whey with reduced lactose content, and  whey protein concentrates and isolate. As a result of the application of appropriate technology, lactose, minerals, as well as whey proteins and their fractions (lactoferrin or lactoperoxidase) can be recovered from whey. Whey preparations are also used as media in  microencapsulation of  sensitive food ingredients (fragrances, dyes, various types of probiotic bacteria) and as edible coatings of foods. Products whose production involves the  above-mentioned preparations include: sausages, hams, pastries, bakery and  confectionery products, fermented dairy products, cheeses, low-fat products, and infant formulas. The functional properties of  whey proteins are mainly used in  their production; they include: solubility, gelling, emulsifying, foaming and water binding properties. In the manufacture of food products, whey and whey preparations can be used as functional additives or for partial substitution of fat and non-fat constituents. Also, partial replacement of fat by high-protein preparations is uniquely associated with a reduction in the caloric value of the final product. In addition, the improvement of  the  functional properties of  food products, i.e. textural properties, seems to be  a  key issue from a  practical stand-

point. Health characteristics i.e. the  hypocholesterolemic, antifungal, antibacterial, anti-inflammatory and  anti-stress activities of whey proteins are also important. CONFLICT OF INTEREST None declared. REFERENCES 1. Alfaifi M.S., Stathopoulos C.E., Effect of egg yolk substitution by sweet whey protein concentrate (WPC), on physical properties of Gelato ice cream. Int. Food Res. J., 2010, 17, 787–793. 2. Aljewicz M., Cichosz G., Kowalska M., Cheese-like products, analogs of  processed and  ripened cheeses, Zywnosc. Nauka. Technologia. Jakosc, 2011, 5(78), 16–25 (in Polish; English abstract). 3. Anand S., Chenchaiah M., Som Nath K., Whey and  Whey Products. 2013, in: Milk and  Dairy Products in  Human Nutrition: Production, Composition and  Health (eds. Y.W.  Park and G.F.W. Haenlein). John Wiley & Sons, Oxford, pp. 477–492. 4. Baldissera A.C., Della Betta F., Penna A.L.B., Lindner. J.D., Functional Foods: a new frontier for developing whey based protein beverages. Semin.-Cienc. Agrar., 2011, 32, 1497–1511. 5. Bolumar T., Toepfl S., Heinz V., Fat reduction and replacement in dry-cured fermented sausage by using high pressure processing meat as fat replacer and  olive oil. Pol. J.  Food Nutr. Sci., 2015, 65, 175–182. 6. Bouzas J., Whey products and  lactose in  confectionery applications. U.S.  Dairy Export Council, Applications Monograph. Confectionery 1999, pp. 1–12. Available at: [www.thinkusadairy. org/assets/documents/Customer%20Site/C3-Using%20Dairy/ C3.7-Resources%20and%20Insights/03-Application%20and%20 Technical%20Materials/WheyLactConfecApplic_English.pdf]. 7. Božanić R., Barukčić I., Jakopović K.L., Tratnik L., Possibilities of whey utilisation. Austin J. Nutri. Food Sci., 2014, 2, 1036. 8. Burrington K., Whey products in baked goods. U.S. Dairy Export Council, Applications Monographs. Bakery 1999, pp. 1–8. Available at: [http://pdf.thepdfportal.net/PDFFiles/61842.pdf]. 9. Bylund G., Dairy Processing Handbook. Tetra Pak Processing Systems. 2003. 10. Chatterton D.E.W., Smithers G., Roupas P., Brodkorb A., Bioactivity of β-lactoglobulin and α-lactalbumin-Technological implications for processing. Int. Dairy J., 2006, 16, 1229–1240. 11. Chung C.S., Yamini, S., FDA’s Health Claim Review: Whey-protein partially hydrolyzed infant formula and  atopic dermatitis. Pediatrics, 2012, 130, E408-E414. 12. Ceglińska A., Pluta A., Skrzypek J., Krawczyk P., Study on the  application of  nanofiltrated whey-derived mineral components in the production of bread. Zywnosc. Nauka. Technologia. Jakosc, 2007, 6(55), 234–241 (in Polish; English abstract). 13. Darewicz M., Iwaniak A., Minkiewicz P., Biologically active peptides derived from milk proteins. Med. Wet., 2014, 70, 348–352 (in Polish; English abstract). 14. De Wit J.N., 2001, Lecturer’s handbook on whey and whey products. European Whey Products Association. Brussels, Belgium. Available at: [http://ewpa.euromilk.org/publications.html]. 15. Ellander A., Harika R.K., Zock P.L., Intake and  sources of  dietary fatty acids in Europe: Are current population intakes of fats

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Whey and Whey Preparations in the Food Industry

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Pol. J. Food Nutr. Sci., 2016, Vol. 66, No. 3, pp. 167–171 DOI: 10.1515/pjfns-2015-0036 http://journal.pan.olsztyn.pl Original article Section: Food Quality and Functionality

Antioxidant Capacity of a Turkish Traditional Alcoholic Drink, Raki Gorkem Yalcin* Department of Analytical Chemistry, Faculty of Pharmacy, Ege University, 35100, Bornova, Izmir, Turkey Key words: raki, beverage, spirit, antioxidant capacity Raki is an aniseed flavoured traditional Turkish alcoholic drink. Antioxidant capacity of raki samples from different commercial brands were evaluated by CUPRAC, DPPH, TEAC and ORAC assays and correlations between these assays and total phenolic content were also investigated. Additionally, the one-way ANOVA and Bonferroni tests were performed to compare differences between values of the samples. Results indicated that different raki samples exhibited different antioxidant capacity and total phenolic content. The mean antioxidant capacity values of samples were in the order of: ORAC>TEAC>CUPRAC>DPPH. The correlations of total phenolic content of samples with their CUPRAC, TEAC and ORAC results were found statistically significant, while DPPH assay showed no significant correlation.

INTRODUCTION Raki is  an aniseed flavoured distilled spirit that is  widely consumed in  Turkey. It  is  often served with seafoods or meze. Raki is described as a spirit that is produced by double distillation of  suma or suma mixed with agricultural based ethanol and  flavouring it  with aniseed (Pimpinella anisum) in the Turkish Food Codex [Anli & Bayram, 2010]. The main raw material of raki is suma, which is a distillate with a maximum 94.5% ethanol content. Raisins, molasses and/or grape must are used for suma production [Yucesoy & Ozen, 2013]. In  the  raki production process, traditional copper alembics with a maximum capacity of 5000 L are used for distillation of suma. After the distillation, distillate is diluted to 45% alcohol. Finally, sugar is added to end product in order to sweeten raki and it is stored for ageing at least one month before bottling [Anli & Bayram, 2010]. Many in  vitro assays have been conducted to evaluate the  antioxidant capacity of  food products and  drinks [Pellegrini et al., 2003; Li et al., 2005; Zulueta et al., 2007, 2009a; Schwarz et al., 2009]. However, determination of antioxidant capacity of  a  particular sample cannot be  performed accurately by any single assay [Ozyurek et al., 2011; Bernaert et al., 2012]. Consequently, at least two assays should be used in order to assess antioxidant capacity accurately [Li et al., 2011; Meng et al., 2011]. Plants are an important source of  natural antioxidants. It  was reported that aniseed had strong antioxidant activity [Gülçın et al., 2003]. Fu et al. [2011] demonstrated antioxidant capacity of  grape samples. The  use of  grapes and/or *  Corresponding Author: Tel.: +90 232 3113992; Fax: +90 232 3885258; E-mail: [email protected], [email protected]

grape based material, aniseed to produce raki may contribute to the ingestion of naturally occurring antioxidant compounds. However, the  antioxidant properties of  raki have not been reported elsewhere. Therefore, antioxidant capacity of raki samples from different commercial brands were analysed in this research and four different assays were tested for antioxidant capacity determination to provide a more reliable investigation. In addition, total phenolic content was also analysed to evaluate correlations between total phenolic content and antioxidant capacity. MATERIAL AND METHODS Samples Seven different commonly consumed raki samples of different brands were purchased from local supermarkets and stored in their original bottles at 4°C. Samples were classified and coded according to their raw materials used in the production: Raki samples produced from dried type grapes (A, B, E, F), fresh type grapes (C, G) and fresh-dried type grapes (D, which is a combination of fresh and dried type grapes). Antioxidant capacity assays The  DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging capacity was determined using the method described by  Tafulo et  al. [2010] with some modifications. Added amount of sample and DPPH reagent, reaction time modifications were applied to the method. Briefly, 0.25 mL of samples were mixed with 2.75 mL of 0.1 mmol/L DPPH. The absorbance at 517  nm was determined after reaction time of 30 min. The standard curve was constructed using Trolox (0.02–0.10 mmol/L) and the results were expressed as µmol/L Trolox equivalent (TE).

© Copyright by Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences ©2  016 Author(s). This is an open access article licensed under the Creative Commons Attribution-NonCommercial-NoDerivs License (http://creativecommons.org/licenses/by-nc-nd/3.0/).

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Antioxidant Capacity of a Turkish Traditional Alcoholic Drink, Raki

CUPRAC (cupric reducing antioxidant capacity) assay was carried out by  using method described by  Apak et  al. [2008] with slight modification. Added amounts of reagents were modified in  the  method. In  brief, 0.75  mL copper (II) chloride (10  mmol/L), 0.75  mL neocuproine (7.5  mmol/L), 0.75  mL ammonium acetate buffer (1  mol/L, pH=7.0), 0.75 mL water and 0.1 mL sample were mixed in a cell. After 30 min, the absorbance at 450 nm was measured. Trolox (0.05–0.50  mmol/L) was used as a  reference standard. Results were expressed as µmol/L TE. For TEAC (trolox equivalent antioxidant capacity) assay, method described by Re et al. [1999] was used with some modifications. Added amount of sample and ABTS reagent, reaction time modifications were applied to the  method. ABTS radical cation (ABTS•+) stock solution was prepared from 7  mmol/L ABTS and  2.45  mmol/L potassium persulphate solutions. The  working solution was prepared by  diluting the  stock solution with phosphate buffer solution (75  mmol/L, pH=7.2) to an absorbance of  0.700±0.003  at 734 nm. Then, 2 mL of ABTS•+ working solution were mixed with 0.1  mL sample. The  absorbance was read at 734  nm, 15 min after the reaction started. A standard curve was obtained by using Trolox (0.005–0.05 mmol/L) and results were expressed as µmol/L TE. ORAC (oxygen radical absorbance capacity) assay was conducted as described by Zulueta et al. [2009b] with minor modification which was modification of value of CTrolox used in  the  following equation. Briefly, 0.25  mL of  samples were mixed with 1.5  mL of  4  nmol/L fluorescein. This solution mixture was incubated at 37°C for 15 min. After this period, 0.25  mL of  160  mmol/L AAPH (2,2’-azobis(2-methylpropionamidine)) was added to start reaction and  fluorescence intensity was read immediately until the fluorescence intensity became less than 5% of the value of the initial reading. Subsequent readings were made every 2 min at the wavelengths of  excitation and  emission of  493  and  515  nm, respectively. ORAC values were calculated by applying the following equation and the results were expressed as µmol/L TE. ORAC=[CTrolox x ( AUCSample – AUCBlank) x k] / / (AUCTrolox – AUCBlank) where CTrolox is  the  Trolox concentration (0.3  µmol/L), k  is  the  sample dilution factor, and  AUC is  the  area under the fluorescence decay curve. The area under the curve (AUC) was calculated according to the following equation: AUC=(f0 – fn+2) + 2(f2+f4+f6+……….+fn+2) where f0 is the initial relative fluorescence intensity at 0 min and f2, f4, f6, …….. fn+2 are the relative fluorescence intensities at times, 2, 4, 6, and n+2 min. Determination of total phenolic content Total phenolic contents (TPC) were determined according to the Fu et al. [2011] with slight modification which was modification of added amount of Folin-Ciocalteu reagent. Briefly, 0.5 mL of the sample was added into 1.0 mL of 1:10 diluted Folin-Ciocalteu reagent. After 4 min, 1.0 mL of saturated so-

dium carbonate solution (about 75 g/L) was added. This solution mixture was then incubated for 2 h at room temperature. After incubation, the  absorbance was measured at 760  nm. A  standard curve was obtained by  using gallic acid (0.02– –0.40 mmol/L) and results were expressed as milligram of gallic acid equivalent per one liter of raki sample (mg GAE/L). Limit of  detection (LOD) and  limit of  quantification (LOQ) LOD and  LOQ were evaluated on the  standard deviation of the response of the blank and the slope using the ratio 3.3σ/S and 10σ/S, respectively, where σ is the standard deviation of the response of 10 blank samples and S is the slope of the calibration curve of the analyte. Statistical analysis All values were analysed by  GraphPad Prism 5  and  Microsoft Excel Software. Three independent aliquots of  the  sample were measured and  all measurements were replicated three times for each sample. The  data were expressed as means±standard deviation (SD). The  one-way ANOVA and Bonferroni tests were used to determine differences among means and  the  differences were considered as significant with pDPPH.  Tafulo et  al. [2010] investigated the  antioxidant capacity of  different beer samples with the  aid of  TRAP (total radical trapping antioxidant parameter), TEAC, DPPH, FRAP (ferric-ion reducing antioxidant parameter), CUPRAC and  ORAC assays. Like results found in the present study, they obtained the highest antioxidant capacity value by ORAC assay, while DPPH assay displayed the lowest value. As can be seen from Table 3, the  mean values of  CUPRAC and  TEAC methods are very close to each other, 113.12  and  119.09  µmol/L TE, respectively. This could be due to the fact that CUPRAC and TEAC are similar antioxidant capacity assays with close reduction potentials. The  correlations between antioxidant capacity and  TPC of samples are evaluated in the present study. The correlation of  TPC of  samples with their CUPRAC, TEAC and  ORAC (0.8071, 0.8196 and 0.7998, respectively) antioxidant capacities were found statistically significant. These significant correlations indicated that phenolic compounds could be  one

TABLE 2. Bonferroni post-hoc test results. Samples

CUPRAC

DPPH

TEAC

ORAC

TPC

A vs B

ns

ns

*

*

*

A vs C

ns

*

ns

*

*

A vs D

*

ns

ns

*

ns

A vs E

*

ns

ns

ns

ns

A vs F

*

ns

*

*

ns

A vs G

ns

*

*

*

*

B vs C

ns

ns

ns

*

ns

B vs D

*

ns

ns

*

*

B vs E

*

ns

*

*

*

B vs F

*

ns

*

*

*

B vs G

ns

*

ns

*

*

C vs D

*

ns

ns

ns

*

C vs E

*

*

*

*

*

C vs F

*

*

*

*

*

C vs G

*

ns

ns

ns

*

D vs E

ns

*

*

*

*

D vs F

*

ns

*

*

*

D vs G

ns

ns

ns

ns

ns

E vs F

ns

ns

ns

ns

ns

E vs G

*

*

*

*

*

F vs G

*

*

*

*

*

*Indicates significant differences at p