Evaluation of the Physicochemical Parameters of Functional Whey Beverages

Whey is a by-product of the manufacture of cheese or curd. It has been used in the food industry for about 20 years; however, interest in the use of whey has recently increased owing to its high nutritional and functional properties. Whey contains a large quantity of nutritionally rich components (1). Biologically active whey proteins, minerals, water-soluble vitamins and lactose remain in whey a er processing (2). Many easily digestible bioactive components derived from whey are under study for their ability to off er specifi c health benefi ts (3,4).


Introduction
Whey is a by-product of the manufacture of cheese or curd.It has been used in the food industry for about 20 years; however, interest in the use of whey has recently increased owing to its high nutritional and functional properties.Whey contains a large quantity of nutritionally rich components (1).Biologically active whey proteins, minerals, water-soluble vitamins and lactose remain in whey a er processing (2).Many easily digestible bioactive components derived from whey are under study for their ability to off er specifi c health benefi ts (3,4).
Whey proteins are of special importance.They represent about 20 % of milk proteins and are characterised by a high digestibility, amino acid score and contain high percentage of branched-chain amino acids (BCAAs), such as leucine, isoleucine and valine.High amounts of these amino acids have long been popular in the industry as a muscle-building supplement for athletes during intensive exercise.Consequently, whey proteins provide excellent nutritional value in foods formulated for kids, adults and elderly people, or as supplements for body health maintenance.β-Lactoglobulin (β-Lg) and α-lactalbumin (α-La) are of the greatest importance and compose more than a half of total whey proteins (50 % β-Lg and 12 % α-La); the rest are: immunoglobulins 10 %, serum albumin 5 % and proteose peptones 0.23 %, lactoferrin (LF) and lactoperoxydase (LP) (1).
ISSN 1330-9862 scientifi c note doi: 10.17113/ b.53.01.15.3763 Whey proteins as functional ingredients refl ect a wide range of potential therapeutic applications.Individual whey protein components and their peptide fragments stimulate the immune system, anticarcinogenic activity and other metabolic features (5).Graf et al. (6) identifi ed 25 intervention trials examining chronic and/or acute eff ects of whey protein supplementation on lipid and glucose metabolism, blood pressure, vascular function and on the musculoskeletal system.However, the evaluation of the effi cacy of bioactive proteins/peptides in animal model and human clinical studies is needed to fully substantiate their role (7).
The nutritional and functional properties of whey are of great signifi cance for the formulation of food products.Whey and whey-derived products, apart from being nutritional ingredients in various foods, can also be used as functional ingredients providing fl avour, texture, colour and aeration properties in a variety of foods (1).Whey proteins can be used as supplements as well as food ingredients due to their unique eff ect on food texture (8,9).Their concentrates are used in the processing of meat and milk products.Soluble whey proteins are used in the manufacture of dietary products.Acid whey is useful for making milk desserts and other confectionary products.Hydrolysed condensed whey is important in the industries of cheese (rico a, mizithra), confectionary and bread baking, while whey and egg protein concentrate is produced as well (1).Fresh whey is also used as a stock for acidic, hydrolysed and pasteurised drinks (kvass, fi zzy drinks, koumiss, etc.), enriched with diff erent additives (sugar, raisins, vegetable oil, syrups, fruit juices, aromatic substances, stabilisers, milk proteins and ω-3 polyunsaturated fa y acids (PUFA)) which are added before pasteurisation (2).Introducing a hydrolysed whey protein concentrate with antihypertensive peptides into beverages appears to create an excellent link between food science and therapeutic nutrition (7).
The physiological function of PUFA is related to heart and vascular diseases.The ω-3 fa y acids are known to be essential for proper development and functioning of brain, retina and nervous system (10).Due to the low current intake levels of ω-3 fa y acids through diet, there is a need for fortifi ed products that can deliver substantial amounts of ω-3 fa y acids (11).
Flaxseed oil is rich in unsaturated fa y acids, recognized as one of the greatest sources of ω-3 fa y acids in nature and having a positive eff ect on human health.Their role in reducing the risk of cardiovascular diseases has been increasingly recognized (12,13).As only three of the saturated fa y acids (SFA) are in fact hypercholesteremic, the PUFA/SFA ratio is not a particularly suitable measure of the atherogenicity or thrombogenicity of the foods (14).The ratio of oleic to palmitic acid (monounsaturated fa y acids (MUFA/SFA) in dietary fats has a regulatory infl uence on certain thrombogenic and fi brinolytic markers during the postprandial state in healthy subjects (15).Consequently, lipid quality indicators that depend on the relative contents of particular groups of fa y acids are atherogenic (AI) and thrombogenic (TI) indices, indicating the global dietetic quality of lipids.
In order to increase the assortment of functional beverages, whey can be used as a source of proteins, and fl ax-seed oil as a source of ω-3 fa y acids.Thus, the aim of this study is to create a functional whey beverage enriched with ω-3 fa y acids and to evaluate its quality.

Materials
Whey beverages containing cold-pressed fl axseed oil with dominant ω-3 α-linolenic fa y acid (C18:3n-3) were used in the experiment.The production of beverages from fresh sour whey, obtained in the manufacture of curd, was done at the technological laboratory of the Food Institute at Kaunas University of Technology, Lithuania, and was approved by milk enterprises: JSC Rivona (Vilnius, Lithuania) and JSC Venitas (Vilnius, Lithuania).
The model system of whey and fl axseed oil was made by enrichment of the whey with 0.2 % of fl axseed oil (JSC Aletovis, Šakiai, Lithuania) containing high amounts of α-linolenic (58 %), linoleic (14 %) and oleic (19 %) fa y acids and by the addition of a stabiliser No 5834 (Palsgaard Industry A/S, Juelsminde, Denmark) consisting of cellulose and pectin in equal parts.The stabiliser was used to prevent precipitation of whey protein in beverages during their thermal treatment.Before its introduction into beverages, the dry mix of stabiliser and sugar in a ratio of 1:10 was dissolved in hot water ((70±5) °C).The content of sugar in fi nal beverages was 7.0 %.The processed beverages, pasteurised at (85±2) °C and cooled to (10±2) °C, were fi lled into 0.5-litre clear glass bo les and stored in refrigerator at (6±1) °C for 30 days for further analyses.

Methods
The sensory parameters (colour, odour and taste) of the prepared whey beverages were evaluated by a panel of eight judges following a fi ve-point hedonic scale (where 1 point means bad, 2 satisfactory, 3 good, 4 very good and 5 excellent) on the 1st, 14th and 30th day of storage.The panellists were selected and instructed according to ISO 8586:2012 (16).The samples were served to the panellists in one session.The acceptability of the beverages was rated based on a scale of points ranging from 'like extremely' to 'dislike extremely' according to LST ISO 4121:2004 (17 Viscosity was analysed with Höppler viscometer KF10 (RheoTec Messtechnik GmbH, O endorf-Okrilla, Germa-ny).The mixture was emulsifi ed by using laboratory mixer (IKA, Labortechnik, Staufen, Germany), at the revolution of 225 s -1 and treatment time of 5 min.
Sedimentation of prepared beverages was monitored according to Towler (21).It was done by centrifugation at 3000×g for 20 min at ambient temperature.Sediment was calculated as a percentage of the total fl uid mass according to the following equation: Sediment in tube= (sediment a er 24 h/fl uid total mass)•100 /1/ The mass fraction of fa y acids in processed whey beverages with fl axseed oil was determined with gas chromatography method, using a fl ame ionization detector.Samples for analysis were prepared according to the standard LST EN ISO 12966-2:2011 (22).Fa y acids were methylated using anhydrous KOH methanol solution.Chromatographic analysis of fa y acid methyl esters was performed using a gas chromatograph Shimadzu The analytical conditions were: column temperature of 60 °C for 2 min, then 13 °C/min to 230 °C, maintaining the temperature for 45 min, carrier gas was nitrogen, injector temperature was held at 250 °C, and fl ame ionization detector temperature was held at 270 °C.
For the identifi cation of fa y acids, 47885-U Supelco ® 37 Component FAME Mix (Sigma-Aldrich Co.) was used.The peaks were identifi ed by comparison of their retention times with those of the standard fa y acid methyl esters.The relative fraction of each fa y acid was expressed as the percentage of the sum of total fa y acids.

Statistical analysis
All the tests were repeated three times.Data were processed using descriptive and statistical analysis methods of SPSS statistical package (SPSS Inc., v. 17, IBM Corporation, Armonk, New York, NY, USA).The number of variables (N), arithmetic averages, their standard deviations (S.D.) and the minimum (min) and maximum (max) values were calculated using descriptive analysis.Statistical processing of scores obtained by sensory evaluation, was done using Excel 2007 (Microso Corporation, Redmond, WA, USA) and paired t-test.For all statistical analyses p≤0.05 was considered statistically signifi cant.

Results and Discussion
As stabiliser was used in processing of the whey beverages with added fl axseed oil, the infl uence of stabiliser on the sensory and physicochemical properties of prepared beverages was analysed.Table 1 shows that the impact of stabiliser on the viscosity and taste of the beverages was statistically signifi cant (p<0.05).However, the added mass fraction of the stabiliser had no eff ect on colour, odour and active acidity of the beverages.
The optimal mass fraction of the stabiliser that ensured the stability of whey protein phase during heat treatment and provided the most acceptable taste of beverages was 0.2 %.The viscosity of the beverages a er adding 0.2 % of the stabiliser increased by 3.6 mPa•s compared with a product without a stabiliser.The taste of the beverages with stabiliser was more acceptable than the watery taste of the product without a stabiliser.This mass fraction of the stabiliser also ensured the absence of sedimentation.
The ω-3 fa y acids are susceptible to oxidation due to the double bonds present in the molecular structure.Therefore, it is important to evaluate the quantitative changes of fa y acids of fl axseed oil-enriched beverages during storage.Table 2 shows the quantitative changes of diff erent fa y acids found in whey beverages with fl axseed oil at (6±1) °C.
The main PUFA of the fl axseed oil is α-linolenic fa y acid C18:3.Its determined mass fraction in the fresh oil was 54.8 %.It is known that the increase of human cholesterol levels is infl uenced by the amount of fa y acids, in this case, myristic, palmitic and lauric acids have the highest impact.The data presented in Table 2 show that there were 4.79 % of palmitic acid and 0.04 % of myristic acid detected in fresh fl axseed oil samples; however, lauric acid was not detected.The fresh whey beverages also contained 18.97 % of physiologically benefi cial oleic acid.The levels of ω-6 fa y acids, including linoleic and arachidonic acids, were lower: 16.58 and 0.06 % respectively.The composition of fa y acids showed that PUFAs were predominant and that the ratio of n-6/n-3 was favourable (0.3).
No signifi cant quantitative changes of fa y acids were detected when the beverages were stored for 14 and 30 days at (6±1) °C.Such results could be the consequence of a low storage temperature and especially of the pH of the medium, as lipases are known to be passive in acid medium.
From a nutritional viewpoint, it can be assumed that the prepared beverages are a good source of whey proteins and ω-3 fa y acids (containing more than 70 % of PUFAs) and according to the results, no oxidation took place during 30 days at refrigeration temperature.Similar results were obtained by Gad et al. (25), who analysed the stability of whey and mango beverages at refrigerator temperature for 15 days.Incorporation of fl axseed oil into beverages is particularly a ractive for development of beverages with specifi c health advantages.The fl axseed oil was reported to reduce susceptibility to cardiovascular diseases and hypertension (26).A er the evaluation of fa y acid composition in whey beverages with fl axseed oil and calculation of AI and TI indices, it was found that AI and TI values were low, which indicates high quantities of fa y acids with antiatherogenic properties in the oil.These indices demonstrate the relationship between fa y acids in food and their contribution to the prevention of coronary diseases (27).Atherogenic index shows the relationship between the sum of the main saturated and that of the main unsaturated fa y acids, the former being considered proatherogenic (favouring the adhesion of lipids to cells of the immunological and circulatory systems), and the latter antiatherogenic (inhibiting the aggregation of plaque and diminishing the levels of esterifi ed fa y acids, cholesterol and phospholipids by preventing the appearance of micro-and macrocoronary diseases).Thrombogenic index indicates the tendency to form clots in the blood vessels.This is defi ned as the relationship between the fa y acids with prothrombogenic (saturated) and antithrombogenic (MUFAs, n-6 PUFAs and n-3 PUFAs) properties (14,28).However, in our case, the atherogenic and thrombogenic indices of prepared whey beverages with fl axseed oil were lower than in the muscles (intramuscular lipids) of diff erent domestic and wild animal species (29), in fi sh and fi sh products (30)(31)(32) and in olive oil (14).
The oxidation processes of fa y acids in the beverages and their dependence on the active acidity were analysed as well.The fa y acids were analysed directly a er making of beverages (1st day) and a er storage for 14 and 30 days at (6±1) °C (Table 3).A er storage of the beverages at refrigeration temperature for 30 days, no statistically signifi cant changes were detected in the oxidation indices (anisidine value (AV) and fa y acidity), although the tendency of increase of peroxide value (PV) was observed.A er 30 days of storage, PV reached 1.56 (p<0.05);however, the maximum limit established by the Order of the Minister of Agriculture of Lithuania No 155, 1999 was not exceeded.
A medium positive correlation was found between the AV and PV (R=0.651;p<0.05).With the increased AV, the fa y acidity also increased (R=0.677;p<0.05).A strong positive correlation was determined between the PV and fa y acidity (R=0.900;p<0.05) during 30 days of storage as well.
During storage for 30 days, the amount of PUFA decreased and the PV and AV increased as a result of weak oxidation process.A strong negative correlation was observed between the amount of PUFA and the anisidine value (R=-0.871;p<0.05), peroxide value (R=-0.728;p<0.05) and the fa y acidity (R= -0.948; p<0.05).To summarise, we can conclude that whey beverages containing fl axseed oil remain stable at pH=4.0 and oxidation a er storage for 30 days at (6±1) °C is weak.
Sensory parameters are the fi rst a ributes to judge the acceptability and overall quality of beverages by consumers.It was found that sensory parameters of the beverages remained acceptable during all 30 days of storage: values of the colour did not change and values of the odour and taste slightly decreased.Similar results were obtained in newly prepared whey beverages analysed by Gad et al. (25).The sensory evaluation study suggests that whey beverages with fl axseed oil retain the overall acceptability with good sensory quality and can be successfully stored at refrigeration temperature.

Conclusions
The analysis showed that functional beverages from whey enriched with fl axseed oil could be produced in milk industry.The use of fl axseed oil containing α-li nolenic fa y acid (54.8 % of total fa y acids) and a stabiliser (0.2 %) had the most positive impact on the sensory properties and physicochemical parameters of functional whey beverages at pH=4.0 during 30 days of testing period.Enrichment of beverages with fl axseed oil may provide the required components for optimum health.Whey beverages as a source of products with low atherogenic index could be useful for human health and are especially rec-ommended for the prevention of cardiovascular diseases.The consumption of whey beverages with fl axseed oil as a product rich in n-3 PUFA may considerably increase in human diets nowadays.

Table 1 .
Infl uence of stabiliser on sensory and physicochemical properties of whey beverages with added fl axseed oil Values for pH and viscosity are expressed as mean±S.D.; diff erences between columns: *p<0.05,**p<0.01;NS=no sedimentation

Table 2 .
Quantitative changes of fa y acids in whey beverages with added fl axseed oil during storage at (6±1) °C Values for fa y acid mass fraction are expressed as mean±S.D.; PUFA=polyunsaturated fa y acids, MUFA=monounsaturated fa y acids, AI=atherogenic index, TI=thrombogenic index

Table 3 .
Dependence of the oxidation and sensory parameters of whey beverages with added fl axseed oil on the storage time at (6±1) °C Values for AV, PV and fa y acidity are expressed as mean±S.D.; diff erences between rows: *p<0.05;AV=anisidine value, PV=peroxide value