Int.J.Curr.Microbiol.App.Sci (2016) 5(8): 134-155

International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 5 Number 8 (2016) pp. 134-155 Journal homepage: http://www.ijcmas.com

Review Article http://dx.doi.org/10.20546/ijcmas.2016.508.016

Whey and its Utilization

Silviya R. Macwan*, Bhumika K. Dabhi, S.C. Parmar and K.D. Aparnathi

Dairy Chemistry Department, SMC College of Dairy Science, Anand Agricultural University, Anand- 388001, Gujarat, India *Corresponding author

ABSTRACT

Whey is a by -product of the dairy industry, which for years was thought to be

insignificant and was either used as an animal feed or it was disposed of as waste. K e yw or ds Considering that over 145 million tons of whey is produced worldwide annually, the desire for new methods to utilise whey can be appreciated. Over the last years Whey, several studies were carried out concerning the importance of whey is nutritional nutritional value and the properties of its ingredients. It is now accepted that main content, value, whey whey , have antimicrobial, antiviral and anti-oxidant properties. Due to the utilization . substantial difficulties encountered in the treatment of whey as a biological waste

and its high potential to be valuable raw material for added value food and bioactive substances production the later tend to be the only accepted and popular Article Info trend of dealing with this dairy industry by-product. Productions of whey proteins Accepted: by ultrafiltration, lactose hydrolysis products, and the use of whole whey or whey 12 July 2016 permeate as a fermentation feedstock are possible options. A large number of Available Online: 10 August 2016 workers have carried out studies on the composition and processing of whey for its use in foods and animal feeds besides studying the nutritive, therapeutic and functional properties of whey.

Introduction amount of material with potential value as a Whey is the liquid fraction that remains food or feed is wasted (Aneja et al., 2002). following manufacture of cheese, chhana, Whey is a serious pollutant as it imposes a paneer and casein. The world production of very high BOD of 30,000- 50,000 mg/lit and whey is estimated at about 165 million chemical oxygen demand of 60,000-80,000 tones. Of which cheese whey contributes mg/lit. Discarding of whey also constitutes a about 95 %. In India, the major source of significant loss of potential nutrients and whey is from the production of chhana and energy and has been looked upon seriously paneer. In the absence of systematic by the environmentalists and technologists survey/statistics the estimated production of due to its potent polluting strength. In whey is about 5 million tons per annum addition the dairy industry suffers from an (Gupta, 2008). The utilization was 75% in economic blow due to several treatment Europe and probably less than 50% in the costs that are incurred in proper disposal of rest of the world and as a result a very large whey. Although several possibilities of 134

Int.J.Curr.Microbiol.App.Sci (2016) 5(8): 134-155 cheese whey utilization have been explored, proteolytic enzymes. The type and a major portion of the world cheese whey composition of whey at dairy plants mainly production is discarded as effluent. Its depends upon the processing techniques disposal as waste poses serious pollution used for casein removal from liquid . problems for the surrounding environment The most often encountered type of whey (Marwaha & Kennedy, 1988; Gonzalez- originates from manufacture of cheese or Siso, 1996). certain casein products, where processing is based on coagulating the casein by rennet, Interestingly, over the last few decades, an industrial casein-clotting preparation dairy industries have applied different containing chymosin or other casein- technologies to process cheese whey coagulating enzymes (Fox et al., 2000). resulting in its separation into its principle Rennet-induced coagulation of casein occurs components, comprising fractions enriched at approximately pH 6.5; this type of whey in proteins, lactose and minerals. It was the is referred to as sweet whey (Table 1). The introduction of new separation technologies second type of whey, acid whey (pH < 5), such as ion exchange, electrodialysis and results from processes using fermentation or membrane filtration, which created addition of organic or acids to possibility of producing a wide range of new coagulate the casein, as in the manufacture products based on whey, including of fresh cheese or most industrial casein functional food ingredients with added value (Jelen, 2003). In general, whey produced and products for pharmaceutical and from rennet-coagulated cheeses is low in medical industries, Nielsen, et al., (2002). acidity, while the production of fresh acid Now-a-days it is no longer considered a cheeses such as ricotta or cottage cheese / waste product but the treasure house of paneer or chhana yields medium acid or acid nutritionally rich whey components. Thus its whey. utilization in the human food chain is now being predominantly favoured due to the Whey from cottage cheese manufacture is economic opportunities provided by some of called “Acid whey” since it is produced the milk nutrients present in whey (Jelen, during acid coagulation of milk proteins and 2002a). Productions of whey proteins by has a low pH and consequently longer ultrafiltration, lactose hydrolysis products, storage life than “sweet whey” which is and the use of whole whey or whey produced by enzymatic coagulation of milk permeate as a fermentation feedstock are proteins i.e. during manufacture of cheddar possible options (Marwaha, 1988). A large and other kinds of cheese (Chandra, 1980). number of workers have carried out studies on the composition and processing of whey The classification of whey based on acidity for its use in foods and animal feeds besides and pH is given in Table 2. studying the nutritive, therapeutic and functional properties of whey. In the production of cheese or casein, about 80 to 90 % of original milk used yields Types and Composition of Whey whey as a by-product, which contains about 50 % of the milk constituents (Jayaprakasha, Whey may be defined broadly as the serum 1992). Whey retains about 45-55 % of the or watery part of milk remaining after milk nutrients comprising serum proteins, separation of the curd, which results from lactose, minerals and . The the coagulation of milk proteins by acid or composition of whey varies depending on

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Int.J.Curr.Microbiol.App.Sci (2016) 5(8): 134-155 the type of coagulation. The pH of cheese world production of liquid cheese whey is in whey ranges from 5.9 to 6.4 and that of the region of 145 million tons (Affertsholt& chhana, paneer and Shrikhand whey Nielsen, 2003). Of this 85 million tons can between 5.3 and 5.6. The Total Solids (TS), be considered to be industrially utilised and fat and content of cheese whey is processed into value added products. The more than that of acid whey, but there is remaining 60 million tons or so is used higher amount of calcium, phosphorus and directly in liquid animal feeds, sprayed on lactic acid in acid whey as compared to fields as fertilizer or just dumped. sweet whey. The typical composition of Industrially processed fraction, about 50 sweet whey and acid whey is in Table: 3. million tons is processed either into edible lactose or dried directly as cheese whey The main components of both sweet and powder, approximately 30 million tons is acid whey, after water, are lactose processed into protein enriched (approximately 70–72% of the total solids), concentrates, referred to as whey protein whey proteins (approximately 8–10%) and concentrates for products containing up to minerals (approximately 12–15%) Gupta 80% protein and whey protein isolates for (2000) (Table 3). The main differences products containing > 90% protein. A between the two whey types are in the further 6 million tons are processed into so- mineral content, acidity and composition of called demineralised blends (Clark, 2004). the whey protein fraction. The acid Based on the global distribution of whey coagulation approach results in substantially utilisation, approximately 60% of whey is increased acidity (final pH approximately industrially utilised, this is built up from 80- 4.5), necessary for casein precipitation. At 90% utilisation in the US and Australasia this low pH, the colloidal calcium contained and only 60% utilisation in Europe. in the casein micelles in normal milk is Industrial processing of whey originating solubilised and partitioned into the whey. from the rest of the world is still at Rennet clotting produces a fragment k- comparatively low levels. In the absence of casein molecule, termed glycomacropeptide systematic data, the predicted value for (GMP), which ends up in whey. GMP whey production in India is 3.0 million constitutes approximately 20% of the whey tonnes per annum (Khamrui & Rajorhia, protein fraction of sweet, rennet-based whey 2000) with major source of whey from the but is not present in acid whey, unless production of chhana and paneer, while renneting is included in the fresh cheese about 50,000 tonnes of whey is made manufacturing process. Other technological available from cheese industry. steps used in the pre-treatment of milk before the main processes may also The United States of America produces influence the composition of whey such as about 5-8 billion pounds (2-6 million that from paneer/chhana resulting in acid tonnes) of acid whey and 45 -I billion whey with very low protein content. pounds (20.5 million tonnes) of sweet whey annually (Clark, 1987). There has been a World Whey Production steady increase in the production of whey powder and lactose over the past three years. Dairy industries all over the world generate Trends in the production of concentrated large amounts of whey per litre of milk whey, whey protein concentrate, reduced processed, depending upon the processes lactose/reduced mineral whey and whey employed, products manufactured. The total blends are variable for this period. Current

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Australian and New Zealand whey not been fully defined. However, whey production is about 3634 and 5874 million protein reduces short-term food intake pounds (1-65 million and 2-67 million relative to carbohydrate and other proteins. tonnes), respectively (Zadow, 1987). On the world scene, the current estimate of whey Whey protein is insulinotropic and whey- production amounts to about 224 billion born peptides affect the renin-angiotensin pounds (102 million tonnes) (Hoogstraten, system. Whey ingestion activates many 1987a). components of the food intake regulatory system. Therefore whey protein has Nutritional Value of Whey potential as physiologically functional food component for persons with obesity and its Whey is a rich source of nutritious co-morbidities (hypertension, type II components and its biological components diabetes, hyper- and dislipidemia). has proven its effects in treatments of several chronic diseases like cancer, It remains unclear, however, if the favorable cardiovascular, HIV etc. As it is effects of whey on food intake, subjective nutritionally too rich it can also be used in satiety and intake regulatory mechanisms in infant, Geriatric and Athletic food. humans are obtained from usual serving sizes of dairy products. The effects Research has been unequivocally established described have been observed in short-term the excellent nutritional and functional experiments and when whey is consumed in properties of whey solids, especially the much higher amounts (Luhovy et al., 2007). whey proteins (Jelen, 1992, Patel et al., 1992). As a first class protein, whey protein Whey proteins have been reported to have is next only to the egg protein in terms of one or more biological functions in food. nutritive value, the biological value, protein These biological properties have efficiency ratio (PER) and net protein significance in the and health of utilization of egg protein and whey protein people. The health and nutritional value of being 104 & 100, 3.6 & 3.8 and 92 & 94 the components of whey include 1) high respectively. Whey proteins are rich in quality nutritional source of amino acids; 2) essential amino acids such as lysine, anti-microbial action; 3) growth methionine and cystine (Renner, 1990). enhancement of beneficial gut microflora, such as Bifidobacteria; 4) immune- Whey protein has potential as a functional enhancing properties; 5) control of specific food component to contribute to the diseases, including cancer; and 6) antitoxin activity. regulation of body weight by providing satiety signals that affect both short-term Whey proteins and long-term food intake regulation (Khamrui and Rajorhia, 1998). Because Whey protein are two types major proteins whey is an inexpensive source of high and minor proteins, major whey proteins are nutritional quality protein, the utilization of Beta-lactoglobulin-65%, Alpha-lactalbumin- whey as a physiologically functional food 25%, serum albumin-8% and minor ingredient for weight management is of proteins/peptides are Glycomacropeptide current interest. At present, the role of (GMP), Bovine serum albumin, lactoferrin, individual whey proteins and peptides in Immunoglobulin, Phospho lipoproteins. contributing to food intake regulation has Whey proteins have a Biological Value

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(BV), of 110, which is higher than the value was used to treat some illnesses, such as for casein, soy protein, beef, or wheat gluten tuberculosis and skin and digestive tract and have a high content of -containing diseases, since the time of Ancient Greece. amino acids such as cysteine and methionine In the 18th century there were specialized (Fox and MacSweeney, 1998). institutions built for curing illnesses with whey which designated the start of detailed Lactose studies of nutritional and therapeutic properties of whey. These so called “whey Lactose, the major component of whey, is cures “were usual in countries like probably the least valuable component and , and at that most difficult to utilize. Lactose comprises time. Whey was also successfully applied about 70% of the total solids of whey (Jelen, for treatments of diarrhea, bile illness, skin 1992). problems, scales in the urinary tract and some intoxication. Minerals Due to high amount of proteins with high Whey is a good source of electrolytes nutritional value these beverages are ideal including sodium and potassium, which are source of energy and nutrients for athletes. required during recovering from diarrhea. Whey proteins are a rich source of branched Minerals such as calcium, , and chain amino acids (BCAA) like isoleucine, phosphorus are present in solution and also leucine and valine. BCAAs unlike other partly bound to proteins. is present in essential amino acids are metabolized trace amounts (Zadow, 1992). Lactose also directly into the muscle tissue and are first promotes absorption of Mg and Zinc ions, amino acids used during periods of exercise which even in trace amount helps in better and resistance trainings. (Sherwood et al., diarrheal management (Ziegler and Fomon, 2007). 1983). Whey protein fractions include also Vitamins lactoferrin - an -binding protein, Glycomacropeptide (GMP) which derives During the manufacturing process, the after cheese making using rennet and is water-soluble vitamins are transferred into naturally free of phenylalanine and alpha- whey in a varying extent: 40-70% of lactalbumin which is a calcium-binding B12; 55-75% of vitamin B6 and pantothenic protein. That way, due to presence of acid; 70-80% of riboflavin and biotin; 80- lactoferrin whey beverages can be used as 90% of thiamine, nicotinic acid, folic acid functional food intended to improve iron and ascorbic acid. In the case of vitamin absorption from food and/or help to keep B12, more of it was transferred into the pathogens from attaching to the intestinal whey when a rennet coagulation rather than walls. That is very important for nutrition of acid coagulation was used (Zadow, 1992). little children and babies. Furthermore, these beverages may improve absorption of Therapeutic Value of Whey calcium what is very important for older population which is often suffering from Whey based beverages target a large scale of osteoporosis. Besides that, a drink with consumers - from old people to little addition of GMP isolate would be a very children. Because of its health benefits, it good source of energy and micronutrients

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Int.J.Curr.Microbiol.App.Sci (2016) 5(8): 134-155 for those suffering from phenylketonuria possesses the vitamins necessary for its (Miller, 2005). utilization. It contains vitamins A, B1, B2, B3, B5, B6, C, D, and E (Onwulata and Whey possesses potent antioxidant activity Huth, 2008). mainly contributed by cysteine-rich proteins that aid in the synthesis of glutathione Utilization of Whey (GSH), a potent intracellular antioxidant, also investigated as an anti aging agent. In United States of America, only about Detoxifying property contributed by Glu- 50% of the total whey production is “dried tathione peroxidase (GSHPx), which is or further processed into whey protein derived from and cysteine, that concentrate, lactose and other products for converts lipid peroxides into less harmful use in formulating human food and animal hydroxy acids and α-lactalbumin, which feed products (Jelen& Le Maguer, 1976; chelates heavy metalsand reduces oxidative Teixeira et al., 1983a; Morr, 1984; Clark, stress because of its iron-chelating 1987). The remainder of the whey, properties. especially from small cheese manufacturing plants, is treated by private or municipal Immunoglobulins and lysozyme in whey waste treatment, fed to livestock or spread provides immunity enhancing benefits to on agricultural land as fertilizer. Although a infants and others. Antihypertensive large percentage of the whey produced in peptides isolated from bovine beta- The is reportedly being lactoglobulin, reduces blood pressure. The processed into products other than whey amino acid precursors to glutathione in powder, Europe in general utilizes only whey might increase glutathione concen- about 50% of its whey production, mainly as tration in relevant tissues, stimulate whey powder (Hoogstraten, 1987b). immunity and detoxify potential carcinogens.α-lactalbumin, high in Processing of whey is limited due to less tryptophan, an ; demand for whey powder and further provides potential benefits include sleep processed whey products, plus the generally regulation and mood improvement under unfavourable economics of whey stress. processing, especially for the smaller sized processing operation (Jelen& Le Maguer, Various minerals like, Potassium, involved 1976). The whey processing industry has in the transmission of nerve impulses and expended considerable effort to improve the muscular contractions; Magnesium, utilization of whey powder, lactose, depolarizes the nerve or muscle causing demineralized whey, whey protein relaxation and help to lower blood pressure concentrate, whey blends and other and supports an alkaline tissue pH; Calcium, products. However, processes for used by the body to maintain an alkaline manufacturing these whey-based products tissue pH, maintain bone density, cell wall are economically feasible only for the larger integrity and nerve impulses. sized processing operations with an Lactoperoxidase, inhibits the growth of iron adequate supply of consistently high-quality dependent bacteria. Lactoferrin, inhibits the whey, and where there is adequate demand growth of bacteria (including pathogenic for the resulting products. The predominant bacteria) and fungi, also regulates iron driving force favouring whey processing is absorption and bio-availability. Whey also the tremendous cost associated with whey

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Int.J.Curr.Microbiol.App.Sci (2016) 5(8): 134-155 disposal through municipal or plant waste of the 1.65 million tonnes of whey treatment facilities (Delaney, 1981). production in Australia is used for pig feed (Zadow, 1987). An additional 5% of total A substantial portion of whey powder, whey whey production in Australia is utilized as concentrate, reduced lactose/ reduced calf milk replacer. The dairy industry in The mineral whey, whey protein concentrate and Netherlands uses about 15000 tonnes of lactose is used in human food product liquid whey and 120000 tonnes of applications, whereas whey blends are used delactosed whey powder for manufacturing mainly in animal feed products. Whey-based calf milk replacer (Hoogstraten, 1987b). blends are formulated to provide a composition similar to that of non-fat dry Whey Disposal milk. Most of the whey concentrate for human food is used in dairy and bakery Approximately 8.7 kg of whey is produced product applications (Morr, 1984; Clark, per 1 kg of cheese manufactured. Sweet 1987). Major portions of whey are used in whey and acid whey, by-products from the dairy, bakery, blends, prepared mixes and manufacture of cheese, cottage cheese and confectionery applications. Most of the acid industrial casein, are extremely potent whey used in human food product pollutants, containing about 35 000 mg/litre applications is produced as dried and biochemical oxygen demand (BOD) and modified whey. 68000 mg/litre chemical oxygen demand (COD) (Jelen, 1979; Hobman, 1984). Whey Powder Whey may also be used for farmland Whey powder is manufactured by irrigation, where it contributes both water concentrating whey to 40-70% total solids, and nutrients to the soil which can promote and spray or roller drying to a moisture plant growth (Oborn& Piggott, 1968). 3.7 content of less than 5%. It is necessary to thousand L of whey contains minerals convert a high percentage of the lactose to equivalent to 5.9 kg potassium sulphate, 36 its a-monohydrate crystalline form to kg ammonium sulphate, 12 kg minimize water absorption and concomitant superphosphate and 6.8 kg calcium defects during storage. Lactose carbonate. This whey disposal approach crystallization is either completed in the works well for near neutral to slightly concentrate prior to drying or, as is most alkaline soils; in acid soils, the added whey commonly done, a two-stage drier is promotes soil compaction, resulting in employed that allows for crystallization inhibition of propagation of soil prior to final drying. Lactose crystallization microorganisms that biodegrade the added is also promoted in this hygroscopic product nutrients from the whey. Spraying is the by rapidly cooling it to less than 100°C as it preferred method for applying whey to the is removed from the drier. soil since it avoids the formation of stagnant pools that inhibit the ability of the soil Animal Feed microorganisms to biodegrade added nutrients. The amount of whey that can be One of the ways to utilize the whey is in the utilized by spreading on land as fertilizer is preparation of formulations of animal feed. limited to about 45-90 tonnes/acre. About 16 % of the 2.67 million tonnes of Exceeding these limits poses a threat to whey production in New Zealand and 28% environmental pollution from run-off and

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Int.J.Curr.Microbiol.App.Sci (2016) 5(8): 134-155 off-odour production. Whey reportedly the manufacture of whey protein improves soil fertility by providing valuable concentrate, or from the manufacture of nutrients, and also by promoting an open, heat-precipitated whey protein, e.g. porous structure that increases water lactalbumin. Whey UF permeate has great infiltration (Gillies, 1974). potential as source material for: (Delaney, 1981; Teixeira et al., 1983b; Chiu & Whey can also be treated by waste disposal Kosikowski, 1985). systems such as outdoor sewage lagoons that utilize aeration and sunlight to assist Hydrolysed Whey and Whey UF microorganisms to decompose protein and Permeate lactose. Waste water containing whey is difficult to treat by conventional processes. Lactose hydrolysis can be catalysed Whey results in a poor settling sludge enzymatically by heating in the presence of following primary and secondary waste strong mineral acids, or by treatment with a water treatment. A waste treatment system cationic ion exchanger in the hydrogen form designed to use primary settling, a two-stage (Jelen, 1979; Zadow, 1984; Harju, 1987). packed tower trickling filter, a final settling, For acid hydrolysis, it is necessary to lower coagulation and sterilization by chlorination the pH of the lactose solution to 1.2-1.5 and sludge dewatering has been described prior to heating to approximately 140 °C. by Gillies (1974). This trickling filter design Hydrolysis of 50-90% of the lactose was as effective for treating whey as for increases sweetness and prevents lactose average industrial effluents. The acidic crystallization in concentrated whey syrups. reaction of whey had no adverse effect on Hydrolysis increases solubility and the system. In fact, lowering the pH of whey decreases the viscosity of whey and UF improved the ability of the process to permeate syrups. Whey syrups can be remove BOD from the waste effluent. concentrated to 60-70% solids in vacuum evaporators without crystallization Deproteinized Whey and UF Permeate problems. However, lactose and lactose hydrolysis products, glucose and galactose, Deproteinized whey is normally become sticky and hygroscopic in spray manufactured by heating whey to driers making it difficult to dry hydrolysed temperatures in the range of 70-80°C to whey and whey permeate syrups. denature the proteins, followed by Concentrated syrups are thus preferred over acidification, and removal of the flocculants powder forms of lactose-hydrolysed whey by decantation, filtration or centrifugation. and whey permeate, even though problems Whey UF permeate is the major by-product from crystallization of galactose and non- of the UF of whey for manufacturing whey hydrolysed lactose in these products are protein concentrate. Deproteinized whey and sometimes encountered. Crystallization whey UF permeate contain about 90% of the problems are diminished in syrups by total solids of the original whey. These hydrolysing about 75% of the lactose. The solids components, mainly lactose, minerals resulting syrup concentrate is partially and non-protein nitrogen, represent a acidified and heated at relatively mild substantial pollution problem for the temperatures to destroy most industry if not utilized (Hobman, 1984). microorganisms and to dissolve residual, This whey fraction is most often derived as non-hydrolysed lactose without causing the UF permeate by-product resulting from Maillard, non-enzymatic browning. The

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Int.J.Curr.Microbiol.App.Sci (2016) 5(8): 134-155 heated product is partially cooled, seeded First of all, the high water content makes with fine lactose crystals and agitated to fresh whey very susceptible to microbial promote the formation of small crystals that spoilage whereby heat treatments are will not settle during storage. needed. On the other hand, whey proteins are heat sensitive and start to denature at Immobilized enzyme technology has been temperatures above 60 ºC whereby a certain utilized to improve the economics of lactose amount of present proteins precipitate after hydrolysis (Anon., 1982; Teixeira et al., the usual thermal treatment of whey (72 1983b; Sprossler& Plainer, 1983). This ºC/15 - 20 sec.). Therefore a lot of efforts technology uses lactase enzyme are made in studying the implementation of immobilized on porous glass beads or ultrasound or membrane processes like porous plastic beads to hydrolyse lactose in microfiltration instead of thermal treatments. UF permeate. The resulting hydrolysate can Ultrasound can also improve solubility of be used as a sweetener in food products or whey proteins (Rezek-Jambraket al., 2008) as a fermentation substrate for producing and in that case the amount of sediment baker’s yeast. formed during storage of whey beverages can be reduced. Besides that, acidification of One striking example has been reported whey down to pH < 3.9 causes whey where the dairy industry has incorporated proteins to become heat resistant and do not lactose hydrolysis into a multifaceted precipitate even during UHT sterilization processing scheme to modify Cottage cheese treatments (Jelen, 2002b). (acid) whey so that it can be used as an ingredient in ice cream mix (Homer, 1986). Relatively high content of minerals in the dry matter of whey presents the next Utilization of Whey Products problem in whey beverage production because these minerals are responsible for In general whey from any source may be undesired salty-sour taste of whey. This used i.e. sweet whey, sour whey or problem is especially encountered in acid powdered whey, but use of powdered whey whey due to higher amount of lactic acid makes the final product unnecessarily whereas the content of minerals (mostly Ca- expensive. Whey or whey derivatives from phosphates and Ca-lactates) is also higher cheese, casein, paneer etc., can be used for due to better solubility. That causes beverage preparation. Whey can be utilized abundant acidity and appearance of clots in in different ways in food industry, but it is the final product and also formation of mostly being dehydrated to whey powder or higher amounts of sediment during heat used for manufacture of whey protein treatments (Tratnik, 2003). concentrates and isolates of lactose or proteins. Whey beverages are considered as However, despite of all difficulties, fresh thirst quencher, unlike most soft drinks, whey processing has proved to be the most whey drinks are light and refreshing but less economical technological solution. acidic than fruit juices and they are said to Therefore many efforts have been made in be more suitable for health as compared to development of beverages with addition of other drinks (Prendergast, 1985). fruit concentrates in order to produce a drink with acceptable sensory properties Several difficulties occur during the especially flavour (Koffi et al., 2005). These processes of whey beverage production. whey beverages are classified into two

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Int.J.Curr.Microbiol.App.Sci (2016) 5(8): 134-155 categories viz. alcoholic beverages and non- decanting, aging, filtering and bottling alcoholic beverages (Palmer and Marquardt, 1978).

Alcoholic Beverages Patel (2012) had developed the self- carbonated whey beverage using Since lactose is the main constituent (70%) the chhana whey. The beverage was of whey dry matter, whey is a very good prepared by inoculating the Kluveromyces material for production of alcoholic marxianus and Lactobacillus helveticus beverages. Alcoholic whey beverages are MTCC 5462 cultures and addition of divided into beverages with low alcohol (7%) then it was incubated at 25°C till content (≤ 1.5%), whey beer and whey wine. acidity reaches 0.5% L.A. and stored at refrigeration temperature for self- Production of whey beverages with low carbonation. alcohol content includes deproteinizing whey, whey concentration, fermentation of Non-alcoholic Beverages lactose (usually by yeast strains Kluyveromyce sfragilis and Saccharomyces Non-alcoholic beverages can be further lactis) or addition of sucrose until reaching classified into fruit and flavouring based the desired alcohol content (0.5 - 1%), beverages and Milk based beverages. flavoring, sweetening and bootling (Sienkiewicz & Riedel, 1990). Thereby, a (a) Fruits and flavouring based beverages certain amount of lactose is being transformed to lactic acid which gives a Processing of whey to beverages began way refreshing sour taste to the end product, back in the 1970, and one of the oldest whey while the rest ferments to alcohol. Some of beverages is Rivella from Switzerland. It is a noted beverages belonging to this category clear, amber coloured liquid with a are “Milone” obtained by fermentation with refreshing acidic flavour. Until today a large kefir culture and whey sparkling wine scale of different whey beverages has been “Serwovit” produced in Poland. developed, which are produced from native sweet or acid whey, deproteinized whey, Whey beer can be produced with or without fresh diluted whey, fermented whey or addition of malt; it can be fortified with powdered whey. minerals or can contain starch hydrolyzates and vitamins. Some of problems observed in Jayaprakashaet al., (1986) suggested a such type of beverages are loss of beer process for whey beverage using foam, undesirable odour and taste due to low deprotenized and clarified whey from solubility of whey proteins and inability of cheese, Chhana or acid casein. The process beer yeasts to ferment lactose because of consisted of adding 6 to 13 % sugar, 0.02 to presence of milk fat. 0.4 % citric acid and flavour at 0.15 to 0.45 ml per litre of whey followed by in-bottle Whey wine contains relatively low alcohol pasteurization or sterilization with or amount (10-11%) and is mostly flavored without carbonation. Beverage with 10 % with fruit aromas. Production of whey wine sugar and orange flavour received highest includes clearing, deproteinazation, lactose score. hydrolysis by β-galactosidase, decanting and cooling, addition of yeasts and fermentation, Reddy et al., (1987) developed an

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Int.J.Curr.Microbiol.App.Sci (2016) 5(8): 134-155 acceptable beverage, deproteinised cheddar beverage from paneer whey by blending cheese whey, lemon juice (8%) and sugar mango, pineapple, lemon and banana pulp or (14%). The shelf life of the product was up juice. Pulp of 4% and juice content ranged to 15 days at room temperature (18-25 °C) from 5% for lemon to 20% for pineapple without much damage to the organoleptic and banana while the whey content ranged quality. from 73 to 87%. Another soft beverage was also prepared from a mixture of paneer Gagrani et al., (1987) added different fruit whey (3parts) and banarsisurkh variety of juices like orange, pineapple and mango at guava extract (1part), sugar (8%) and lemon the rate of 10%, 15%, and 15% respectively colour. to whey for the manufacture of fruit based beverage. The reported mango based Macedo et al. (1998) manufactured cultured beverage to be superior than others with whey milk beverage using buffalo milk respect to colour, flavour, mouthfeel and cheese whey, cow skim milk and soy milk physical characteristics like sedimentation, with six combinations of Lactobacillus casei turbidity, viscosity etc. subsp. shirota, L.acidophilus, B. longum and B. adolescentis. The result indicated that the Acid whey has been found most suitable for mixture of 35, 35 and 30%, respectively of blending with fruit juices to produce cloudy skimmed cow milk, buffalo cheese whey fruit drinks (Tuohy et al., 1988). A pH of and soy milk was suitable as a low cost less than 4.0 is recommended to prevent substrate for the growth of co-culture L. protein coagulation during pasteurization. casei subsp. Shirota and B.acidolescentis, The pH reduction can be achieved by resulting in a product of good taste and addition of lactic acid or other organic acids. flavor with a high number of these desirable The pectin esterase is inactivated at 85-95 organisms. °C/15-40 sec to prevent loss of cloudiness, which is required in fruit juices such Iniquez and Vera (1999) developed a products possess a shelf life of 2 weeks at formulation for flavoured beverage using refrigeration temperature. buffalo milk whey, and concentrated pineapple juice or orange (4.5 - 5.0%) shelf Gandhi (1989) patented acid whey-lactic life of the beverage was 7-8 days at 4 °C. fermented noncarbonated beverage Shukla et al., (2000) developed a ready to produced from cheese whey or paneer whey, serve beverage from whey by the addition of heated (85 to 90 °C / 20 min) and cooled to 10% sugar and 30% litchi juice. 40 °C inoculated with L. acidophilus culture (@ 2%). The final product had 0.8 to 0.85 % Khamrui (2000) used cheddar cheese whey (LA) acidity. for the development of ready to serve kinnow juice beverage. The process Jayaprakasha (1992) prepared whey drinks consisted of mixing kinnow juice from deproteinized and clarified cheddar concentrated 23 °C Brix by reverse osmosis, cheese whey with the addition of with concentrated fresh cheddar cheese sugar(10%), citric acid (0.4%) and orange, whey (45%) total solids along with other pineapple, mango or raspberry flavouring ingredients sugar 7%, Pectin 0.05% and and in – bottle pasteurization or sterilization CMC 0.15%. The formulation on with or without carbonation. reconstitution with three times water gave a good beverage. Singh et al., (1994) developed an acceptable 144

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Besides that, addition of many other fruits This suggests the possibility of producing like concentrates of apple, pear, peach, beverages from whey with similar sensory apricot and cherry has also been applied. profiles to those of fermented milk drinks or The addition of berries which are known as with some flavor attributes of drinking a good source of iron and antioxidants have yogurt, following manufacturing procedures proved to be very useful. That is especially conventionally used for milk (Gallardo- important in production of whey beverages Escamilla et al., 2005). with improved nutritional value. Best example for supporting this thesis is Drgalic et al., (2005) studied survival and Brazilian group of scientists who have growth of probiotic strains Lactobacillus developed a whey drink flavoured by acidophilus La-5, Bifidobacterium bifidum addition of strawberry concentrate and Bb-12 and Lactobacillus casei Lc-1 in fortified with ferrous bisglycinate. They reconstituted whey for 28 days of cool have proved that long-term consumption of storage. All strains have shown good this drink had an impact on reduction in the survival during storage time of fermented prevalence of anaemia in children and beverages. The beverage fermented by adolescents (Miglioranza et al., 2003). probiotic strain Bb-12 obtained lower sensory score than the other two beverages Duric et al., (2004) studied the effect of fermented by strains La-5 and Lc-1. quality of whey fruit juice (orange, pear, peach and apple) blends. The pH of the Brandao et al., (2006) developed fermented beverage was adjusted to 3.5 using citric symbiotic drink using whey, soy milk and acid. The quality of blends mostly depended whey protein concentrates in different on the sucrose content and dry matter combinations. Four formulae were content. They concluded that peach–whey developed containing whey, soy milk and beverage containing 6% of dry matter and sugar (A1), soy milk, whey protein 2% of sucrose as well as having pH 3.5 was concentrate, sugar and inulin as a dietetic of best quality. soluble fiber (A2), soy milk and sugar (A3), soy milk, whey, inulin, sugar and whey Deosarkar (2004) manufactured beverage protein concentrate (A4) and inocuated with from lactose hydrolyzed permeate with L. acidophilus bacteria. The differences mango pulp as flavor. The level of sugar were observed in the fat and protein content. was added at 5%, guar The results demonstrated that formula A3 gum at 0.1% and sodium benzoate at 0.03%. was accepted more in comparison to other The pH of the beverage was maintained at formulae. 4.0 using citric acid solution (20%) and pasteurized (72 ºC/15 sec) product was Sahu et al., (2006) developed whey based packed in preformed polyethylene pouches mango- herbal (lemongrass) beverage with and stored safely up to 35 days under different concentration of lemon grass refrigerated storage conditions (7±1 °C). distillate by making use of paneer whey. In There are even some indications that the preparation of beverages the proportion fermentation of whey using yoghurt culture of mango pulp (12%), sugar (8%), water (Lactobacillus delbrueckii ssp. bulgaricus (48%), paneer whey (32%) were kept for and Streptococcus thermophilus) produces a constant while the volume of lemongrass more intense yoghurt flavor compared to the distillate was varied from 0 -2.5 percent. one obtained when skim milk is fermented. The beverage prepared with the addition of

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1.5 percent lemongrass distillate was found sterilization was done at 121 °C for 10 to be the acceptable formulation. Stored minutes and the pH was maintained at 5.1 beverage having the properties of 14.93° B and Bottles were cooled at room temperature TSS, 3.98 pH, 23.01 percent total , and then stored under refrigerated conditions 5.02 percent reducing sugars, 0.211 percent (7±1 °C).The prepared beverage during acidity and 1.5 percent lemongrass distillate storage study showed that there is an obtained the average sensory score of 8.33 at increasing trend in the TSS, acidity, and the end of 60 days of storage period which reducing sugar and a decreasing trend in the was highest among all other combinations of pH and ascorbic acid but total sugar has lemon grass distillates. non-significant effect during storage.

Dubey et al., (2007) prepared whey-guava Divya and Archana (2009) prepared whey beverages using paneer whey. The volume guava beverage by heating the whey at 45 of guava pulp (25%), sugar (10%) and ºC and addition of sugar (10%), guava pulp paneer whey (65%) were kept constant (25%), guar gum (0.05%), sodium alginate while the pasteurization temperatures and (1%) and potassium meta bisulfate (100 timings were varied from 60°C – 70°C for ppm). The mixture was filtered, bottled and 15-35 minutes. However, whey-guava crown corked and the beverage was in bottle beverages pasteurized at 70°C for 35 pasteurized at 70 ºC for 35 min and cooled minutes was found to be best in terms of and stored. sensory quality after 45 days and pH, acidity, protein, total sugars and reducing Cruz et al., (2009) developed the butter sugars found to be high than that of the other cheese whey and acerola juice beverage. samples. This beverage contains good amount of due to presence of vitamin C rich Choudary and Sandey (2009) developed Brazilian fruit acerola. Combination of whey based mango- herbal (cardamom) 30:70 mixture of Butter cheese whey and beverage (WBMH) with different acerola juice was found to be more concentration of cardamom extract by using acceptable. The beverage was prepared by chhanawhey. Mango pulp of langra variety heating the whey to 72 ºC for 15 sec and was used in the preparation of beverage. The addition of sugar (12%), diluted acerola level of sugar (8%) and mango pulp (12%) juice (1:3) and pectin (0.5 %). The mixture was kept constant while, the volume of was homogenized, filtered, crown corked, in cardamom extract was varied from 0 to 3%. bottled pasteurized (85 ºC for 10 min), The sterilized beverages were kept in cooled (25 ºC) and stored at 5 ºC. refrigerated condition for a storage period of one month. The beverage prepared with the Madhavi (2009) developed synbiotic whey addition of 2% cardamom extract was found drink using whey, sugar, inulin and orange most acceptable in sensory characteristics up fruit juice. For beverage preparation to 30 days at refrigeration temperature. probiotic culture of L. rhamnosus MTCC Naik et al., (2009) prepared Whey Based 5462was used. The beverage was made Watermelon Beverage (WBWB) by using fermented whey, 10% sugar, 3% blending watermelon juice (15%), sugar inulin and 10 % orange fruit juice. The (7%) and different concentration of Betel product will remain acceptable up to 28 leaves distillate (0, 1, 2, 3%) into chhanna days. whey (78-75 %).The beverage was In-bottle

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Yadav et al., (2010) prepared the whey propionic acid, as an unnamed component of based banana herbal beverage using banana soured milk has been known since the days juice (10%), sugar (8%) and menthaarvencis when man first herded animals. Lactic acid extract (2%). The whey was heated to 45 ºC and its derivatives are widely used in the and other additives are added and after food, pharmaceutical, leather, and textile proper mixing and filtration, filtrate was industries. Recently, there has been an bottled and crown corked. Shelf life of 15 increased interest in lactic acid production, days at refrigerated temperature was since it can be used as a raw material for achieved by in bottle pasteurization in production of polylactic acid, a polymer boiling water for 30 min. used as a specialty medical and environmental-friendly biodegradable Pandian et al., (2011) developed mango plastic. Of the 80,000 tonnes of lactic acid flavoured sweetened whey drink using produced worldwide every year, about 90% whey, sugar (12%) and mango pulp (4, 5 is made by LAB fermentation and the rest is and 6%). The whey was heated to 45 ºC and produced synthetically by the hydrolysis of sugar and mango pulp was added and after lactonitrile (Panesar et al., 2007) proper mixing and filtration, bottles were filled and crown corked. The beverage was Whey from dairy industry although low in pasteurized at 63 ºC for 30 min. sugar content constitutes an economical raw material for lactic acid production (Shahbaz Milk based whey beverages et al., 1996).

Wroblewska (1977) developed a cultured whey-milk beverage namely “Rewit” from Currently, a high fraction of generated milk (2% fat), rennet whey and using Cheese Whey is managed by membrane starters (Streptococcus and Lactobacillus). processes, mainly, ultrafiltration. The “Rewit” contained 30 to 50ml concentrated obtained permeate has a low protein content apple or orange juice. Sabikhi and and an elevated lactose and mineral salts Thompkinson (2007) developed whey based concentration, both aspects are Lassi using whey and buffalo milk. advantageous in lactic acid fermentation. As Combination of CMC, tri sodium citrate and a consequence, several works have been pectin was used as stabilizer in processing. carried out aimed at obtaining lactic acid Fermentation was carried out using the lactic after ultrafiltration of Cheese Whey. Highest acid bacteria. The Lassi was UHT treated lactic acid production rate was obtained with and packed in tetrabricks under aseptic L. helveticus cultivated in whey permeate, condition which gave the shelf life of 150 with corn steep liquor (CSL) as the nitrogen days at 30 ºC. source. Lactic acid productivity of 9.7 g/L/h using L. helveticusstrain milano has been Utilization of Whey for Biomass/Lactic obtained in continuous fermentation of Acid Production whey-yeast extract permeate medium. In a work with L. salivarumYE supplementation Since lactose is the major component of was replaced by in situ treatment of whey solids, in addition to water-soluble fermentation medium with proteolytic vitamins, minerals and proteins, numerous microorganisms. (Mollea et al., 2013) biotechnological processes have been developed to utilise whey to make useful However, expensive culture media, which products of industrial importance, such as contain natural complex organic nitrogen lactic acid. Lactic acid or α-hydroxy sources such as yeast extract, malt extract 147

Int.J.Curr.Microbiol.App.Sci (2016) 5(8): 134-155 and/or polypeptone, are necessary for the developed and different conditions were cultivation of lactic bacteria because optimized for lactic acid production. nutritional requirement of lactic bacteria is Maximum yield of lactic acid (4.4 %) was very complicate. Lactococcuslactis recovered from shake medium in the especially has numerous growth presence 0.1% NaCI. 0.1 % MgS04.7H2O. requirements. The growth mechanism and 0.1 % NH4H2P04, 2% molasses. 0.1 % yeast the nutritional requirement of lactic acid extract and 2.0% corn steep liquor after 48 bacteria have not been clarified yet. hours incubation at 35 ºC and pH 5.5 on Recently, many workers have been studying orbital shaker (Shahbazet al., 1996). the effects of nutrients (Amrane et al., 1993; Ledesma et al., 1977) on the growth and 16 isolates of Lactic acid producing bacteria product formation of lactic bacteria. Among isolated from various biological sources the nutrients contained in the culture were inoculated in whey water (1% medium for lactate fermentation, the inoculum) and kept at 37 °C in the shaker at nitrogen source such as yeast extract seems a speed of 150 revolutions per minute for 36 to be very important for cell growth and acid h. Lactic acid production was estimated after production. Some workers have reported the 36 h and the strains 4a, 12a and 15b showed promotion effect of yeast extract on the lactic acid production of which 12a growth of lactic bacteria (Aeschlimann et produced the highest concentration. The al., 1990; Orberg et al., 1984). However the amount of Lactic acid produced by 12a was growth promotion effect of these complex 0.62 g L-1 under unadjusted condition nitrogen sources have not been completely which is comparable to previously reported clarified yet (Tanaka et al., 1995). Growth strains in enriched medium (Shiphrahet al., medium of milk whey for the production of 2013). lactic acid by Lactobacillus acidophilus was

Table.1 Typical composition of whey

Components Sweet whey Acid whey Chhana/ paneer whey Total solids (%) 7.0 7.0 6.5 Fat (%) 0.3 0.1 0.5 Protein (%) 0.9 1.0 0.4 Lactose (%) 4.9 5.1 5.0 Ash (%) 0.6 0.7 0.5 (Gupta, 2000)

Table.2 Types of whey based on acidity and pH

Type of whey Titratable acidity (%) pH Sweet whey 0.1 – 0.20 5.8 – 6.6 Medium acid whey 0.20 – 0.40 5.0 – 5.8 Acid whey 0.40 – 0.60 4.0 – 5.0 (Bund and Pandit, 2005)

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Table.3 Composition of different types of whey

Sweet whey Medium acid whey Acid whey CM BM CM BM Casein Composition cheddar cheddar Paneer Shrikhand chhana chhana whey cheese cheese whey whey whey whey (HCl) whey whey Solids (%) 6.35 6.87 6.06 6.25 6.40 5.95 6.96 pH 6.10 6.40 5.60 5.50 5.70 4.60 NA Lactose (%) 5.00 5.01 5.03 4.86 4.90 4.30 NA Protein (%) 0.90 0.98 0.30 0.72 0.89 0.70 0.38 Fat (%) 0.06 0.34 0.13 0.10 0.10 NA 0.43 Ash (%) 0.59 0.54 0.60 0.57 0.44 0.72 NA Lactic acid (%) 0.13 0.14 0.21 0.19 0.20 0.25 0.24 Calcium (ppm) 448 501.5 710.65 526 446.3 1,298 NA Phosphorus (ppm) 486 441.5 560.5 493.7 533.3 1,770 NA CM : Cow milk; BM : Buffalo milk; NA : Not available (Khamrui and Rajorhia, 1998)

Continuous mix batch bioreactors were used showed the need for a complex of nutrients to investigate the effects of various for Lactobacillus helveticusfor the growth concentrations (0, 5, 10 and 15 g/L) of two and product formation and the need to nutrients (Yeast extract and Lactamine AA) supplement cheese whey with some on the growth of Lactobacillus helveticus commercially available growth supplements. (ATCC 15009) and the production of lactic Several Studies showed that lactic acid acid from cheese whey. The results from this productivity of most Lactobacilli is study showed that yeast extract was superior significantly improved by the addition of to Lactamine AA in its influence (improved yeast extract, amino acids, protein yield and conversion efficiency) when used concentrates, hydrolysates, vitamins and as nutrient during batch fermentation inorganic compounds such as (NH4)2HPO4 experiments of lactic acid production from and (NH4)2HPO4 (Amrane and Prigent, cheese whey (Ghaly et al., 2003). 1998; Demirci et al., 1998; Champagne et al., 1992; and Cheng et al., 1991). Other Industrial fermentation of lactic acid may be studies showed the need to supplement limited by the availability of micro and cheese whey with some commercially macronutrients, which are required by lactic available growth supplements such as corn acid producing microbes for cellular growth steep liquor, yeast extract, casamino acids, and maintenance. Micronutrients are peptone, neopeptones, cane molasses and predominantly metallic ions, which are trypticase (Cheng et al., 1991; Gupta and required in trace quantities as cofactors in Gandhi, 1995; and Roy et al., 1986, Ghaly et enzymatic reactions, whereas macronutrients al., 2003). include nitrogen, phosphorus, potassium, sodium and sulfur and are needed mainly for Some studies report the use of mixed the synthesis of cellular material (Amrane cultures in lactic acid production with and Prigent, 1998). Roy et al., (1987) and synergistic effects. Other research groups Aeschlimann and Von Stocker (1990) tried to improve LA production using r

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E.coli harbouring an inducible expression Biotechnol. Lett., 32: 398-402. plasmid containing D-lactate dehydrogenase Affertsholt, T., Nielsen, W.K. 2003. Walk encoding gene of Lactobacillus plantarum this whey. Dairy Industries or using metabolic engineering of LAB, International, 31-32. fungal or yeast systems, but all of these Amrane, A. and Prigent, Y. 1993. strategies, if compared with the use of mixed Influence of media composition on cultures, involve higher costs due to genetic lactic acid production rate from engineering studies and the need of whey by Lactobacillus helveticus. sterilization (Mollea et al., 2013). Biotechnol. Mt. 15: 239-244. Amrane, A. and Prigent, Y. 1998. Lactic LAB have been immobilised by several acid production rates during methods on different supports (calcium different growth phases of alginate, k-carragenane, agar and Lactobacillus helveticusand whey polyacrylamide gels) and the immobilised supplemented with yeast extract. systems have been investigated for lactic Biotechnol. Lett., 20: 379-383. acid production from whey. A two-stage Aneja, R.P., Mathur, B.N., Chandan, R.C. process was used for continuous and Banerjee, A.K. 2002. Heat acid fermentation of whey permeate medium coagulated Products. Tech of Indian with L. helveticus immobilised in k- Milk Products. Delhi, India. Cited carrageenan/ locust bean gum, which from:www.indianmilkproducts.com. resulted in high lactic acid productivity (19– Anon. 1982. Corning/Kroger combines 22 g L−1 h−1) (Mollea et al., 2013). technology to exploit lactose- hydrolyzed whey. Food Dev. 16:34- L. caseiwas immobilized in Ca pectate gel. 5. A high lactose conversion (94.37%) to lactic Brandao. 2006. XXII Inter american acid (32.95 g L−1) was achieved and the cell Congress of Chemical Engineering system was found highly stable, no decrease and Argentinian Congress of in lactose conversion to lactic acid was Chemical Engineering. “Innovations observed up to 16 batches (Mollea et al., and management for sustainable 2013). development.” Cited fromhttp://www.aaig.org.ar/formcon Whey is a very interesting product due to its gress2006/papers/14b/14b-640.pdf components. Their properties, functions and Bund, R.K. and Pandit, B.A. 2005. chemistry structure make whey a great base Isolating proteins from whey, for the creation of a series of new products Modern Food Processing, 1: 36. or an ideal alternative compound to more Champagne, C.P., Martin, N., Couture, R., traditional ones. In terms of whey utilization Gagnon, C., Jelen, P. and Lacroix, there are so many things that can be done C. 1992. The potential of instead of treating whey as a waste. immobilized cell technology to produce freeze-dried, phage- References protected cultures of Lactobacillus lactis. Food Res. Int., 25: 419-427. Aeschlimann, A. and von Stockar, U. Chandra, B.N. 1980. Utilization of whey 1990. The effect of yeast extract obtained in the manufacture of supplementation on the production chhana as an ingredient in cereal of lactic acid from whey permeate foods and beverages. M. Tech thesis by Lactobacillus helveticus. 150

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study on whey based banana herbal

(Menthaarvensis) beverage.

How to cite this article:

Silviya R. Macwan, Bhumika K. Dabhi, S.C. Parmar and K.D. Aparnathi. 2016. Whey and its Utilization. Int.J.Curr.Microbiol.App.Sci. 5(8): 134-155. doi: http://dx.doi.org/10.20546/ijcmas.2016.508.016

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