Whey-based fermented beverage technological process review.
Probiotics have been taking value over the last years due to its benefits in human health. Researchers have been looking for options in order to increase probiotics consumption, and one of the more nutritional choices is to use whey as a substrate in fermented beverages. Whey is a by-product liquid obtained during cheese processing. It is an economic source of protein, which provides multiple properties in foods. The main objective of this chapter was to carry out a complete review of important researches related to whey-based fermented beverages production. Researches show that probiotic micro-organisms have the ability to grow in whey properly, in such a way that they reach high concentrations, needed to achieve the probiotic effect that consumers are looking for. Certain substances, such as fruit pulps and carboxymethyl cellulose, have been used to improve viscosity, flavor among other important characteristics. Sensorial evaluations have been performed in order to assess consumers’ impression, and they have been pleasantly accepted. Average shelf-life is 21 days. Through this review, it is known that whey is an excellent alternative to increment probiotic consumption, not only because it is an outstanding substrate for probiotic micro-organism’s growth but also due to its excellent sensorial characteristics.
- fermented beverage
- acid lactic bacteria
- organoleptic characteristics
During the last decades, the use of probiotics has been increasing, due to their important benefits in human health. Kollath, in , first defined the term “probiotic,” when he suggested the term to denote all organic and inorganic food complexes as “probiotics,” in contrast to harmful antibiotics, for the purpose of upgrading such food complexes as supplements. In 1965, Lilly et al.  used the term “probiotic” to describe those substances secreted by an organism that stimulate the growth of another. Since then, this definition has been evolving remarkably, so that today, probiotics are defined as microbial dietary supplements, viable, selected, which when are introduced in sufficient amount, affect human organism beneficially through their effects on the intestinal tract . On the other hand, Vasudha and Mishra  define them as alive microbial supplements, which beneficially affect the host by improving its intestinal microbial balance.
Dairy products have become a healthy alternative to increase probiotics consumption, developing fermented beverages based on milk, whey or their mixture. Whey has been less used than milk. However, it has wonderful physico-chemical characteristics that make it an excellent substrate to be used in the development of fermented beverages.
Whey is a green translucent liquid obtained by separating milk clot in cheese making process . Its composition and characteristics depend on the technological process used and the type of milk. It is composed of 5% lactose, 93% water, 0.85% protein, 0.53% minerals, and 0.36% fat .
Its characteristics correspond to a fluid of yellowish green color, turbid, fresh taste, weakly sweet, acidic, with a content of nutrients of 5.5–7% that come from milk. It retains about 55% of total milk ingredients like lactose, soluble proteins, lipids, and mineral salts . Whey is a by-product of high energetic and nutritional quality. For human being, it serves as an important source of vitamins, proteins, and carbohydrates.
Some statistical studies indicate that a significant portion of this waste is discarded to tributaries, resulting in an environmental problem due to its high biochemical oxygen demand. It physically and chemically affects the soil structure, decreasing the yield of agricultural crops, and polluting water because it depletes dissolved oxygen .
For the reasons explained above, dairy industry has been looking for alternatives for the use of this by-product, which is a high pollutant; however, it has a great nutritional value. Among the products of successful acceptance are fermented dairy drinks, refreshing beverages , protein concentrates , infant formulas , and others.
The processing of whey for beverages production began in the 1960s, and Rivella was the first fermented drink prepared from whey, made in Switzerland . Whey products improve texture, reduce flavor and color, emulsify, stabilize, improve flow properties, and show many other functional properties that increase the quality of the products .
The main objective of this chapter is to carry out a complete review about fermented beverages based on whey inoculated with probiotics micro-organisms that have been produced around the world over the last years, focusing specially in important aspects such as sensorial and microbiological quality, shelf-life, and probiotic effects, showing that probiotics consumption can be increased through the use of whey as a substrate in this type of formulation, promoting it as a useful dairy by-product due to its excellent sensorial characteristics and its contribution in high quality organoleptic foods.
2. Relevant aspects related to the use of whey as a substrate for the fermented beverages formulation
2.1. Whey physico-chemical characterization
The knowledge of whey physicochemical characterization is an important step in the use of this by-product in the dairy industry for different industrial processes. For this reason, most of the studies related to the use of whey propose a physico-chemical characterization in order to evaluate whether it meets the standards required to be used in technological processes.
In a very recent research, Molero et al. , carried out a physico-chemical characterization of whey obtained by cheese making process applying an artisanal method. It consisted of the determination of pH, titrated acidity, total solids, fat, protein according to the Venezuelan Standard COVENIN, and determination of lactose and minerals by analytical difference. The values obtained were statistically analyzed using a statistical package. The results classify whey as sweet, with excellent nutritional characteristics and attractive to be used in food technology for probiotic production, protein-fermented beverages, among other applications.
In an interesting research, Tirado et al.  carried out a physico-chemical characterization of whey derived from the production of coastal cheese. Fat analyzes were performed by Gerber method, lactose by the Lane and Eynon method (AOAC 923.09, 920.183b); the protein was analyzed by the Kjedahl method (AOAC 920,152); total solids by spectrophotometry; pH was determined by the method established in AOAC 945.10/90 and the acidity expressed as a percentage of lactic acid according to the Colombian Technical Standard. The values obtained were: fat 0%, lactose 3.69%, protein 2.29%, total solids 6.28%, acidity 0.08% lactic acid, and pH 6.5.
Linares et al.  showed similar results in the physico-chemical characterization of sweet whey samples, obtaining a pH of 6.84; acidity titrated of 0.11% (% lactic acid); protein between 0.6 and 1%; and ash 0.6%. De Paula et al.  performed a physico-chemical characterization of whey obtained from the manufacture of coastal cheese. This characterization was carried out using the following methods: acidity (AOAC 947.05/90), pH (AOAC 981.12/90), soluble solids (AOAC 932.12/90), total solids (AOAC 925,105/90), and lactose (FIL 28a/74). The following results were obtained: acidity (% lactic acid) 0.11; pH 6.58; total solids 6.83%, protein 0.98%; fat 0.4%; and lactose 4.54%.
Similarly, Montero et al.  carried out a whey fermentation with
In other studies, Londoño et al.  developed a fermented drink of fresh cheese whey inoculated with
On the other hand, Miranda et al.  carried out a physico-chemical characterization of sweet and acid whey produced in the cheese complex of Bayamo (Cuba). The authors determined acidity, pH, density, and fat content, following the guidelines of the Ministry of Agriculture of Cuba; lactose was determined by the phenol-sulfuric method; dry extract, crude protein, calcium and phosphorus were tested according to internationally recommended methods. The acid whey was distinguished by a lower pH and a higher acidity than the sweet. No significant differences were observed between the two varieties of whey for the remaining characteristics tested. All of them were within the specifications of quality established by the Cuban norm. Low acidity of whey benefits its quality, because it allows a better use for human and animal feeding. It is great important to know about dry extract in the evaluation of the quality of cheese whey as raw material, because it would indicate its water content: a greater amount of water makes whey has less nutritional value.
In this order of ideas, Sepúlveda et al.  developed a fermented beverage with the use of fresh whey with the addition of Maracuyá pulp. For this purpose, a physico-chemical characterization was performed, determining pH, viscosity, total solids, protein content, fat, and ash by AOAC methods; lactose was determined by the reactive method of Teles; calcium, sodium, and potassium were determined using the spectrophotometric method of atomic absorption. When comparing composite ranges obtained with data reported by Amiot et al. , Scott , Posati and Orr , and Morales et al. , similarities were observed with measures reported for total solids, lactose and protein; under these levels of composition a substrate for fermentation was guaranteed, influencing in the performance of beverage processing properly. On the other hand, fatty content in whey is directly linked to the cheese manufacturing conditions. The values obtained in this trial were considerably lower than that reported by Scott , who states that fat content for sweet whey ranges from 0.2 to 0.7%. The pH obtained was slightly higher than that reported by Spreer  for this type of whey.
From the studies explained above, it is a fact that whey’s physico-chemical characteristics vary depending on the composition of the milk, the cheese making process and the type of cheese, which could determine the ultimate destination of this by-product.
2.2. Microbial cultures used to produce fermented beverages based on whey
The cultures most likely used are lactic acid bacteria (LAB), which play an important role in fermentation processes. They are widely used in food industry because of their involvement in texture, taste, smell and aroma, and development of fermented foods .
LABs may be contained in a group of micro-organisms named lactic cultures or starters . They are used in dairy industry for fermented milks production, cheeses, butter, and other products that are required to be fermented . LABs were referred to as probiotics in the 1960s.
The scientific interest in bacteria as protective agents against different diseases comes from the observation of Metchnikoff, who at the beginning of the twentieth century, emphasized the longevity and good health of the Bulgarian peasants, who consumed large quantities of yoghurt .
The observations of multiple scientists such as Trapp et al.  assumed that consumption of large quantities of foods rich in lactic acid bacteria, eliminated toxin-forming bacteria, while raising the proportion of lactic acid bacteria and intestinal flora, improved health and increased life expectancy. Since then, and throughout almost a hundred years of study, various authors have endeavored to know different functions of beneficial micro-organisms that populate the digestive tract.
Lilly et al.  used the term “probiotics” to describe those substances secreted by an organism that stimulates the growth of another, as opposed to the term “antibiotic,” understood as any chemical compound used to eliminate or inhibit the growth of infectious organisms. Parker  was the first to use “probiotic” referring to organisms and substances that contribute to intestinal balance.
The definition of probiotics has evolved remarkably, so that today, they are defined as viable, microbial selected dietary supplements that, when they are introduced in sufficient amounts, affect the human organism through their effects on the intestinal tract . Probiotics must meet some basic requirements to be selected in the development of commercial probiotic products. The most important requirements are: the probiotic micro-organism survives in the product, the physical and genetic stability during product storage is guaranteed, and all its essential properties that evidence its health benefits after consumption, are maintained during manufacture and storage of the product . Laws et al.  states that the essential criteria for primer selection include acidification, aroma, taste, stability, and texture.
Many researches have been carried out with this class of micro-organisms, producing drinks of high microbiological and sensorial quality. Following the same idea, Molero et al.  formulated a probiotic fermented beverage based on whey, using a mixed culture of
On the other hand, Linares et al.  evaluated the effect of different proportions of citrus pulp on the sensorial acceptability of a fermented and protein drink made from residual whey. For this purpose they used a lyophilized lactic culture of
In another study, Fiorentini et al.  evaluated the influence of different combinations of probiotic bacteria and different fermentation temperatures on the physico-chemical characteristics of fermented lactic beverages based on soybean and whey. For this purpose, a lyophilized probiotic culture was used, composed of
In other studies, Pescuma et al.  developed fermented functional beverages based on whey, using lactic acid bacteria: strains of
Katechaki et al.  performed a research related to thermal drying of
Similarly, Londoño et al.  worked on a fresh cheese fermented drink formulation, inoculated with
In another research, Gallardo et al.  evaluated taste and sensation in the mouth of beverages made from whey with addition of hydrocolloids. The functionality of hydrocolloids at low concentrations is that it enhances viscosity and prevents particles sedimentation. It also contributes to the microstructural properties of meals, based on its ability to confer structure to the continuous phase of the substrate, which depends on their solubility in water and/or their intermolecular associations . Beverages were prepared with a commercial yoghurt starter culture, which consisted of
Hernández et al.  worked on the preparation of a probiotic drink based on whey, using cultures of
Following the same idea, Dalev et al.  evaluated the sensory quality of whey-based probiotic beverages. A probiotic culture was prepared with strains of
Sepulveda et al.  prepared a fermented beverage with the use of fresh whey with the addition of Maracuyá pulp, using a traditional lactic acid culture, in a 1:1 ratio of
Kéfir has also been used as a starter culture in the production of beverages from whey [49, 50, 51]. Kefir is made by inoculating milk with kefir grains. This grain is irregular and its size varies from 3 to 35 mm in diameter, contains lactic acid bacteria (
Yeasts, such as
2.3. Technological process followed for whey-based probiotic fermented beverages production
Table 1 summarizes the review of whey-based fermented beverages. It tells the technological process used in drinks manufacture. It is interesting to observe how the use of probiotic micro-organism plays an important role. The tendency is to use this type of bacteria and the reason is its potential benefit to human health.
|Author||Micro-organism||Special additives||Fermentative process|
|Molero et al. [13, 34]||Whey was pasteurized at 65°C for 20 min and cooled to 38°C. Inoculation of micro-organisms. Fermentation was carried out controlling pH until 4.5|
|Tirado ||Mixture of whey, skim milk powder and sugar was pasteurized at 63°C for 30 min and cooling to 43°C. Inoculation of micro-organisms in equal proportions. Fermentation was carried out for 3 h at 40°C controlling pH up to 5.|
|Linares et al. ||Lactic culture: ||Citrus fruit pulps||Fruit juice mixed with whey, previously pasteurized at 70°C for 30 min and fermented at 42°C for 5 h. White sugar was added in order to standardize at 14 °Brix. The beverage obtained was pasteurized at 80°C for 15 s and packed at the same temperature|
|Martínez et al. ||Pasteurized maracuya pulp||Five different treatments with different Maracuya pulp percentages (5; 7,5; 10; 12 y 15%) and final value of 14 °Brix|
|Vela et al. ||Mango pulp (||150 ml of pasteurized whey was taken and inoculated with |
|Teixeira et al. ||Kéfir grains||Kefir grains washed with distilled water and inoculated in 250 ml of whey, with a temperature of 25°C for 72 h|
|Fiorentini et al. ||Probiotic freeze-dried cultured of ||Water-soluble soybean extract||Whey powder was dissolved in water in a 1:1 ratio right before use. Mixture of 40% whole milk, 30% whey of mozzarella cheese, 30% water-soluble extract of soybean and 10% of sugar. Heat treatment at 90°C for 5 min, cooling to fermentation temperature (37°C). Incubation until reaching a pH between 4.5 and 5. Cooling at 20°C, homogenized, distributed in plastic bottles and stored at 7°C for 21 days|
|Legarová et al. ||Commercial yoghurt starter culture: ||Incubation at 43°C and cooling at 4°C.|
|Katechaki et al. ||Drying was carried out in convection ovens at 35, 45, and 55°C for 10 h. After drying remaining moisture was removed with further drying at 102°C. Fermentation was performed at 37°C for 3 days|
|Pescuma et al. ||Incubation a 37°C for 24 h|
|Padín and Díaz ||Organic solvents||Reconstituted whey powder at 20% w/w. 100 ml of reconstituted whey 10% v/v were inoculated with a |
|Montero et al. ||100 ml of culture was taken to inoculate 900 ml of whey in a beaker and incubated for 24 h at 39°C. Fermented whey was poured into 9 l of fresh whey and fermented 24 h at 39°C in a convection oven. The 10 l of fermented whey were poured into 90 l of fresh whey and rested 24 h at room temperature|
|De Castro et al. ||Oligofructose||Pasteurized milk with commercial sucrose was heat treated at 95°C for 5 min while liquid whey with oligofructose was heated to 65°C for 30 min. The temperature of the mixture was lowered to 40°C. The beverage was made with the addition of the lyophilized culture at 8.3 mg/100 ml. Fermentation occurred at 40°C. pH of 4.6 was monitored. The beverage was then cooled to 4°C|
|Londoño et al. ||Inverted sugar syrup; maracuyá pulp; carboxymethyl cellulose (CMC)||Inoculation was performed maintaining a pH of 5.8 and stirring for 3–5 min. Subsequently, the beverage was flavored with the addition of maracuya pulp, packed and stored at 4°C|
|Hernández et al. ||Reconstituted whey (7%) with addition of 7% sucrose and 0.4% pectin. Three treatments were applied by inoculation of probiotic strains in different ratios. Incubation at 37°C and storage at 4°C for 30 days|
|Gallardo et al. ||Commercial yoghurt starter culture: ||Hydrocolloids||Fermentation was carried out by inoculation (2% v/v) with the starter culture at 42°C until reaching a pH of 4.6|
|Dalev et al. ||Soy milk||Equal amounts of whey and soy milk. Fermentation at 37°C for 24 h until reaching a pH of 4.4–4.6. Drinks were cooled and supplemented with processed fruits|
|Oliveira et al. ||Probiotics cultures: yoghurt culture of ||Complete and skimmed pasteurized milk mixed with skim milk powder to obtain 130 g/l of total solids and 26 g/l of fat, supplemented with 20 g/l of casein hydrolyzate. Heat treatment at 90°C for 10 min cooled to 4°C and stored 24 h prior to use. Fermentation with incubation at 42°C until reaching a pH of 4.3|
|Sepulveda et al. ||Maracuyá pulp (||It was incubated 2 or 3 h, maintaining the temperature until reaching a pH of 4.6. Fermentation was stopped with a fast cooling of 4°C. Agitation to ensure that maracuya pulp and vitamin dosage were well incorporated into the mixture|
|Cóndor et al. ||Fermentation at 30°C for 7 days, evaluating the fermentation kinetics with the reading of oBrix and pH|
In the development of these beverages, authors have used additives in order to improve some organoleptic characteristics, for example oligofructose, hydrocolloids, processed fruits, and others. Regarding technological process, fermentation is the essence of the drink production. However, it can be changes related to raw material (e.g., whey powder, liquid whey, combination with soymilk or whole milk), prior bacteria isolation, among others. Dairy industry has thus diversified methods for producing whey-based beverages. In the following sections, the acceptance of these drinks can be evidenced based on the sensorial evaluations and their probiotic character based on the viable count.
2.4. Fermented whey-based beverage sensorial quality
In the process of making dairy drinks, it is essential that they have adequate sensory properties to ensure they are accepted by consumers. Sensory quality researches have been increased over the recent years.
In this order of ideas, Molero et al.  carried out a sensory evaluation of probiotic fermented beverages based on whey. Four treatments were developed using combinations of two stabilizers, carboxymethyl cellulose and unflavored gelatin and two starter cultures:
Similarly, Valencia et al.  carried out a sensorial evaluation to nutritional drinks based on pumpkin and whey, enriched with oats and passion fruit. They evaluated 12 beverage formulations considering color, aroma, taste, and acceptability. They performed the test with a panel of 26 school-aged children; each child received three 100 mL samples. They used a questionnaire with expression faces of pleasure or displeasure, corresponding with a hedonic scale of 1–5, being 1 the lowest score and 5, the highest score. They found significant differences between the results obtained for the samples analyzed, but all of them were very well accepted by the panelists.
De Paula et al.  performed a sensory evaluation of fermented beverage fermented from whey with and without Maracuyá pulp. They performed an order-preference test, using a panel of 59 consumer catheters, using note 1 for the most preferred and 5 for the least preferred. They coded the samples and presented them randomly in 50 mL beakers, finding that the combination of whey with passion fruit flavor was the most preferred beverage. In addition, they indicated that the panelists described the product as very good, novel, and interesting.
In another research, Vela et al.  evaluated a probiotic beverage based on whey with addition of Mango and Almond pulp, in a ratio of 1:1, using
Legarová et al.  evaluated whey-based fermented beverages using the commercial yoghurt culture of
Similarly, De Castro et al.  analyzed the sensorial acceptance of whey based fermented beverages with different concentrations of oligofructose (2 and 5%). They evaluated the samples after 72 h of preparation, using: (a) an untrained panel of 36 people, who were asked which drink they liked the most or did not like; and (b) an untrained panel of 50 people in one test of acceptability, using a structured hedonistic scale of 9 points (1—I do not like anything, 9—I like it very much) and a test of intention to buy using a scale of 5 points (1—definitely not going to buy it; 5—I will definitely buy it). Panelists preferred 2 and 5% oligofructose beverages compared to the control drink (without oligofructose), where acidity was an attribute mentioned by 41% of judges. Furthermore, in terms of acceptability, the average score for both drinks was over 7 points, evidencing that the variation in the oligofructose content added did not affect acceptance of the beverages.
On the other hand, Londoño et al.  carried out a sensorial evaluation of a fermented beverage made from fresh whey, using
Gallardo et al.  used a highly trained panel in the sensory evaluation of a whey-based fermented beverage with and without addition of hydrocolloids, with commercial yoghurt as an inoculum (0.02%).They performed the sensory evaluation under controlled conditions, following the ISO Standard 8589 (1988), using samples of 30 mL, in triplicate, on separate days. They used a 100 mm unstructured linear scale to measure the sensation of panelists leaving the sample for a maximum of 3 s before swallowing. According to the results, the addition of hydrocolloids affected the perception of the viscosity of fermented beverages substantially, evidencing the lack of a greasy sensation in the mouth.
In other studies, Hernández et al.  performed the sensory analysis on a reconstituted whey-based probiotic drink (7%) and pasteurized (80°C, 3 min) using three combinations (treatments) of
Following the same idea, Dalev et al.  conducted a qualitative sensory analysis of fermented whey and soy milk beverages to five probiotic beverages based on whey and soy milk: (1) unfermented soy milk (control drink), (2) fermented drink with equal volume of whey and soy milk. Starter culture:
They performed the sensory evaluation using the Quantitative Description Analysis (QDA) method . They selected descriptors or attributes to be evaluated and used a 10 cm unstructured linear scale, shown in monitors, converting the results on a numerical scale (from 0 to 10 units) expressing them in conventional units. They employed a panel of six people (four women and two men) trained according to the International Standards (ISO 1993), with at least 1 year of experience in descriptive tests of different foods, who received varied samples of 20 mL, each in triplicate. The results showed highly significant differences in attributes such as soy milk odor, cereal odor, fermented taste, strawberry odor, sweet taste, and after taste. They concluded that the addition of processed fruits helps to improve the characteristics of the beverages substantially, and therefore, the qualification as organoleptic quality. Soy milk has been used to prepare products such as yoghurt, but its poor organoleptic characteristics have been responsible for a very low acceptance by consumers.
2.5. Whey-based fermented beverages shelf-life
It is a fact that there are physico-chemical factors that can influence micro-organism survival in fermented beverages, being the most important acidity, temperature, oxygen concentration, type of inoculum and storage conditions. Theoretically, it is expected that for a reasonable time, the product will maintain the characteristics that define it as probiotic, so that quality can be guaranteed to the consumer. For this reason, many researches have evaluated the beverages shelf-life.
In this order of ideas, Fiorentini et al.  performed a viable lactic bacteria count in fermented beverages prepared from whey and soybean addition, after 7, 14, and 21 days storage under refrigeration at 7°C. They performed a selective count of
Similarly, Hernández et al.  performed a viable micro-organisms count to a beverage made from whey inoculated with
In other research, Sepúlveda et al.  carried out an evaluation of the physicochemical characteristics during the storage of a beverage prepared with whey with addition of Maracuyá pulp. As the days went by, a decrease in pH and an increase in acidity were observed, suggesting a shelf-life of no more than 21 days. Oliveira et al.  performed a microbiological analysis of four whey-based drinks inoculated with probiotic cultures at 1, 7, 14, 21, and 28 days of storage at 4°C. They used MRS-bile agar for counting
Cóndor et al.  performed a microbiological evaluation of a whey-based beverage using immobilized
Whey has been used for probiotic fermented beverages development significantly. It has excellent physico-chemical characteristics that make it become an excellent substrate, allowing probiotic bacteria growth in such a way that they reach high concentrations, achieving the probiotic effect and all of its health benefits. On the other hand, whey-based probiotic beverages have extraordinary organoleptic characteristic and they are widely accepted by consumers. An average shelf-life of these beverages is 21 days under refrigeration conditions, ensuring probiotics benefits during this period of time. Throughout this review, it is shown that probiotic consumption can be increased through the use of whey not only due to its excellent physico-chemical characteristics, but also due to its ability to develop beverages with high sensorial characteristics and due to its excellent acceptance.