Proteins are the important dietary supplement and are a main source of amino acids. Milk is considered the most important nutritional supply for all organisms and contains vitamins, calcium, minerals, and protein casein which gives its characteristic white colour and bioactive peptides that are promising for health. In recent years, much interest has been focused towards probiotic food products that contain microorganism which are beneficial to health. Whey which is derived from milk is the most beneficial components and used in cheese making which is in high demand worldwide, and thus consists of water, lactose, proteins, vitamins, and bioactive peptides, lacto-globulin, lacto-ferrins, immunoglobulin's, and may act as antioxidant, antibacterial and chelating agent, It has also been used in treatment of cancer, HIV , hepatitis B and many more. Lactic acid is commonly used as a starter strain all over the world to produce nutritional compounds in milk products such as yogurt, cheese, fermented milk and etc. The most common lactic acid producing bacteria is lactobacillus. Lactic acid bacteria are gram positive facultative anaerobic bacteria that tend to survive even after pasteurization but do not grow after pasteurization and therefore are used in fermentation that produce lactic acid and other organic compounds and can be divided in two groups, mesophilic and thermophilic lactic acid bacteria having an optimum growth temperature between 20°C and 45 °C. Thermophilic bacteria are best known for yogurt type products and are used as ripening agent in cheese and have a growth temperature at around 55°C (Wouters, et al, 2002). Recently many milk protein derived bioactive compounds are generated using lactic acid bacteria in many industries for reducing many health risks such as chronic disease and to boost immune system.
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Proteins derived from milk and whey
Breakdown of major milk proteins are only released and activated during digestion and therefore Lactic acid bacteria (LAB) are used to produce these bioactive peptides from dairy products in order to provide nutritional value and develop better improved healthy food products. LABS are the first bacterial strain to be used in food industry to convert fermented sugars to lactid acid and are also responsible inhibiting microbes that cause food spoilage thus preserving milk and its products by anti-microbial factors such as ethanol, diacetly, bacteriocins, carbon dioxide and hydrogen peroxide. It senses peptides such as nisin by quorum sensing system. The activity of peptide released from milk proteins is entirely based on amino acids sequence and composition, and the size varies from 2-20 amino acid residues, and can reveal many functional properties and therefore these milk bioactive peptides are considered the most important for targeting various health problems (e.g. heart, bones, teeth, immune system, digestive system, etc). Nisin is produced by certain acid producing cultures, such as Lactococcus lactis (formerly known as Streptococcus lactis). Present study has found that if whey containing nisin, is introduced into a fermented dairy culture, could inhibit uncoupled formation of acidic products. Although dairy workers have tried incorporated nisin alone or complete nisin-producing cultures into fermented dairy products in attempts to inhibit uncoupled acid production, but the experiment was not unsuccessful. The addition of nisin or culture (containing both curds and whey) are not effective in inhibiting uncoupled acid production. Few bacterial strains of LAB (e.g lactococci) may produce nisin during fermentation of whey, which may also have the ability to produce other bioactive peptides such as lactate, which may be beneficial for preserving food. According to Nauth et al. (2000) lactococci culture that produces nisin may provide the provide effect which may prevent uncoupled production of acid in yogurt or yogurt like products, but it was found that when yogurt was added to whey derived from dairy culture of nisin producing lactoccocus microorganism, it inhibited acid prodution.
The generation of bioactive peptides during milk fermentation with dairy starter cultures is now well known. Peptides with different bioactivities have been found in a number of dairy products, such as various cheese varieties and fermented milk. According to Korhonen (Korhonen, 2009) these proteins may be beneficial to reduce the risk of obesity and diabetes, few products that contain these peptides are already released in market to be consumed. These biologically active peptides are produced from milk proteins. Similarly, according to Panesar. (2008), lactose unitilsing yeast (Kluyveromyces sp.) are very important source of β-Galactosidase enzyme production (temperature and pH had effect on enzyme production), which is commercially and industrially used for various purposes such as to produce low acid (low lactose) dairy products to meet the dietary requirement for people that are lactose intolerant. Methods involved in producing these peptides are, enzymatic hydrolysis (glycolysis), fermentation of milk with proteolytic starter cultures, proteolysis ( casein) by enzymes derived from micro organism (Pescuma et al, 2008).
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Enzymatic hydrolysis is the most common way of producing bioactive peptides from milk, where the whole protein is hydrolysed. Digestive enzymes are said to produce biologically active peptides . Pepsin, trypsin and chymotrypsin are the major enzymes responsible for release of peptides, calcium-binding phosphopeptides (CPPs), antibacterial, immune-modulatory and opioid peptides from different casein (a-, b- and j-casein) and whey proteins, e.g., a-lactalbumin (a-la), b-lactoglobulin (b-lg) and glycomacropeptide (GMP). Dairy starter cultures are highly proteolytic and are used in microbial fermentation to produce bioactive peptides. Various chemical and biochemical processes are involved in cheese flavor formation. Main organisms for fermentation as starters are lactic acid bacteria (LAB), e.g., Lactococcus lactis, Lactobacillus species, Streptococcus thermophilus, Leuconostoc mesenteroides. However, other cultures are also used, such as Propionibacterium in the case of Swiss-type and Maasdammer-type of cheeses, and various aerobic cultures (e.g., Brevibacterium, Arthrobacter, Staphylococcus, Penicillium, Debaromyces) for surface-ripened cheese . Mesophilic lactobacilli, originating from the milk environment might grow in the dairy products and thereby be a source of enzyme activities leading to the formation of flavours. LAB is responsible for the conversion of lactose fermentation to lactate, although pyruvate may also be converted to flavour compund (diacetyl, acetoin, acectic acid, etc which give yogurt and cheese its characteristic flavour. Lipolysis leads to generation of free fatty acids, which can be precursors of flavour compounds, although Lab is known to contribute little to lipolysis. In cheese formation conversion of casein by LAB is very important to form further flavour compounds of cheese and inorder to break peptides that are responsible for bitter taste ( Smit et al, 2005).
According to many studies one of the common bacterial strains used for production of bioactive peptides is Lactobacillus. Yogurt, cheese starter bacteria and commercial probiotic bacteria are also commonly used industrially and commercially to produce various kinds of useful bioactive peptides in fermented milk and may also lead to antioxidative properties. Milk has low concentration of free amino acids and peptides, while the selected starter culture entirely depend on proteolytic system and degrades milk protein producing free amino acids and peptides which are taken up by surrounding cells. Amino acids and peptides are responsible for giving food its specific flavour characteristics e.g. sweet, bitter, sour, malty, and etc (refer to table 1.1 for description of key flavour compounds)
Banana, malty, chocolate-like
Malty, powerful, cheese
Fresh cheese, breathtaking, alcoholic
Rancid, sweat, cheese, putrid
Sweaty, butter, cheese, strong, acid
Pungent, sour milk, cheese
Fruity, buttery, ripe fruit
Yoghurt, green, nutty, pungent
Table 1.1 Description of major flavour compounds (Smit et al, 2005)
According to Chammas et al. (2006) different starter bacterial strains are used in milk and whey fermentation to produce desired product as each strain is specific. Therefore the main properties of microbes in milk fermentation are acidification, increased amount of proteins, texture improvement and flavour production. Dianan et al. (2003) in one of her study showed that goat milk which has lower casein compared to cow's milk, can be fermented by using probiotic starter bacterial culture with low acidification activity (Streptococcus thermophilus, Lactobacillus acidophilus, and Bafidobacterium). It was shown that adding whey protein in the goat milk during fermentation increased protein level which made the product more favourable in terms of texture, appearance, viscosity, flavour, taste, and aroma. Fermentation of milk with a commercial starter culture mixture of five lactic acid bacteria strains followed by hydrolysis with a microbial protease increased inhibitory activity of the hydrolysate. The sequence of the peptide (diacetylactis) obtained from b-casein was Ser-Lys-Val-Tyr-Pro- Phe-Pro-Gly-Pro-Ile which were stable to pH, digestive enzymes, and acidification. It is known that during cheese ripening great variety of peptides are formed most of which wield biological activities and biological activity is dependent on cheese ripening period; For example in gouda cheese it is kwon that the peptides production slows down after a certain time ( after 13 weeks). Around 28 peptides produced showed various bioactivities e.g., mineral-carrying, antimicrobial, antihypertensive and immunostimulatory activities.
Furthermore, Pescuma et al. (2008) shows that β-Lactoglobulin is the main whey protein and lactic acid bacteria is used as the starter culture in fermentation which may depend on proteasees, peptidases and amino acids and reduced lactose content in whey producing lactic acid and compounds that are responsible for texture, appearance, arome, flavour and viscosity. Few LAB species such as S. thermophilus tend to inhibit allergic proteins in milk. LABs use casein proteins as a source of amino acid. Protolysis hydrolysis of β-casein involves the breakdown of proteins by L. lactis by an extracellular cell wall bound proteinase PrtP. Uptake of large peptides is the crucial step in proteolysis which are realised by PrtP and catalysed by ATP binding protein (Konings, et al,2000).
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In Garneau et al. (2002) and Vuyst et al. (2007) study, bacteriocins produced by LAB are biologically active proteins which are used as food preservatives for their longer life..Various strains of Lab are able to produce bioactive molecules such as, acetic acid, ethanol, diacetyl, formic acid, fatty acids, and bacteriocins, which serve as antimicrobial peptides that contribute to health, food preservation and safety. Bactreriocins are industrially very important and are produced in high amount by LAB compared to food fermentation which could be due to factors such as temperature, pH, and nutrient availability. Bacteriocins produced can be used as food additives, e.g. nisin is made in purified form.
Preservation of food for long life is important and safe for humans . Lactic acid bacteria are good source of producing different bioactive compounds during fermentation. Production bioactive compounds such as β-D-galactosidase can be very costly. Lactic acid bacteria and probiotics can be easily battered by temperature and this could lead to protein loss or low shelf life and therefore methods should be explored to overcome this and therefore to retain the stability of lactic acid bacteria in fermented milk so that nutritional level is not affected. In view of the current global trend of increasing prevalence of obesity and related diseases, diabetes, in particular, more experimental research should be focused on natural dietary bioactive peptides which can induce safety and reduce insulin resistance