Module 13. Functional foods
Lesson 47
MILK INGREDIENTS
AS NUTRACEUTICALS
47.1 Introduction
Milk is considered as nature’s perfect food, that besides meeting the nutritional requirements for the people of all ages also possess wide array of bioactive components. In recent years nutraceuticals and bioactive molecules present in milk have attracted a lot of attention from researchers, nutritionist, medical practitioners, and consumers alike. Since time immemorial, dairy products have been an integral part of human diet. Milk is the only food, which has got the power to sustain life in all the stages of development, and is considered an important part of a balanced diet. It is known to possess 500 different compounds, most of them having certain unique nutritional and disease preventing ability. Besides being a source of quality proteins and energy–rich fat, it contains important micronutrients like calcium, potassium, sodium, magnesium and vitamins, which are vital for overall development of the human body. Also, several health attributes are associated with milk or its constituents.
· Role of calcium in controlling hypertension and colon cancer
· Protective role of carotenenoids and conjugated Linoleic acid (CLA) against cancers
· Butyric acid, the short chain fatty acid has been shown to regulate cell growth and enhance the anti-tumor activities
Certain minor milk components either naturally occurring or formed during processing have also been endowed with many unique health benefits. Examples include lactoferrin, lactulose, galacto-oligosaccharides (GOS), conjugated linoleic acid (CLA), β-lactoglobulin, and bioactive peptides. Some of the important classes of functional dairy foods and nutraceuticals are listed below:
Table 47.1 Examples of functional components in milk and milk products
|
Class/Components |
Source |
Potential Benefit |
Probiotics
|
||
|
Lactobacilli, Bifidobacteria |
Fermented dairy products like dahi, yoghurt, lassi, cheese |
Improve gastrointestinal health and systemic immunity |
|
Fatty Acids |
||
|
Conjugated Linoleic Acid (CLA) |
Fat rich dairy products, fermented milk products |
Anti-cancer, Anti-atherosclerosis and anti-diabetics |
|
Whey Proteins |
||
|
β-Lactoglobulin |
Milk, Whey |
Enhanced synthesis of glutathione, a natural antioxidant |
|
Lactoferrin |
Whey, Colostrums |
Anti-bacterial, increase bioavailability of iron |
|
Prebiotics |
||
|
Lactulose |
Heated milks, Synthesized from lactose |
Bifidogenic factor, improve GIT conditions in infants, laxative, prevent allergy |
|
Galacto-oligosaccharides (GOS) |
Fermented foods, Galactosyltransferase activity of microbes |
Promote growth of probiotic bacteria, anticancer, increase mineral bioavailability |
|
Bioactive Peptides from Milk Proteins |
||
|
Caseino-phosphopeptides (CPP) |
Fermented milks, Proteolysis of casein in GI tract |
Mineral binding specially calcium |
|
Casomorphins |
Proteolysis of α & β- casein |
Increased intestinal water & electrolyte absorption, increased GI transit times |
47.2 Milk Proteins and Derivatives as Therapeutic Components
Proteins are the building blocks of the growing tissues and inadequate quantity may impair the physical and mental development of the individuals. The nutritional quality of dietary protein is essentially related to its amino acid composition as well as to the availability of these amino acids. In this respect milk proteins have a high content of essential amino acids. The body requirement of proteins varies with age and milk proteins have long been considered as food protein for young ones. Apart from being a rich source of essential amino acids, milk proteins contribute to the sensory attributes and consistency of meat, dairy and bakery products. Furthermore many milk proteins possess specific biological properties which make them potential ingredients of health promoting foods.
The milk protein consists of numerous specific proteins that is primarily composed of casein. Casein constitutes about 80% of total milk proteins, remaining 20% are whey proteins. The major whey proteins in milk are β-Lactoglobulin (β-Lg), α-Lactalbumin (α-La), bovine serum albumin (BSA), immunoglobulin (Ig) and proteose peptones (PP).
Table 47.2 Comparative presentation of various milk protein fractions is
|
Protein fractions |
Concentration in milk |
|
|
Buffalo |
Cow |
|
|
α-s1 Casein (g/100 ml) |
1.44 - 1.8 |
1.0 |
|
α-s2 Casein (g/100 ml) |
0.22 – 0.28 |
0.26 |
|
β-Casein (g/100 ml) |
1.26 - 1.58 |
0.93 |
|
κ-Casein (g/100 ml) |
0.43 - 0.54 |
0.33 |
|
β-Lactoglobulin (mg/100 ml) |
0.39 |
0.32 |
|
α-Lactalbumin (mg/100 ml) |
0.14 |
0.12 |
|
Lactoferrin (mg /L) |
5 – 32 |
5 – 28 |
|
Bovine Serum Albumin (mg/100 ml) |
29 |
40 |
|
Immunoglobulins (mg/100 ml) |
9.8 |
38 |
|
Proteose-peptone (μg/100 ml) |
172 |
220 |
47.2.1 Milk casein
Casein is composed of several almost similar kind proteins which form a multi-molecular granular structure called casein micelles. They contain casein molecules, water; salt specially calcium and phosphorus and certain enzymes. Casein micelles contain 4 types of casein molecules namely α s-1, α s-2, β and ǩ-casein which are bound together by amorphous calcium phosphate. Milk casein exhibits excellent digestibility and unique amino acid composition. Nutritionally casein is known to improve the bioavailability of certain vital minerals including calcium, phosphorus, iron and zinc. However casein on hydrolysis yields various physiologically active peptide. These bioactive peptides are most sought milk derived nutraceuticals nowadays.
47.2.2 Whey proteins as prospective nutraceuticals
Proteins can be separated from whey using ultrafiltration with or without diafiltration technologies. During this process low molecular weight compounds (lactose, non-protein nitrogen, vitamins and minerals) are removed from whey to permeate. The remaining proteins are, in turn, concentrated in the retentate. In general, whey proteins are globular, smaller in size, and heat denaturable. Whey proteins have high nutritional and functional properties and are capable of fulfilling the diverse attributes to satisfy different forms of utilization.
Whey proteins are rich source of all essential amino acids. The Biological Value of 104 and Protein Digested Corrected Amino Acid Score (PDCAAS) of 1.0 for whey protein are quite high. The proportion of sulphur containing amino acids i.e. cysteine and methionine is reported to be higher than that of meat, soy and casein. Whey proteins provide more than 100% of the requirement for sulphur amino acids for growing human being, whereas plant protein is limiting in these amino acids. Tryptophan, which acts as building block for niacin, is present in higher amount in whey proteins. Therefore, whey protein based functional foods can be developed for different groups.
47.2.2.1 β-Lactoglobulin (β- Lg)
It is major whey proteins present in the milk bovine but absent in human milk. In bovine milk it comprises of 10% of the total milk protein or about 50% of whey protein. β-Lg has a free sulfhydryl group which is responsible for its interaction with ĸ and α s-2 casein through the formation of disulfide bridges on heating. β-Lg is very resistant to proteolytic enzymes of stomach and due to this unique property, β-Lg act as a resistant carrier of retinol (provitamin A) across the gastro-intestinal mucosa. β-Lg is rich source of essential amino acid cysteine, and it stimulates the synthesis of glutathione in the liver. Interaction of β-Lg with К-casein is of great significance as these interactions are relevant to allergenicity problems in certain individuals.
β-Lg possess numerous sites for binding of minerals, fat soluble vitamins and lipids and can be utilized for incorporation of antioxidant vitamins into low fat products.
47.2.2.2 α-Lactalbumin (α-La)
It is second most prevalent protein in the whey and represents to about 3% of the total milk protein or about 13% of total whey protein. This is the most heat resistant and the smallest protein having a molecular weight of 14,146. Its molecule has 4 disulfide linkages. α-La is a metallo-protein as it is strongly associated with calcium ion. Biologically α-La is required for the synthesis of lactose which is the principal source of energy for newborns. The a-La contains 2-3 times more tryptophan than an average protein. In body, tryptophan is converted into 5-hydroxytryptophan and then to 3-hydroxytryptamine (serotonin). Inadequate 1evel of serotonin in the brain has been linked to depression, obesity, insomnia and chronic headache.
47.2.2.3 Immunoglobulin (Ig)
These are minor blood proteins that are passed on to mammary gland and secreted into milk. The level of Ig is highest in colostrum but continuously decreases during advancing lactation period. Various immunoglobulins include IgG, IgA and IgM that impart passive immunity to new born.
47.2.2.4 Lactoperoxidase (LP)
Lactoperoxidase (LP) is considered as naturally occurring antimicrobial enzyme present in milk. Enzyme is known to catalyze the peroxidation of thiocyanate (SCN-) resulting in generation of intermediates products that interact with membrane bound protein and alters its permeability. The concentration of LP is more in buffalo milk than in cow milk, however to induce antimicrobial effect exogenous addition of 12 ppm thiocyanate and 10 ppm of H2O2 in raw milk is required.
47.2.2.5 Lysozyme
Lysozyme is an antimicrobial enzyme found in milk. The concentration of lysozyme in colostrum and normal milk is about 0.14-0.7 and 0.07-0.6 mg/l, respectively. Milk lysozyme is active against a number of Gram-positive and some Gram-negative bacteria. There seems to be a synergistic action of lysozyme and lactoferrin against many bacteria.
47.2.2.6 Lactoferrin
Lactoferrin is a dominant whey protein in milk and plays an important role in iron uptake in the intestine. The concentration of lactoferrin in bovine colostrums and milk is about 1.5-5 mg/ml and 32-50 mg/l, respectively. Lactoferrin are single chain polypeptides of about 80,000 Dalton containing 1-4 glycans, depending on the species. Lactoferrin exhibits both bacteriostatics and bactericidal activity against a range of microorganisms. Lactoferrin also causes the release of lipopolysaccharides molecules from outer membrane of the Gram-negative bacteria and acts as an antibiotic. The occurrence of lactoferrin in biological fluids like milk, tear, saliva and seminal fluids suggested that it could have a role in the non-specific defense against invading pathogens.
47.2.3 Antioxidative potential of
whey proteins
Dietary whey proteins have a number of putative, biological effects when ingested. The ability of whey proteins to increase the level of natural anti-oxidants within the body and possibly in stabilizing DNA during cell division is emerging as a premier contribution to population health. Possible modes of action maybe biochemical, including levels of sulphur containing peptide, glutathione and the influence of protein on fat metabolites generated in gut, or immunological or a combination of both. The anticarcinogenic properties of whey proteins are related to compounds rich in sulphur containing amino acids, methionine and cysteine. They contain γ-glutamyl-cysteine residue, which makes cysteine readily available for synthesis of glutathione, a strong xenobiotic deactivating and anti-neoplastic agent. Methionine is utilized for glutathione synthesis in times of cysteine deficiency and it also acts as methyl donor. Hypomethylation of DNA is an important risk factor for cancer at number of sites. Glutathione is, believed to act as an antioxidant, anticarcinogenic and in stabilization and repair of DNA.
Experimental animals fed with four different proteins namely whey, soybean, casein and red meat, as sources of protein, were administered with injection of the carcinogen, dimethylhydrazine. Whey protein-fed animals showed the lowest incidence of colon cancer. Experiments in rodents indicate that the antitumor activity of the dairy products lies with protein fraction and more specifically in the whey protein component of milk.
The anticarcinogenic activity of whey proteins can be attributed to their ability to induce bio-synthesis of folic acid, vitamin B12, riboflavin, retinol and vitamin D. Binding of iron by lactoferrin makes this potential pro-carcinogenic unavailable for intestinal damage. Binding of vitamin B to proteins makes them more bio-available and protects them for being utilized by intestinal microorganisms.
Whey protein isolates (WPI) has been used to treat HIV patients because immunoglobulin and BSA present in it, may stave off this disease.
47.2.4 Bioactive peptides as therapeutic components
Dietary proteins or their precursors may occur naturally in raw food materials, exerting their physiological action directly or upon enzymatic hydrolysis in vitro or in vivo. Several dietary proteins, can act as a source of biologically active peptides. These peptides remain inactive within the parent protein, and are released during gastrointestinal digestion or food processing. Once liberated, the bioactive peptides may provide different functions in vitro or in vivo. Such peptides can be released during hydrolysis by digestive or microbial enzymes. Microbial enzymes from LAB have demonstrated to be able to liberate theses peptides from milk proteins, in various fermented milk products. Upon oral administration, bioactive peptides may affect the major body systems- namely the cardiovascular, digestive, immune and nervous systems. For this reason, the potential of certain peptides sequences to reduce the risk of chronic diseases or to boost natural immune protection has aroused a lot of scientific interest over the past few years. These beneficial health effects may be attributed to known peptide sequences exhibiting, e.g., antimicrobial, antioxidative, antithrombotic, antihypertensive and immunomodulatory activities. The activity of peptides is based on their inherent amino acid and composition and sequence. The size of active sequences may vary from 2-20 amino acid residues, and many peptides are known to possess multi- functional properties. Milk proteins are considered the most important source of bioactive peptides and an increasing number of bioactive peptides have been identified in milk protein hydrolysates and fermented dairy products. The release of various bioactive peptides from milk proteins through microbial proteolysis has been reported
47.2.4.1 Peptides with opoid activity
Opiates are drugs containing opium, with basic substance morphine in it. They have been used since ancient times in medicine to relieve pain and induce sleep. Opoid peptides, are defined as peptides having both an affinity for an opiate receptor and opiate like effects inhibited by naloxone. The major exogenous opioid peptides, b-casomorphins, are fragments of the b-casein sequence 60 – 70. Whey proteins contain opioid – like sequences, obtained from whey namely a-La and b-Lg, in their primary structure. These peptides have been termed a and b-lactorphins. Proteolysis of a-lactalbumin with pepsin produces a-Lactorphin, and while digestion of b-Lactoglobulin with pepsin and then with trypsin, or with trypsin and chymotrypsin, yields b-lactorphin.
47.2.4.2
Peptides with Angiotensin – I-converting enzyme inhibition activity
Angiotensin, a blood polypeptide exists in two forms, the physiologically inactive angiotensin – I and the active angiotensin – II. The inactive form is converted into active by angiotensin – I converting enzyme (ACE), which is a key enzyme in the regulation of peripheral blood pressure. ACE plays a major physiological role in the regulation of local levels of several endogenous bioactive peptides. Casokinin sequences have been found in all casein fractions, but as1 – and b - caseins, in particular, are rich in ACE inhibitory sequences.
47.2.4.3 Anti-thrombotic peptides
Thrombosis is defined as the formation or presence of a blood clot within a blood vessel. Plasma protein called as fibrinogen is produced in the liver and is converted into fibrin, necessary for platelet aggregation. Milk peptides are known to inhibit this platelet fixation. Hydrolysis of bovine k-casein by chymosin constitutes the first stage of milk clotting. In this reaction, one bond (Phe105 – Met106) of k-casein is rapidly hydrolyzed, leading to the release of an insoluble N–terminal fragment (para - k - casein; residues 1 - 105) and a soluble C–terminal fragment (caseinomacropeptide; residues 106 - 169) from which a series of tryptic peptides active in platelet function has been characterized. These peptides are called as Casoplatelins.
47.2.4.4 Antimicrobial peptid
Hydrolysis of lactoferrin by pepsin produces hydrolysates with greater antimicrobial potency. The generated peptide is known as lactoferricin. The iron binding capacity of the hydrolysates is lost, but the antimicrobial activity is not affected by the addition of iron. These results indicate that the antibacterial activity of these lactoferrin hydrolysates is not dependent on iron.
47.2.4.5 Mineral binding peptides
It is well known that casein – derived phosphorylated proteins enhance vitamin D – independent bone calcification in rachitic infants. The extent of phosphorylation is dependent on the casein type. These confer to the proteins the ability to chelae calcium, which is related to their level of phosphorylation; thus αs2> αs1> β> κ. These phosphorylated fragments are believed to play a crucial role in protecting the milk gland against calcification by controlling the calcium phosphate precipitation. Enzymatic hydrolysis of casein using enzymes results in formation of several Caseinophosphopeptides (CPPs). The calcium chelating activity of CPP – fragments in vitro has been attributed to the role of phosphoserine residue in stabilizing the colloidal calcium phosphate of casein micelles.
47.3 Probiotic Dairy Foods
Human gastrointestinal tract (GIT) harbours more than 100 trillion microorganisms belonging to 400 different bacterial species. The number of microbial cells present in GI tract is almost 10 times than the rest of the body cells. A delicate balance exists between beneficial and harmful bacteria present in GIT and any disturbance may lead to abnormalities. About 70% of the body’s immune system is localized in GIT. Incorporation of beneficial bacteria into foods to counteract harmful organisms in the GIT has been the most visible component of this new area. Such microorganisms are termed as “Probiotics”. There is a growing scientific evidence to support the concept that beneficial gut microflora may provide protection against gastrointestinal disorders including gastrointestinal infections, inflammatory bowel diseases, and even cancer.
Probiotics are defined as “living micro-organisms, which upon ingestion in certain numbers exert health benefits beyond inherent basic nutrition”. But interest in this area was initiated by Metchnikoff more than 100 years ago. The concept of probiotic can be traced to the end of the 19th century when Döderlein, for the first time attributed the inhibition of growth of pathogens to lactic acid production by bacteria. As per FAO/WHO definition: “probiotics are live microorganisms which when administered in adequate amounts confer a health benefit on the host”.
The term ‘‘probiotic’’ includes a large range of microorganisms, mainly bacteria but also yeasts. Because these bacteria can stay alive until the intestine and provide beneficial effects on the host health, LAB, non-LAB and yeasts can be considered as probiotics. Some examples of probiotics microorganisms are as follows:
Table 47.3 Probiotic microorganisms
|
Lactobacillus Spp. |
Bifidobacterium spp. |
Other Lactic Acid Bacteria |
Other Species |
|
L. acidophilus |
B. adolescentis |
Enterococcus faecium |
Saccharomyces boulradii |
|
L. casei |
B. animalis |
E. faecalis |
Bacillus cereus |
|
L. cellobiosus |
B. longum |
Streptococcus thermophilus |
|
|
L. fermentum |
B. brevi |
|
|
|
L. lactis |
B. bifidum |
|
|
|
L. helveticus |
B. infantis |
|
|
|
L. reuteri |
B. lactis |
|
|
|
L. brevis L. plantarum |
|
|
|
|
L. curvatus |
|
|
|
47.3.1 Mode of action of probiotics
Although the mechanisms of action of probiotics are largely unknown at the molecular level, a probiotic can act in a number of ways, including the following:
1. By direct interaction within the gut lumen with the complex ecosystem of the gut microbiota, they may produce substances like acids, CO2, H2O2 and bacteriocins that inhibit the growth of harmful microbes,
2. By interaction with the gut mucus and the epithelium, inducing barrier effects, assist digestive processes, enhance mucosal immune and enteric nervous system;
3. Through signaling to the host beyond the gut to the liver, systemic immune system, and other potential organs such as the brain.
The basis for selection of probiotic micro-organisms include safety, functional aspects (survival, adherence, colonization, antimicrobial production, immune stimulation, antigenotoxic activity and prevention of pathogens) and technological details such as growth in milk and other food base, sensory properties, stability, phage resistance and viability. Fermented milk products being a 'live' food, is potentially an excellent vehicle for these beneficial microbial cultures. Several attempts have been made to manufacture probiotic milk products like probiotic dahi, probiotic cheese, probiotic yoghurt and yoghurt drinks. Probiotic dahi developed at NDRI containing Lactobacillus acidophilus and Lactobacillus casei was found to delay the onset of glucose intolerance, hyperglycemia, dyslipidemia, and oxidative stress in high fructose induced diabetic rats.
47.4 Fortified Milk Products
Milk in its natural form is almost unique as a balanced source of man’s dietary need. The various steps in processing and storage have a measurable impact on some specific nutrients. Milk also provides a convenient and useful vehicle for addition of certain nutrients to our diet and has following benefits:
· Easier quality control measure implementation
· Wider consumption by all age groups
· Cost is affordable by target population.
· Higher stability and bioavailability of the added micronutrients
· Addition of fortificants usually caused minimum change in colour, taste and appearance.
Liquid milk fortification with vitamins A and or D is mandatory in several countries. β-carotene is added as a colour-enhancing agent to some milk products such as butter. Dried milk is often fortified with vitamins A and D, calcium, and iron. Milk based infant formula and weaning foods are fortified with a range of vitamins, minerals, and other nutrients such as polyunsaturated fatty acids. Powdered milk used for complementary feeding in Chile is fortified with vitamin C, iron, copper and zinc. However, the milk fortification usually impaired its sensory and processing quality characteristics.
47.4.1 Fortification of milk & milk products with vitamins
Under ambient conditions the water soluble vitamin C and vitamins of the B-complex group such as thiamin, riboflavin, vitamin B6, niacin, pantothenic acid, folic acid, biotin and vitamin B12 are powdered and thus relatively easy to add in milk and other dairy products. The fat soluble vitamins which include vitamin A, D, E and K, exist either as an oil emulsion or as crystals, which may cause processing difficulties during the manufacture of certain fortified dairy products.
One of the problem encountered with the vitamins, is their limited stability in presence of heat, humidity and oxygen. Among the water soluble vitamins, vitamin C, folic acid, vitamin B6 and vitamin B12 are the less stable. In case of fat soluble vitamins vitamin A, D and E are least stable. In order to improve the stability of these vitamins, a number of different coating technologies have been developed such as microencapsulation. When two or more vitamins are added to a food product at the same manufacturing stage, this is commonly done in the form of premix or as blend. Premix is a homogenous mixture of desired vitamins in a dry powder from, whereas a blend is the same for the fat soluble vitamins, but in an oily form.
47.4.2 Fortification of milk and milk products with minerals
Selection of an appropriate mineral fortificant is based on its organoleptic considerations, bioavailability, cost and safety. The colour of iron compounds is often a critical factor when fortifying milk and milk products. The use of highly soluble iron compounds like ferrous sulfate often leads to the development of off-colours and off-flavours due to reactions with other components of the food material. Infant cereals have been found to turn grey or green on addition of ferrous sulfate. Off-flavours can be the result of lipid oxidation catalyzed by iron. The iron compounds themselves may contribute to a metallic flavour. Some of these undesirable interactions with the food matrix can be avoided by coating the fortificant with hydrogenated oils or ethyl cellulose.
Bioavailability of iron compounds is normally stated relative to a ferrous sulfate standard. The highly water-soluble iron compounds have superior bioavailability. Bioavailability of the insoluble or very poorly soluble iron compounds can be improved by reducing particle size. Unfortunately this is accompanied by increased reactivity in deteriorative processes. The problem of low bioavailability of some of the less reactive forms of iron is often circumvented by the use of absorption enhancers like, ascorbic acid, sodium acid sulfate and orthophosphoric acid, added along with the fortificant.
