Module 8. Antimicrobial substances in milk

Lesson 29

ANTIMICROBIAL SUBSTANCES IN MILK

29.1 Introduction

The germicidal properties of milk have been known for years but only recently the progress is made in characterizing some of the antimicrobial substances in milks of different species (i.e. cow and buffalo.Normal raw milk contains different levels of inhibitory substances that restrict the normal development of certain bacteria and sometimes may even kill these. The degree of inhibitory activity varies considerably in milks of different species viz., cow, buffalo, human (as the maximum activity present in human milk), in milks of same animal at different milking. The activity may even vary between milk samples collected from different quarters of udder of same animal.There are two groups of antimicrobial substances in milk.

1. Inherent or naturally occurring substances in milk

2. Non inherent compounds secreted in to milk from other sources

29.2 Naturally Occurring Anti Microbial Substances

These compounds may be specific and non-specific in conferring protection to the new born. In other words the specific factors are produced in response to specific antigen, whereas non specific may have less specific anti-microbial activity.

29.2.1 Specific factors

A. Immunoglobulins

B. Complement

C. Macrophages

D. Bifidus factor

29.2.2  Non specific factors

A. Iron binding protein like lactoferrin

B. Lysozyme

C. Lactanins

D. Lactoperoxidase

E. Vitamin binding proteins and fatty acid

29.3  Specific factors

29.3.1  Immunoglobulin (Ig)

These factors are responsible for immunological protection of newborns. Although in man, prenatal immunity is conferred primarily through the transmission of immunoglobulins from mother across the placenta to the foetal circulation. Buffalo and cow rely on the transfer of immunoglobulins from colostrums into new borne’s circulation post-natally, when gastro-intestinal tract is permeable to intact protein molecules.

IgA is the chief immunoglobulin of milk and confers passive immunity. In human milk, the main Ig is called secretory IgA. It is present in colostrums in concentrations up to 50mg per ml which drops to a level of 1milligram per milliliter in mature milk. The sites of action of IgA are restricted, since this Ig cannot be absorbed from infant gut in significant quantities.

29.3.1.1  Functions of S IgA

· Neutralizes toxins and viruses

· Inhibits bacterial adhesions

· Prevents absorption of food antigens

· Suppresses bacterial growth

· Aids in phagocytosis by bringing about attachment of bacteria to macrophages.

In human milk anti Escherichia coli IgA has been identified that is active against infantile diarrhea.

29.3.2  Complement

Small amounts of nine components of complement have been detected in human milk and these are also associated with the bactericidal properties of milk. These also add to the defense mechanism due to lysis of a variety of potentially pathogens.

29.3.3  Macrophages

Viable leucocytes are present at a concentration of 2 x 10⁶ cells per ml in human colostrums, falling to 2percent of this value in normal milk. About 80 percent of the cells in colostrums are macrophages that are associated with phagocytosis i.e. engulfing and subsequent killing of bacteria many of which are pathogenic in nature.

29.3.4  Bifid us factor

A bifidus factor is a compound that specifically enhances the growth of bifidobacteria in either a product or in the intestines of humans. Several products have been marketed as bifidogenic factors, such as several prebiotics and methyl-N-acetyl D-glucosamine in human milk.Bifidus factor supports the growth of lactobacillus,beneficial bacteria that protect the baby against harmful bacteria by creating an acidic environment, where it cannot survive.

29.3.4.1  Beneficial effects

In breast-fed infant mother’s milk having bifidus factor leads to growth of bifidobacterium. This in turn has these beneficial effects

· Inhibition of enteropathogenic Eschericia coli, Shigella spp. and other pathogens due to production of lactic and acetic acids.

· Supports the growth of Bifidobactriumspp. lactobacillus spp and other probiotic is a beneficial bacteria that protects baby against harmful bacteria by creating an acidic environment.

· Acidic pH in stools that is necessary for absorption of calcium, Fe and phosphorus.

· Synthesis of Vitamin B-complex that helps in infant growth.

In infants receiving baby foods, the following adverse effects can be resulted

· Over growth of Eschericia coli in intestines.

· Increase in alkaline pH of stools due to over growth of Eschericia coli.

· Decrease in synthesis of Vitamin B12.

In such cases, the bacteroids conjugate with bile-salts in intestines and reduce their function in fat absorption.

29.4 Non Specific Factors

29.4.1  Lactanins

The concentration of lactanin fractions viz lactanin 1 (L₁) and lactanin 2 (L₂) in milk varies. The bovine colostrums are rich in L₁ and normal milk contains higher quantities of L₂ fraction. A third fraction L₃ has also been detected in raw milk. This differs from L₁ and L₂ on the basis of its source and inhibitory action.

There is a partial inactivation of L₁ at 60 ˚C per 30 minutes. On heating raw milk to 70 ˚C per 20 minutes at pH 7.0, L₁ is completely destroyed leaving behind L₂ fraction in relatively higher concentration. It is proved that these two compounds L₁and L_2, when interact are more bactericidal than either one alone.

Lactanin is mostly active against Streptococcus pyogenes. The ability of these microbesto grow in udder is due to the fact that lactanin has little activity under anaerobic conditions prevailing there. Addition of glutathione, cystein and other reducing substances in milk reduces the anti microbial action of lactanins in milk. However, other factors in milk are stimulatory for the growth of Streptococcus pyogenes in udder that causes mastitis.

29.4.2  Lysozyme

It is present in different amounts in milks of different species. The average lysozyme content of human milk (30mg per 100 ml) is 3000 times more than that of bovine milk. Such significant difference in the lysozyme content may have physiological or nutritional implications on infant feeding. Breast fed infants have greater resistance to enteric infections than those fed on artificial foods, and lysozyme is possibly associated with the mechanism of natural immunity and local resistance.

Most of the Gram positive bacteria are very sensitive to the lytic action of lysozyme due to the hydrolysis of ß 1-4 glycosidic linkage of the peptidoglycon component of their cell wall. However Gram negative bacteria are normally lysozyme resistant.

29.4.3  Lactoferrin (Iron binding protein)

Lactoferrin (previously known as lactotransferrin or lactosiderophilin) is one of the important anti microbial substance in milk. It is a red glycoprotein that resembles blood serum transferring and is found in varying concentrations in milks of different species. Human milk has a higher amount of this protein than cow or buffalo milk. The concentration of lactoferrin in human and buffalo colostrums is very high (1.2 g per 100 ml and 100mg per ml, respectively) and falls during lactation to 160mg per 100 mland 50 ml per ml, respectively in mature milk.1 to 4percent of lactoferrin in human milk is saturated with iron. It is mainly a whey protein (70percent found in whey 30percent bound to casein and fat).

The inhibitory effect of lactoferrin is based on its ability to squeeze iron out from the medium, thereby making it unavailable to the microbes that compete with the lactoferrin for limited amount of iron as an essential growth factor. Its bactericidal action is potentiated by adding iron invitro or due to the high citrate and low bicarbonate concentrations, as in bovine milk.

It has antibacterial property against microorganisms likeStaphylococcus aureus, Staphylococcus albus, Pseudomonas aeruginosa, Vibrio cholerae, Clostridium tyrobutyricum, Bacillus stearothermophilus,and Bacillus subtlis.

Apolactoferrin (iron free lactoferrin) at 0.02mg per ml concentration in milk inhibits the growth of Enterobacter aerogenes and Eschericia coliboth.

29.4.4  Lactoperoxidase

Lactoperoxidase is present in cow milk in considerable amount (30mg/ml) but is absent in human milk. This enzyme has been found to be identical with L₂ fraction of lactanin. In bovine raw milk the inhibitory action of this enzyme is due to formation of an antimicrobial system or LP system under normal conditions.

The secretion of thiocyanate component in milk is governed by nutrition of the animal. Hydrogen peroxide can be contributed either by polymorpho-nuclear-leucocytes or by hydrogen peroxide producing udder micro flora, e.g. Streptococci

The lactoperoxidase system is bacteriostatic to microorganism like Group B and Group N Streptococci, whereas it is bactericidal for Group A Streptococci, E.coli, Salmonella typhimurium etc. It protects calf against enteric problems and may have some role in providing defense mechanism to mammary gland against infections.

Lately attempts have been made to exploit the LP system as a method of preservation (i.e. cold sterilization) for raw milk. Unlike in conventional method of preservation by heat treatment, there is no inactivation of antimicrobial substances and other heat labile constituents of raw milk (like certain vitamins). In these efforts, the two components of LP system viz. thiocyanate and hydrogen peroxide are added from outside to attain their balanced proportions.

29.4.5  Vitamin binding proteins

There are certain proteins present in milk of some species that bind with some essential vitamins firmly, thereby making bacteria starved (that are fastidious for such vitamins). These proteins particularly bind with Vitamin B₁₂ and Folate in milk. In milk of both cow and human the binding capacity of folate binding protein is far in excess of natural folate content. The same is true for Vitamin B₁₂in human milk but in cow milk there is little or no excess of unsaturated binding protein. Hence, in presence of unsaturated binding proteins, the growth of microbes requiring vitamin B₁₂and folate is inhibited. The vitamin binding proteins have following functions:

· Regulate bacterial growth in gastro intestinal tract of suckling animals by denying the vitamins to specific bacteria

· Aid in the absorption of vitamins in the gut

· Conserve vitamin B₁₂by aiding the re-absorption of vitamin B₁₂lost in bile

· Play a role in the mammary gland prior to milk secretion

29.4.6  Fatty acids

Milks of different species also contain free fatty acids in appreciable concentrations. Generally, medium chain fatty acids, especially octonoic and deconoic acids and some polyunsaturated chains fatty acids are more active against Gram positive microorganisms. Lower fatty acids after being freed from the glycerides of milk fat are inhibitory to many bacteria including lactobacilli and lactic streptococci. Free fatty acids in milk result either from the action of lipase naturally present in milk or from the lipolytic activity of certain microbes. Although, many microorganisms require fatty acids, as nutrients for growth, but when their concentration exceed certain limits in medium growth is strongly inhibited.

29.5  Non Inherent Anti Microbial Substances

Antibiotics, sanitizers and other chemical substances used at some stage in dairy industry may gain access in to milk and exert antimicrobial effect there.

29.6  Significanceof Anti Microbial Substances in Milk                                         

29.6.1 Health benefits

The primary significance of the antimicrobial substances detected in milk can be related to their possible role in conferring protection to mammary gland and neonates in mammals. The new borne therefore should essentially be given the colostrums, which is very rich in most of these antimicrobial factors. ‘Breast feeding is the best for infants’ derives its substance from the abundance of antimicrobial factors in mother’s milk, as against animal milk based infant formulae.

29.6.2 Preservation of milk

Although, the presence of antimicrobial factors cannot solely be dependent upon to prevent or even to retard significantly the development of mixed population of microbes in raw milk, some microbes get markedly affected. However, an artificial exploitation of milks natural antimicrobial system called lactopeoxidase system, for developing a cold sterilization like process, can prove effective in preservation of raw milk. Such a process holds great promise under tropical farm conditions in countries like India.

29.6.3 Processing problems

Where raw milk is used for product making, e.g. in cheese making, the acid development in cheese vat may be retarded by inhibition of starter activity due to the presence of antimicrobial substances. In starter culture propagation, the heating of milk (80˚C) is essential as it inactivates factors that may interfere with the normal starter growth.