PRESERVATION OF BIOCHEMICAL METHODS WITH SPECIAL EMPHASIS ON ENZYMES - Food preservation technology

1.0          Food preservation and requirement of bio-chemical control methods

In the production of food, it is crucial to take proper measures for ensuring its safety and stability during the shelf-life. Food preservation is carried out to maintain the quality of raw material and physicochemical properties as well as functional quality of the product whilst providing safe and stable products.

Despite improved manufacturing facilities and implementation of effective process control procedures such as Hazard Analysis and Critical Control Points (HACCP) in the food industries, the number of food borne illnesses has increased. Nowadays consumers favor food with few chemical preservatives. As a result there is increased interest in the preservation through biochemical methods because of their safe association with human foods. Several metabolic products produced by these enzymes have antimicrobial effects, including organic acids, fatty acids, hydrogen peroxide and diacetyl.

2.1          Glucanases

Glucanases are the extra cellular enzymes that break down a glucan, a polysaccharide made of several glucose sub-units. As they perform hydrolysis of the glycosidic bond, they are hydrolases which is secrete by Lysobacter enzymogenes and it is characterized for its propensity to lyse fungi and other micro organisms. Glucanases are capable of degrading the major cell wall components of fungi and oomycetes. L. enzymogenes also produces other factors, such as antibiotics that are antagonistic to the growth of microbes.

Functions 

This enzyme is commonly used as a preservative in wine industry where the marine-derived Williopsis saturnus was found to produce very high killer toxin activity against the pathogenic yeast Metschnikowia bicuspidate which is used in wine industry and isolated from the diseased crab. But the purified β-1,3-glucanase from W. saturnus had no killer toxin activity but could inhibit activity of the toxin produced by the same yeast. In contrast, the toxin produced had no β-1,3-glucanase activity.

Mechanisms of the inhibition may be that the β-1,3-glucanase competed for binding to β-1,3-glucan on the sensitive yeast cell wall with the toxin, causing decrease in the amount of the toxin bound to β-1,3-glucan on the sensitive yeast cell wall and the activity of the toxin against the sensitive yeast cells.

Mode of action

Enzyme systems for yeast cell lysis are usually a mixture of several different enzymes, including one or more beta (1-3) glucanase (lytic and nonlytic), protease, beta (1-6)  glucanase, mannanase and chitinase, which act synergistically for lysing the cell wall.

Enzymatic cell lysis of yeast begins with binding of the lytic protease to the outer mannoprotein layer of the wall. The protease opens up the protein structure, releasing wall proteins and mannans and exposing the glucan surface below.

The glucanase then attacks the inner wall and solubilizes the glucan. In vitro, this enzyme cannot lyse yeast in absence of reducing agents, such as dithiothreitol or b-mercaptoethanol, because the breakage of disulphide bridges between mannose residues and wall proteins is necessary for appropriate exposition of the inner glucan layer. When the combined action of the protease and glucanase has opened a sufficiently large hole in the cell wall, the plasma membrane and its content are extruded as a protoplast.

In osmotic support buffers containing 0.55–1.2 M sucrose or mannitol, the protoplast remains intact, but in dilute buffers it lyses immediately, releasing cytoplasmic proteins and organelles, which may themselves lyse. Meanwhile, proteins released from the wall and the cytoplasm could be subject to attack by product-degrading protease contaminants in the lytic system or in the yeast cells themselves.

 2.2         Phenoloxidases

Phenoloxidases (POs) are a group of copper proteins including tyrosinase, catecholase and laccase. In several insects and crustaceans, antibacterial substances are produced through the PO cascade, participating in the direct killing of invading microorganisms. However, although POs are widely recognised as an integral part of the invertebrate immune defense system.

Functions

Among immune defence mechanisms, phenoloxidases are a group of copper proteins including tyrosinase, catecholase and laccase, which are the rate limiting enzymes in melanisation, and play an important role in immune defence mechanisms in invertebrates. In these organisms, phenoloxidases exist as an inactive form, prophenoloxidase.

Mode of action

Pathogen associated molecular patterns (PAMPs), such as peptidoglycans or lipopolysaccharides from bacteria, or β-1,3-glucans from fungi, are recognized by pattern-recognition receptors (PRRs). This will trigger the activation of a cascade of serine proteases that activates polyphenol-activating enzymes (PPAs), and therefore, the conversion of the pro-enzyme prophenoloxidase into phenoloxidase.

The three types of phenoloxidases can oxidise o-diphenols, such as L-3,4-dihydroxyphenylalanine (L-DOPA; catecholase activity). However, among these three enzymes, only tyrosinases can hydroxylate monophenols, such as L-tyrosine (monophenoloxidase activity), and only laccases can oxidise m- and p-diphenols, or aromatic compounds containing amine groups, such as p-phenylenediamine (PPD; laccase activity).

Different roles have been attributed to phenoloxidases, especially in haemolymphatic immune defence mechanisms, and phenoloxidase-generated reactive compounds are known to contribute to the destruction of microbial cells.

2.3          Lactoferrin

Lactoferrin is a non-haem iron binding protein that is part of the transferring protein family, along with serum transferrin, ovotransferrin, melanotransferrin and the inhibitor of carbonic anhydrase, whose function is to transport irons to blood serum.

Some of the numerous properties of lactoferrin, related to its protective functions, can be attributed to its iron binding activity, whereas other properties of lactoferrin are independent.

There are three forms of lactoferrin depending on its iron saturation; apolacto ferrin (iron free), monoferric form (one ferric ion) and hololactoferrin (binding two Fe³⁺ ions). The tertiary structure of hololactoferrin and apolactoferrin is different.

Four amino acid residues are very important for iron binding (histidine, twice tyrosine and aspartic acid), while an arginine chain is responsible for binding the carbonate ion.

Antibacterial activity and mechanisms of action of lactoferrin

The effect of lactoferrin was demonstrated against many bacteria, such as B.subtilis , clostridium spp, micrococcus sp, etc. which can attach themselves to the host cell. Lactoferrin has been also shown to exert anti-microbial activity against some yeasts and fungi such as C.albicans, C.krusei, etc.

Bacteriostatic activity

Lactoferrin ability to bind free iron can inhibit growth of many species of bacteria (and fungi). A lack of iron inhibits the growth of iron-dependent bacteria such as E.coli. In contrast lactoferrin may serve as iron donor, and in this manner, support the growth of some bacteria with lower iron demands such as Lactobacillus spp or Bifido bacterium spp. generally considered as beneficial.

                Bacteriocidal activity

Independent from iron binding and involving the basic N-terminal region of lactoferrin. Lactoferrin can disrupt or possibly even penetrate bacterial cell membranes, and that the isolated N-terminal basic peptides, named lacto ferricins, were more potent than the intact protein.

                Additional antibacterial activities

Biofilm formation, which represents a colonial organization of bacterial cells, is a well studied phenomenon where bacteria also become highly resistant to host cell defense mechanisms and antibiotic treatment. Lactoferrin play an important role in the innate immunity by blocking the biofilm development by Ps. Auruginosa. At concentrations lower than those killing or preventing the growth and with iron chelating activity, lactoferrin stimulates twitching, a specialized form of surface motility, causing the bacteria to wander across the surface instead of forming clusters of biofilm.

                Antifungal activity

Lactoferrin shows a significant antifungal activity by its ability to bind and sequester environmental ion. And the lactoferrin can kill some fungus by altering the permeability of the cell surface, as it does with bacteria.

Applications of lactoferrin in industry

Lactoferrin is already used in a wide range of products including infant formulae, sport and functional foods.

• Milk based infant formulae – improved resistance against pathogens and oro-gastro-intestinal microflora, anti oxidant
• Yoghurt  – improved resistance against pathogens, anti infection and oro-gastro-intestinal microflora, anti oxidant
• Health supplement – aid in iron absorption. Eg :- for pregnant women, immune aid

2.4          glutathione peroxidase

All milks contain a certain amount of somatic cells represented by polymorphonuclear cells (PMN), lymphocytes and macrophages. In bacterial infection and other inflammation processes affecting the mammary tissue, the number of somatic cells in milk increases, especially the PMN level. During mastitis, PMN cells migrate from the peripheral blood into milk, through the mammary epithelium. In many countries, somatic cell count (SCC) is used as an indicator for the hygienic milk quality. An increased SCC in a bulk tank milk indicates that a significant proportion of milk originates from mastitis cows.

More than 140 different microorganisms are recognized to cause mastitis. They are classified into four different groups: contagious, environmental, opportunistic and others. Most mammary gland infections are caused by only a few types of bacteria, including streptococci (Streptococcus agalactiae), staphylococci (Staphylococcus aureus) and coliforms (Corynebacterium bovis).

Functions

Glutathione-peroxidase (GPx) is widespread in the cytoplasm of animal cells. The function of this enzyme is to protect cells against the damaging effects of peroxides, as part of an antioxidant enzymatic system. Milk contains low levels of GPx, more than 90% being represented by extra cellular form. The function of this enzyme in milk is not yet fully known, it is the only known enzyme that fixes 30% of total selenium (Se), an important element of diet. It is also known that milk GPx varies according to species and diet.

Mode of action

When bacteria invade and colonize the mammary gland, macrophages respond by initiating the inflammatory response, attracting polymorphonuclear (PMN) cells in milk to kill bacteria. More than 90% of somatic cells found in infected glands are neutrophils (PMN). Antibacterial activity of neutrophils is mediated via reactive oxygen species (ROS).

Glutathione peroxidase is an antioxidant enzymes in milk, it catalyses the reduction of different peroxides aided by glutathione or other reducing substrates. The average value for GPx activity in normal milk was 23 U.ml^-1. Adding glutathione peroxidase will increase the activity of GPx and preserve the milk in a more effective way by great activity.

2.5          lacto peroxidase

Refrigeration is the most commonly used method to stop or retard the deterioration of milk on its way from the farm to the dairy industry. The lactoperoxidase system (LPS) has been introduced as an alternating way of preserving milk.

The lactoperoxidase system

It consists of lactoperoxidase (LS) and two substrates; thio cyanate ions (SCN^-) and hydrogen peroxide (H₂O₂).

                Lactoperoxidase

Lactoperoxidase is a glycoprotein consisting of a single peptide chain containing 612 amino acid residues. This enzyme is an oxidoreductase and catalyses the oxidation of thiocyanate at the expense of hydrogen peroxide to generate intermediate products with antimicrobial properties against bacteria, fungi and viruses.

                Thiocyanate ion

Thiocyanate ions are present in mammary, salivary and thyroid glands and their secretions, in organs such as the stomach, kidney and in fluids such as synovial, lerebral, cervical, and spinal fluids.

                Hydrogen peroxide

Hydrogen peroxide is not normally detected in raw milk, but it may be generated endogenously, for example, by polymorphoneuclear leucocytes in the process of phagocytosis, in addition many lactobacilli, lactococci, and streptococci produce sufficient hydrogen peroxide under aerobic conditions, to activate LPS.

Antibacterial activity of lactoperoxidase

The oxidation of the thio groups (-SH) of enzymes and proteins is of crucial Importance in the bacteriostatic and/or bacteriocidal effect of the LPS; the structure damage of cytoplasmic membranes by the oxidation of –SH groups results in a leakage of potassium ions, amino acid and peptide into the medium, thus the uptake of glucose, amino acids, purines, pyrinidines in the cell and synthesis of proteins. DNA and RNS are also inhibited.

On the other hand, the anti microbial activity of LPS can be inhibitedby reducing agents containing –SH groups, such as cysteine, glutathione, mercapto-ethanol, dithiothreitol, and sodium hydro sulphite either by direct binding to the haem group or by scavenging OSCN^-.

3.0          References

• Application of Alternative Food-Preservation Technologies to Enhance Food Safety and Stability by Antonio Bevilacqua et al.
• Enzymatic lysis of microbial cells by Oriana Salazar and  Juan A. Asenjo
• Enzyme-Assisted Processing Increases Antimicrobial and Antioxidant Activity of Bilberry by RIITTA PUUPPONEN-PIMIA et al.
• Food preservation by Nicholas J.Russel and Grahame V.Gould
• http://prof.dr.semih.otles.tripod.com/enzymesused/theroleof/theroleof.htm
• http://stalischem.wordpress.com/2010/04/25/major-enzyme-applications-in-food-industry/
• http://www.deepdyve.com/lp/elsevier/xylan-chitosan-conjugate-a-potential-food-preservative-Aqig5Q0HK2
• http://www.hindawi.com/journals/er/2010/862537/tab2/
• Involvement of -Glucans in the Wide-Spectrum Antimicrobial Activity of Williopsis saturnus var. mrakii MUCL 41968 Killer Toxin by Cyril Guyard et al.
• Mutagenesis of β-1,3-Glucanase Genes in Lysobacter enzymogenes Strain C3 Results in Reduced Biological Control Activity Toward Bipolaris Leaf Spot of Tall Fescue and Pythium Damping-Off of Sugar Beet by Jeffrey D. Palumbo et al.
• New Methods of Food Preservation By Grahame W. Gould
• Preservation and fermentation: past, present and future by R. Paul Ross et al.