How cancer spread? (pt.2)

Angiogenesis is required for tumors to grow beyond a few millimeters in diameter

Tumors release signaling molecules that trigger angiogenesis- that is, growth of blood vessels- in the surrounding host tissues, and these new vessels are required for tumors to grow beyond a tiny, localized clump of cells.

Two experiments showing the requirement for angiogenesis.

(a) Cancer cells were injected into an isolated rabbit thyroid gland that was kept alive by pumping a nutrient solution into its main blood vessel. The tumor cells, alive but dormant in the isolated thyroid gland,  fail to link up to the organ's blood vessels, and the tumor stops growing when it reaches a diameter of roughly 1-2 millimeters.

(b) Cancer cells were either injected into the liquid-filled anterior chamber of a rabbit's eye, where there are no blood vessels, or they were placed directly on the iris. Tumor cells in the anterior chamber, nourished solely by diffusion, remain alive but stop growing before the tumor reaches a millimeter in diameter. In contrast, blood vessels quickly infiltrate the cancer cells implanted on the iris, allowing the tumors to grow to thousands of times their original mass.

Blood vessels growth is controlled by a balance between Angiogenesis activators and inhibitors

When normal cells were placed in the chamber surrounded by a filter , they do not stimulate blood vessels growth. In contrast, cancer cells produce molecules that diffuse through the tiny pores in the filter and activate angiogenesis in the surrounding host tissue.

The main angiogenesis-activating molecules are proteins called vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF), normal cells as well as cancer cells produce these proteins. When cancer cells release these proteins into the surrounding tissue, they bind to receptor proteins on the surface of the endothelial cells that form the lining of blood vessels. This binding also 

secrete protein-degrading enzymes called metalloproteinases (MMPs). The MMPs break down the ECM, thereby permitting the endothelial cells to migrate into the surrounding tissues. As they migrate, the proliferating endothelial cells become organized into hollow tubes that develop into new blood vessels.

Also, for angiogenesis to proceed, these proteins must overcome the effects of angiogenesis inhibitors ,like angiostatin, endostatin and thrombospondin, that can restrain the growth of blood vessels. A finely tuned balance between the concentration of angiogenesis inhibitors and activators determines whether a tumor will induce the growth of new blood vessels. When tumors trigger angiogenesis, it is usually accomplished by an increase in the production of angiogenesis activators and a simultaneous decrease in the production of angiogenesis inhibitors.

Cancer cells spread by invasion and metastasis.

 The ability of cancer cells to spread is based on two distinct mechanisms:

Invasion refers to the direct migration and penetration of cancer cells into neighboring tissues, whereas, metastasis involves the ability of cancer cells to enter the bloodstream (or other body fluids) and travel to distant sites, where they form tumors- called metastases.

The events following angiogenesis can be grouped into three main steps.

First, cancer cells invade surrounding tissues and penetrate through the walls of lymphatic and blood vessels, thereby gaining access to the bloodstream. Second, the cancer cells are transported by the circulatory system throughout the body. And third, cancer cells leave the bloodstream and enter various organs, where they establish new metastatic tumors. If cells from the initial tumor fail to complete any of these steps, or if any of the steps can be prevented, metastasis will not occur.

Changes in cell adhesion, motility and protease production allow cancer cells to invade surrounding tissues and vessels.

Several mechanisms make the invasion behavior possible. The first involves changes in the cell surface proteins that cause cells to adhere to one another. Such proteins are often missing or defective in cancer cells, which allows cell to separate from the main tumor more readily. One crucial molecule is E-cadherin, a cell-cell adhesion protein whose loss underlies the reduced adhesiveness of many epithelial cancers. Cancer cells lack E-cadherin.

A second factor underlying invasive behavior is the increased motility of cancer cells, which is stimulated by signaling molecules produced by surrounding host tissues or by the cancer cells themselves. Some of these signaling molecules can act as chemoattractant that serve as guiding signals that attract migrating cancer cells. Activation of the Rho family GTPases (Rho, Rac and Cdc42) plays a central role in the stimulation of cell motility that leads to invasion and metastasis.

Another trait is the ability of cancer cells to produce proteases that degrade protein-containing structures that form the backbone of the basal lamina, that would otherwise act as barriers to cancer cell movement.  One such protease is plasminogen activator, an enzyme that converts the inactive precursor plasminogen into the active protease plasmin. The plasmin performs two tasks: (a) It degrades components of the basal lamina and the ECM, thereby facilitating tumor invasion. (b) it cleaves inactive precursors of matrix metalloproteinases into active enzymes. These proteases allow cancer cells to penetrate basal lamina, facilitating its migration until they reach tiny blood or lymphatic vessels.

Relatively few cancer cells survive the voyage through the bloodstream.

The bloodstream is a relatively inhospitable place for most cancer cells and fewer than one in a thousand cells survives the trip to a potential site of metastasis.

 An experiment designed to find out that if these successful cells are simply random tumor cell population or they are especially well suited for metasizing. In this exp., mouse melanoma cells were injected into the tail vein of a mouse. After a small number of metastases arose in the lungs, cells from these lung tumors were removed and injected into another mouse. When this cycle was repeated ten times, the final population of melanoma cells produced many more lung metastases than were produced by the original cells. This means that  Initial melanoma consisted of a heterogenous population of cells with differing properties and that repeated isolation and reinjection of cells derived from successful metastases gradually selected for those cells that were best suited for metastasizing. 

Cell populations that are derived from a single initial cell, are called clones.

Blood flow patterns and organ- specific factors determine the sites of metastasis.

Although the bloodstream carries cancer cells throughout the entire body, metastases develop preferentially at certain sites. One factor responsible for this specificity is related to blood- flow patterns. Based solely on size considerations, circulating cancer cells are most likely to become lodged in capillaries. After becoming stuck there, cancer cells penetrate the walls of these tiny vessels, enter the surrounding tissues, and seed the development of new tumors. As the first capillary bed by the cancer cells will be in the lungs, lungs are the most frequent site of metastasis.

For cancers of the stomach and colon, cancer cells entering the bloodstream are first carried to the liver, where the vessels break up into a bed of capillaries. The interactions between cancer cells and the microenvironment in the organs they are delivered to gives the reason why cancer cells grow best at particular sites. For e.g., prostrate cancer commonly metastasizes to bone. Bone cells produce specific growth factors that stimulate the proliferation of prostrate cancer cells. 

The immune system influences the growth and spread of cancer cells.

Immune system can attack and destroy foreign cells. Of course, cancer cells are not literally foreign but they often exhibit molecular changes that might allow the immune system to recognize the cells as being abnormal. Individuals treated with immunosuppressive drugs can develop many cancers. Most of the cancers that occur at increased rates in people with AIDS are caused by the viruses. 

Tumors are heterogeneous populations of cells that express different antigens. Those cells containing antigens that elicit a strong immune response are likely to be attacked and destroyed, while cells that either lack or produce smaller quantities of such antigens are more likely to survive and proliferate. So as tumors grow, there is a continual selection for cells that elicit a weaker immune response.

Some cancer cells produce molecules that kill or inhibit the function of T lymphocytes. Tumors may also surround themselves with dense supporting tissue that shields them from immune attack.

Cancer cells simply divide so quickly that the immune system cannot kill them fast enough to keep  

tumor growth in check. The larger a tumor grows, the easier it becomes to overwhelm the immune system. Cells with weaker immune response are selected for tumor growth. 

The tumor microenvironment influences tumor growth, invasion and metastasis.

Tumor behavior is influenced by interactions between tumor cells and the surrounding tumor microenvironment. For example, angiogenesis is triggered by growth factors released by tumor cells that act on normal endothelial cells in the surrounding tissue. Proteases produced by both facilitate invasion by degrading the components of ECM. Mobility and direction is influenced by signaling molecules. Penetration and then metastases is stimulated by growth factors.

The tumor microenvironment can also contain cells and molecules that hinder invasion and metastasis. For e.g., TGF-beta, a potent inhibitor of proliferation for many cell type. Sometimes even the cancer cells start secreting TGF-beta themselves, which inhibits the growth of surrounding normal cells and allows cancer cells to reproduce and invade surrounding tissues more rapidly because of the decreased competition from neighboring cells.  

CANCER CELLS

Diseases in which tissues grow and spread unrestrained throughout the body, eventually choking off life describe the term "cancer". Cancers can originate in almost any organ. Depending on the cell type involved, they are grouped into several different categories: Carcinoma, which account for about 90% of all cancers, arise from the epithelial cells, for example, lung, breasts and colon cancer. Sarcomas, develop from supporting tissues such as bone, cartilage, fat and muscle. Lymphomas (tumors that grow as solid masses of tissue) and Leukemias (cancer cells proliferate mainly in the blood) arise from cells of blood and lymphatic origin.
The danger posed by the disease comes from a combination of two properties: 
the ability of cells to proliferate in an uncontrolled way and
 their ability to spread through the body.

Tumor arises when the balance between cell division and cell differentiation or death is disrupted.

A cancer is an abnormal type of tissue growth in which some cells divide and accumulate in an uncontrolled, relatively autonomous way, leading to a progressive increase in the number of dividing cells. The resulting mass of growing tissue is called a tumor (or neoplasm). 

Cell differentiation is the process by which cells acquire the specialized properties that distinguish different types of cells from each other. As cells acquire these specialized traits, they generally lose the capacity to divide. Cell division is carefully balanced with cell differentiation and death so that no net accumulation of dividing cells takes place. 

For example in the skin the new cells are generated by the basal layer giving rise to two cells with different fates. One cell stays in the basal layer and retains the capacity to divide, whereas other cell loses the capacity to divide and moves toward the outer skin surface. Eventually it dies and is shed from the outer skin. This arrangement ensures that there is no increase in the number of dividing cells. 

In tumors, this finely balanced arrangements is disrupted and cell division is uncoupled from cell differentiation and death.  This give rise to two cells that both continue to divide, thereby feeding a progressive increase in the number of dividing cells. As the dividing cells accumulate, the normal organization and function of the tissue gradually become disrupted, giving rise to tumor.

Based on the differences in their growth patterns, tumors are classified as either benign tumor or malignant. Benign tumors grow in a confined local area and are rarely dangerous. In contrast, malignant tumors are capable of invading surrounding tissues, entering the bloodstream, and spreading to distant parts of the body. 

The term 'cancer' refers to any malignant tumor- that is, any tumor that can spread from its original location to other sites.

 Cancer cell proliferation is anchorage independent and insensitive to population density

Normal cells don't grow well in culture if they are suspended in a liquid medium or in a semisolid material such as soft agar. But when provided with a solid agar they can attach to, normal cells become anchored to the surface, spread out, and begin to proliferate. In contrast, cancer cells grow well not only when they are anchored to a solid surface but also when they are freely suspended in a liquid or semisolid medium. Cancer cells are therefore said to exhibit anchorage-independent growth.

Normal cells get self- destructed by apoptosis in the absence of cell surface proteins called integrins. Triggering apoptosis in the absence of proper anchorage is one of the safeguards that prevents normal cells from floating away and setting up housekeeping in another tissue. Because cancer cells are anchorage independent, they circumvent this safeguard.

Cancer cells also differ from normal cells in their response to crowded conditions in culture. Once the monolayer stage is reached, cell division stops in case of normal cells. This phenomenon is referred as density dependent inhibition of growth. Cancer cells exhibit reduced sensitivity to this phenomena and thus do not stop dividing even when the monolayer stage is reached. instead, cancer cells continue to divide and gradually begin piling up on one another.

Cancer cells are immortalized by mechanisms that maintain telomere length.

When most normal cells are grown in culture, they divide a limited number of times. But cancer cells exhibit no such limit and continue dividing indefinitely, behaving as if they were immortal. This is because, when normal cells divide, the telomeric DNA sequences are lost from the ends of each chromosome every time DNA replicates. Most cancer cells accomplish this feat by producing telomerase, the enzyme that adds telomeric DNA sequences to the ends of DNA molecules. Another alternative method employs enzymes that exchange DNA sequence information between chromosomes. By one mechanism or other, cancer cells maintain telomeric length above a critical threshold and thereby retain the capacity to divide indefinitely. 

Defects in signaling pathways, cell cycle controls, and apoptosis contribute to uncontrolled proliferation.

Cell proliferation is regulated by protein growth factors that bind to cell surface receptors and activate signaling pathways within the targeted cells. While cells do not usually divide unless they are stimulated by the proper growth factor, cancer cells circumvent this restraining mechanism through alterations in signaling pathways that create a constant signal to divide. 

Normal cells become arrested at the restriction point that controls the progression from G to S phase under suboptimal conditions. However, cancer cells continue to proliferate, escaping this point. if conditions are extremely adverse, they will eventually die at random points in the cell cycle rather than arresting in G1. This abnormal behavior occurs because cell cycle controls do not function properly in cancer cells. Cancer cells are also unresponsive to internal conditions, such as DNA damage.

Cancer cells have various ways of blocking the pathways that trigger apoptosis; this allows them to survive and proliferate under conditions that would normally cause death. 

Urothelial carcinoma

SERUM PROTEINS

Proteins are the most abundant compounds in your serum ( the rest of your blood when you remove all the cells). They are the basic components of enzymes, many hormones, antibodies, and clotting agents. Proteins act as transport substances for hormones, vitamins, minerals, lipids, and other materials. In addition, proteins help balance the osmotic pressure of the blood and tissue. Osmotic pressure is part of what keeps water inside a particular compartment of your body. Proteins play a major role in maintaining the delicate acid-alkaline balance of your blood. Finally, serum proteins serve as a reserve source of energy of your tissues and muscle when you are not ingesting an adequate amount.
The major measured serum proteins are divided into two groups, albumin, and globulins. There are four major types of globulins, each with specific properties and actions.
A typical blood panel will provide four different measurements - the total protein, albumin, globulins, and the albumin globulin ration.

BLOOD PROTEINS:

1. Albumins = 3-5 g/dL (55%) - Create and maintain oncotic pressure; transport insoluble molecules
2. Globulins = 2-2.5 g/dL (38%) - participate in immune response
3. Fibrinogen = 0.2- 0.45 g/dL (7%) Blood coagulation
4. Regulatory proteins (<1%) Regulation of gene expression
5. Clotting factors (<1%) Conversion of fibrinogen into fibrin

TOTAL PROTEIN

Because the total protein represents the sum of albumins and globulins, it is more important to know which protein fraction is high or low than what is the total protein.

Optimal Range = 7.2 to 8.0 g/dL

Total protein may be elevated due to:

• Chronic infection (including tuberculosis)
• Liver dysfunction
• Collagen vascular disease (Rheumatoid arthritis, Systemic lupus, Scleroderma)
• Hypersensitivity states
• Dehydration (diabetic acidosis, chronic diarrhea etc.)
• Hemolysis
• Cryoglobulinemia
• Alcoholism
• Leukemia (cancers that usually begin in the bone marrow and result in high no. of abnormal WBCs )
• Multiple myeloma (a cancer of plasma cells. Plasma cells develop from B lymphocytes. Their primary function is to produce antibodies. In people with multiple myeloma the malignant plasma cells produce only one type of intact (whole) immunoglobulin in large amounts and/or produce an excess of only one of the light chains, or rarely heavy-chains only types)

Total protein may be decreased due to:

• Malnutrition (insufficient intake and/or digestion of proteins)
• Malabsorption (celiac disease- autoimmune disorder that can occur in genetically predisposed people where the ingestion of gluten leads to damage in the small intestine)
• Liver disease (insufficient production of proteins)
• Diarrhea (loss of proteins through GI tract)
• Severe burns (loss or proteins through the skin)
• Hormone imbalance that favor breakdown of tissue
• Loss through the urine in severe kidney disease (proteinuria)
• Low albumin
• Low globulins
• Dilution of protein due to extra fluid held in the vascular system (during pregnancy)

ALBUMIN:

Albumin is synthesized by the liver using dietary protein. Its presence in the plasma creates an osmotic force that maintains fluid volume within the vascular space. A very strong predictor of health; low albumin is a sign of poor health and a predictor of a bad outcome.

Optimal Range: 3 to 5 g/ 100ml

Albumin levels may be elevated due to:

• Dehydration (actual)
• Congestive heart failure\
• Poor protein utilization
• Glucocorticoid excess (can result from taking medications with cortisone effect, the adrenal gland overproducing cortisol, or a tumor that produces extra cortisol like compounds)
• Congenital

Albumin levels may be decreased in :

• Hypothyroidism
• Chronic debilitating disease (e.g., RA)
• Malnutrition (protein deficiency)
• Dilution by excess water (drinking too much water, termed as 'polydipsia' or excess administration of IV fluids)
• Kidney losses (nephrotic syndrome)
• Protein losing- enteropathy (protein is lost from the GI tract during diarrhea)
• Skin losses (burns, exfoliative dermatitis)
• Liver dysfunction (the body is not synthesizing enough albumin and indicates very poor liver function)
• Insufficient anabolic hormones such as growth hormone, DHEA, testosterone etc.

GLOBULINS:

Globulins are proteins that include gamma globulins (antibodies) and a variety of enzymes and carrier/transport proteins. The specific profile of the globulins is determined by protein electrophoresis (SPEP), which separates the protein according to size and charge. There are four major groups that can be identified : gamma globulins, beta globulins, alpha 2- globulins and alpha- 1 globulins. Once the abnormal group has been identified, further studies can determine the specific protein excess or deficit. Since the gamma fraction usually makes up the largest portion of the globulins, antibody deficiency should always come to mind when the globulin level is low. Antibodies are produced by mature B lymphocytes called plasma cells. While most of the other proteins in the alpha and beta fractions are made in the liver.

Optimal Range: 2.3 to 2.8 g/dl

Optimal Range (alpha-globulin) : 0.2 to 0.3 g/L

Optimal Range (beta-globulin ): 0.7 to 1 g/L

Globulin levels may be elevated in:

• Chronic infections (parasites, some cases or viral and bacterial infection)
• Liver disease (biliary cirrhosis, obstructive jaundice)
• Carcinoid syndrome
• Rheumatoid Arthritis
• Ulcerative colitis
• Multiple myelomas, leukemias, Wald Enstrom's macroglobulinemia
• Autoimmunity (systemic lupus, collagen disease)
• Kidney dysfunction

Globulin level may be decreased due to :

• Alpha 1 antitrypsin deficiency (emphysema)
• Acute hemolytic anemia
• Liver dysfunction 
• Hypogammaglobulinemia/ Agammaglobulinemia (immune disorder; reduction of types of gamma globulins)

A/G (ALBUMIN/GLOBULIN) RATIO:

The liver can function adequately on 20% of liver tissue, thus early diagnosis by lab methods is difficult. A reversed A/G ratio may be a helpful indicator. With severe liver cell damage, the prolonged prothrombin time will not change with ingestion of vitamin K. The proper albumin to globulin ration is 2:1. When < 1.7, there is maybe a need for increasing stomach acidity. When > 3.5 there may be a need for stomach acidity and pepsin.

Optimal Range: 1.7 to 2.2

A/G Ratio may be elevated in :

• Hypothyroidism
• High protein/ high carbohydrate diet with poor nitrogen retention
• Hypogammaglobulinemia (an immune disorder characterized by a reduction in all types of gamma globulins, therefore low globulins)
• Glucocorticoid excess (can be from taking medications with cortisone effect, the adrenal gland overproducing cortisol, or tumor that produces extra cortisol-like compounds, low globulin)

Decreased A/G Ratio common causes:

• Liver dysfunction
• Overproduction of globulins, such as seen in multiple myeloma or autoimmune diseases
• Increased globulins (without an increase in albumin) due to infection
• Inflammation
• Underproduction of albumins, such as may occur with cirrhosis (scarring of the liver)
• Increased loss of albumin from the circulation, as may occur with kidney disease
• Inflammation (negative acute phase protein-protein whose concentration increase or decrease during inflammation. Albumin, transferrin, transthyretin, retinol-binding protein, antithrombin, transcortin. The decrease of such proteins may be used as markers of inflammation).

With a low total protein that is due to plasma expansion (dilution of the blood), the A/G ratio will typically be normal because both albumin and globulin will be diluted to the same extent.

METHODS OF ESTIMATION :

•  Precipitation method: Precipitate out globulin by addition of 28% sodium sulfite by virtue of their high mol. weight (100- 1000 kDa). The major protein remaining in serum will be albumin which can now be quantitated by biuret. Then subtract albumin from total protein to calculate globulin.
• Enzymatic method: The principal is based on the fact that LDH isoenzyme-5 (LDH-5) binds specifically to Dextran- blue (DB). Albumin inhibits the binding of LDH-5 with DB. The absence of LDH activity in DB fraction after gel filtration indicates the presence of albumin in sample and vice versa.
• Immunoenzymatic Assay: The human albumin assay is a two-site immunoenzymatic assay using as amplified biotin/ streptavidin detection system for maximum sensitivity. Samples containing human albumin are reacted in microtiter strips coated with an affinity purified capture antibody. A second biotinylated anti-HSA antibody is reacted forming a sandwich complex of solid phase antibody- albuminbiotinylated antibody. After a wash step to remove any unbound reactants the strips are then reacted with streptavidin labeled with alkaline phosphatase. Another wash sequence is performed followed by the addition of p-nitrophenyl phosphate (PNPP) substrate. The amount of hydrolyzed substrate is read on a microtiter plate reader and will be directly proportional to the conc. of human albumin present. Accurate quantitation is achieved by comparing the signal of unknowns to HSA standards assayed at the same time.

BCG METHOD:

Bromocresol green (BCG) is a dye which is used as a pH indicator in applications such as growth medium for m/o and titrations.

In acidic pH (below 3.8) BCG will ionize to give the monoanionic form (yellow), that further deprotonates at higher ph (alkaline solution, above pH 5.4) to give the dianionic form (blue).

INTENDED USE: For the determination of albumin conc. in human serum or plasma.

PRINCIPLE OF BCG:

The method is based on the specific binding of BCG, an anionic dye, and the protein at acid pH produce a color change of the indicator from yellow-green to green-blue with the resulting shift in the absorption wavelength of the complex. The intensity of the color formed is proportional to the conc. of albumin in the sample.

Albumin is cationic at pH of 4.3, therefore it interacts with the anionic dye of BCG to form a blue-green colored complex. The conc.is determined by measuring absorbance at 630 nm. 

ENZYME IMMOBILIZATION (pt. 2)

APPLICATIONS OF IMMOBILIZED ENZYMES:

A) FOOD INDUSTRY:

• Immobilized enzymes are of enormous importance for the processing of food sample. The food industry requires purified enzymes, however during the process of purification the enzymes denature. Therefore, I.E are used as the immobilization makes the enzyme stable. The application of concerned enzyme in the immobilized state has greatly enhanced the scope of lactose hydrolysis, whey processing, skimmed milk production, amino acid production etc.
•  A number of processes have been introduced in recent times. Among these are the use of immobilized cells containing glucose isomerase for the production of high fructose syrup. Glucose amylase for the production of glucose from starch is frequently used in an immobilized state. The enzyme beta- galactosidase from yeast is now being used in immobilized form for splitting of lactose to glucose and galactose. Large scale process for the immobilization of invertase has been developed for the splitting of sucrose to glucose and fructose.
• A number of processes for amino acid production have been developed using immobilized enzymes or cells. The penicillin acylases are intracellular enzymes produced by several m/o (both bacteria and fungi). This group of enzymes is involved mainly in the industrial production of 6-aminopenicillanic acid . In addition. penicillin acylase can also be employed in other useful bio- transformations, such as peptide synthesis. 

B) ANTIBIOTIC PRODUCTION

The prime challenge for the industrial execution of enzymatically produced biologically relevant antibiotics is the advanced performance of chemical processes used for antibiotic production. Enzyme based routes offer many advantages over the conventional chemical methods, like its an environment friendly approach as it evades organic chloride solvents, and it works at room temp. The antibiotic penicillin can be produced by using an immobilized enzyme from the culture of penicillium production cultures in fermentation.

C) MEDICINE:

The medical field has been transformed in the area of reliability, precision, manpower and time due to the introduction of IE in the form of proteins, receptors, signal molecules, antibodies etc. The major advantage of enzyme immobilization in biomedical is that the free enzymes are consumed by the cells and thus are inactive for prolonged use, however, the IE remains stable to stimulate the growth and to repair the defect of the concerned tissue. The enzymes when combined with the biomaterials provide biological function. Even in the case of cancer therapy the enzymes are delivered to the oncogenic sites with the new improved methods.

D) ANALYTICAL BIOCHEMISTRY:

An immobilized system can be used in the development of a precise and sensitive biochemical assay. The basic principle is that the enzyme acts on the substrate and a decrease in substrate conc. , increase the product conc. can be followed, using one of many assay methods. The reaction can be followed spectrophotometrically or photometrically.

E) BIODIESEL:

Biodiesel is mono-alkyl  esters of long chain fatty acids. Biodiesel is produced by the esterification reaction involving triglycerides and alcohol in the presence of catalyst. Recently, lipases catalyzed trans-esterification reactions have became attractive for biodiesel production since the glycerol (by-product of the reaction) can be easily removed and the recovery of bio-diesel is easy. However, the high production cost of lipases is the major hindrance in the commercial production (large scale) of biodiesel. The objective behind the use of IE for biodiesel production is to make the process more economical by reducing the cost of enzyme production as the IE could be reused for many cycles.

F) BIOREMEDIATION:

The discharge of waste water containing high conc. of toxic and reactive dyes is a well established problem. The conventional methods (chemical and microbial) of dye degradation have their own limitations. Recent studies have indicated that an enzymatic approach has involved much attention in the removal of these toxic pollute from the aqueous solutions (like lake, pond and river). Currently, some enzymes (e.g., peroxidases) have been immobilized on some inexpensive support have been reported to be highly effective in decolorizing reactive textile dyes compared to their soluble counterparts as the IE could be used for multiple cycles.

G) TEXTILE INDUSTRY:

The textile industry has clearly demonstrated with time the potential and effective application of enzymes such as cellulases, amylases, cutinases etc. These enzymes are microbial in origin and are of immense importance in the textile industry has switched from harsh chemical methods, causing pollution (damage to fabric, to mild enzymatic processes. However, the processing of fabrics with enzymes require high temp. and high pH, and the free enzyme are unstable under such reaction conditions. Hence, enzyme immobilization came into picture, as it not only provides stability to the enzyme, it also brings down the working cost of the enzyme.

ENZYME IMMOBILIZATION (Pt.1)

Enzymes are biological catalyst that promotes the transformation of chemical species in living systems. From biotechnological view, they are of immense importance and diverse potential. However, for large extent commercialization of these bioderived catalyst, enzymes  reusable factor becomes mandatory. Failing which they would no longer be economical. Moreover, most enzymes are relatively unstable, there cost of purification is high, it is technically very difficult to recover the active enzyme from the reaction mixture after use and maintenance of their structural stability during biochemical reaction is also very high challenging. Enzymes can catalyzes reactions in different states- they can act as individual molecules in reactions, they can be attach to surfaces or they can work in aggregates with other entities. The attachment or rather "immobilized" state of enzyme has been of interest for industrial purpose.
 The term "immobilized enzymes" refers to "enzymes physically confined or localized in a certain defined region of space with retention of their catalytic activities and which can be used repeatedly and continuously".

METHODS OF ENZYME IMMOBILIZATION:

1. IRREVERSIBLE :

As the name suggests during this process of Irreversible enzyme immobilization, enzyme when binds to the support cannot be retrieve back after the process is complete. That is to say that there is a complete loss of enzyme activity or the support becomes inactive when the support and enzyme are detached.

• COVALENT BONDING: 

  It is a conventional method for immobilization. This method can be achieved by attachment of the concerned enzymes to the support material through covalent linkage. In most instances, the immobilized procedure consists of at least two steps : (a) activation of the support material and (b) enzyme attachment . The covalent binding is usually formed between the functional group of the support material and the enzyme surface that contains the amino acids residues. The Amino acid residues in the covalent binding are the sulfhydryl groups of cystine, hydroxyl group of serine and threonine. In labs, a wide variety of support have been used for enzyme immobilization. for e.g., charcoal, cellulose, synthetic polymers. One prime advantage of this method is that because of the stable nature of bond formed between enzyme and support material (matrix), the enzyme is not realized into the solution upon use i.e., the enzyme should not be present in the product. However, in order to achieve high levels of bound activity the amino acid residues essential for catalytic activity should not be involve in the covalent linkage to the support matrix. However, because of the covalent nature of the bond, the support material has to be discarded together with the enzyme once the activity decays. The attachment between the enzyme and the support material could either be a direct linkage or through a spacer arm. The potentiality of using a spacer arm is that it provides a greater degree of mobility to the enzyme thus, resulting in higher activity than direct attachment.

• ENTRAPMENT:   

  It is caging of enzymes by covalent or non-covalent bonds within the gels or fibers such that it allows the substrate and products to pass through but retains the enzymes. In this approach, an enzyme is added to the solution of monomers before the gel is formed. Gel formation is very initiative either by altering the temperature or by adding a gel inducing chemical. As a result, the enzyme becomes trap in the gel. This method differs from the covalent coupling methods described above as over here the enzyme is not bound to matrix or membrane but rather is entrapped inside the gel. The advantage of entrapment method is that it is quick, inexpensive or requires mild conditions for reaction process. Moreover, the support matrix protects the enzyme from contamination of microbes, proteins and other enzyme. However, the practical use of this method is limited by restriction of mass transfer through membrane or gels. Therefore, entrapment of enzyme within gel or membrane is a convenient method for use in processes involving low molecular weight substrates and products. Efficient entrapment has been achieved with alginate-gelatin-calcium-hybrid carriers which prevents enzyme leakage and provide increased mechanical stability.

• ENCAPSULATION:

  It provides large surface area to enzyme to contact with the substrate. This method is useful because several enzymes can be immobilized in a single step. Enzymes are immobilized  within micro-capsules. These are prepared from organic polymers. The membrane encloses the enzyme and remains semi-permeable to the substrate and products. This is an easy and economical technique. The stability of a biocatalyst in a solution is an important aspect of this technique. This method is not applicable for high molecular weight substrates. Leakage of enzymes takes place through  micro-capsules. Using this technique. capsules of suitable size can be obtained. The diffusion limitation can be also controlled.

• CROSS-LINKING:

  This  method is based on the formation of covalent bonds between the enzyme molecules by means of multi-functional reagents leading to 3-D cross-linked aggregates. Among the most popular cross-linkers are glutaraldehyde aliphatic diamines. Cross-linking can be both intra- and inter molecular. The major advantage of cross-linking is that very less deabsorption of enzyme takes place as it is strongly bound. This method is best used in conjunction with other methods. However, the major disadvantage of it is that it causes significant chances to active site. thus, resulting in partial loss of enzymes activity,. 

  2. REVERSIBLE :

  Contrary to irreversible enzyme immobilization, during reversible enzyme immobilization, the enzyme under mild conditions can be separated from the support after the process is complete. This is due to the type of interaction involved (non-covalent bonds- hydrophobic interactions, Hydrogen bonding, Vander Waals etc.) in the formation of enzyme-support complex. This method of enzyme immobilization is highly economical because of the advantage presented by it. The support can be regenerated and attached to fresh enzyme for the next cycle of the process. The cost of the support is a prime aspect in the overall process economics.

• ADSORPTION:

  Besides being an easy method to perform with broad applications, this method offers high enzyme-loading. The catalyst went under suitable reaction conditions like temp., pH, incubation time would be absorbed on to the surface of support by simple mixing of the two. Further to this, all the loosely and unbound enzyme could be removed by washing and the stable enzyme support complex can be directly used. Since no reactive forces are involved in the process. Therefore, there are no conformational changes in the enzyme on immobilization. the enzyme can be adsorbed on the matrix by one of the following ways:

(a) NON - SPECIFIC : This is the one of the simplest method for enzyme immob. which is based on                                           ionic binding or physical adsorptions. During ionic binding the enzyme are bound to support matrix by sol-linkages, whereas in the case of physical adsorption it is the non-covalent bonds i.e., H-bonds, Vander Waal forces through which the enzyme is bound to the matrix. The nature of forces involved in no-specific adsorption results in an immob. process that can be reversed by changing the conditions that influence the strength of interaction. Based on the charges of the matrix and protein, strong bonds are formed between the two. However, the enzyme is not distorted and it usually reserves the catalytic activity of enzyme. Such methods are therefore economically attractive but may differ from problems such as enzyme leakage from the matrix when the interactions are relatively weak.

(b) IONIC BINDING: The basic principle of ionic binding is the protein-ligand interactions which                                           involve the salt linkages. Although this method is reversible and simple but in general, it is difficult to find conditions under which the enzyme remains both strongly bound and fully active. The nature of this type of non-covalent immob. could be reversed by changing the polarity, temp. or ionic strengths. However, problems may arise from the use of a highly charged support, when the substrate or product themselves are charged.

(c) HYDROPHOBIC INTERACTIONS:  The bases of this method is not the chemical bonds                                                                                     between the enzyme and support but its an entropically governed interaction that defines this method of immob. The foundation of this method depends on experimental variable like salt conc. , pH, temp. ionic strength. The degree of interaction depends on the chemical nature (specifically the hydrophobicity) of support and the enzymes . The extent of substitution and the size of hydrophobic ligand governs the strength of interaction between the support and enzyme.

(d) AFFINITY BINDING: It is the immob. of enzyme on to the matrix through specific interactions.                                               This method exploits the specificity of the enzyme towards its support (matrix) under different physiological conditions. The remarkable selectivity of the interaction between the matrix and enzyme is a major benefit of the method. Two methods are being followed in affinity immob. either the matrix is pre-coupled to an affinity ligand for target enzyme or the enzyme is conjugated to an entity that develops affinity towards the matrix.

• CHELATION OR METAL BINDING:

  In this method, mainly Zirconium or titanium salts are used and often this method is known as 'metal-linked immob.' The metal hydroxyl or its salt is  precipitated on the support material by either neutralization or heating. The metal salts or hydroxides have the potential to bind with the nucleophilic groups present on the surface of the support material because of the stearic factors, it is impossible for the matrix to occupy all co-ordination positions of the matrix. Therefore, some of the positions remain free to co-ordinate with groups from the enzyme. From practical and operational point of view, this method is simple and specific activities of immob. enzymes by this method have been reported to be relatively high. The major advantage with this method is that the carrier and the enzyme can be separated and hence regenerated by altering the pH. Thus making it a reversible process.

Differentiate between seed fermentor and production fermentor:

SEED FERMENTOR:

1. Used to raise the inoculum.
2. Uses inoculum medium
3. Capacity is 10-100L.
4. More than one fermenter  may be used to get desired amount of inoculum.
5. Cells are grown in exponential phase and harvested. Main purpose is to get active cell.
6. Medium and fermentation conditions are optimized to get maximum cell density and this also adapt the cell to new environment.

PRODUCTION FERMENTOR:

1. Used for production of metabolites.
2. Uses production medium.
3. Capacity- 1000- 100,000 L
4. One large fermenter is used.
5. Cells are grown mainly to get the desired product (stationary or primary )
6. The main aim is to get high yield of product.

Differentiate between carriers and channels.

CARRIERS:

1. Carriers are proteins that transport molecules down their electrochemical gradient. Hence, they do not require energy.
2. Carriers protein transport molecules.
3. Carriers proteins flip between two conformations.
4. Carrier proteins can mediate both active and passive transport.
5. Solute molecules are bound to one side and released from the other side.
6. Have low transport rates of 10^4 ions per second.
7. Consists of alternative solute-bound conformations.
8. Carrier proteins are glycoproteins.
9. Carrier proteins are synthesized in the free cytoplasmic ribosomes.
10. Carrier transport water soluble molecules across the membrane as well as the insoluble molecules.
11. Specificity of carrier proteins is due to an ion selectivity filter.
12. E.g., of Carrier protein mediated active transport : Na+/ K+ ATPase and SR Ca2+ ATPase

CHANNELS:

1. Channels involve proteins that transport molecules against their electrochemical gradient, hence requires energy provided by ATP hydrolysis.
2. Channel protein transport ions.
3. The position of channel proteins is fixed.
4. Channel proteins can mediate only passive transport.
5. Solute molecules diffuse through the pores.
6. Have high transport rates of 10^8 ions per second.
7. Channel proteins do not bind with solute molecules it transport.
8. Channel proteins are lipoproteins.
9. Channel proteins are synthesized in the ribosome bound to rough endoplasmic reticulum.
10. Channel transports only water soluble molecules across the membrane.
11. Specificity of channel proteins is due to specific binding sites to which the solute molecules bind.
12. E.g., of channel protein mediated passive transport: Voltage gated channel and Ion gated channels.

Differences between active and passive transport

ACTIVE TRANSPORT:

1. Active transport uses ATP to pump molecules against the concentration gradient. Transport occurs from a low conc. of solutes to high conc. of solute. 
2. Requires cellular energy.
3. Types of transport include endocytosis, cell membrane/ sodium potassium pump and exocytosis.
4. Function : Transport molecules against the conc. gradient so more of the substance is inside the cell (i.e., a nutrient) or outside the cell (i.e., waste) than normal. Disrupts equilibrium established by diffusion.
5.  Types of particles transported : Proteins, ions, large cells, complex sugars
6. Requires carrier proteins. Matrix or permeases of the membrane are not involved.
7. It is highly selective.
8. It is rapid process.
9. It is unidirectional.
10. It is reduced or stopped by O2 deficiency.
11. Metabolic inhibitors stop active transport.
12. Decrease in temp. decreases it.
13. E.g., Phagocytosis, pinocytosis, Na+/K+ pump, secretion of a substance into the bloodstream

PASSIVE TRANSPORT:

1. Movement of molecules occurs down the conc. gradient. It goes from high to low conc. in order to maintain equilibrium in the cells.
2. Does not require cellular energy.
3. Types of transport includes diffusion, facilitated diffusion and osmosis.
4. Function : Maintains dynamic equilibrium of water, gases, nutrients, wastes etc. between cells and extracellular fluid; allows for small nutrients and gases to enter/exit. No net diffusion/osmosis after equilibrium is established.
5. Types of particles transported: Anything soluble in lipids, small monosaccharides, water, O2, CO2, hormones etc.
6. Carrier proteins are not involved. It takes place through matrix/channels/permeases.
7. It is partly non-selective. All diffusible substances can be transported according to their conc. gradient.
8. It is comparatively slower process.
9. It is bidirectional
10. Unaffected by O2 content.
11. Metabolic inhibitors do not influence passive transport.
12. Not affected by temp.
13. E.g., Diffusion, facilitated diffusion, osmosis

Differences between ligand gated ion channels and voltage gated ion channel

LIGAND GATED ION CHANNELS:

1. These channels are cell membrane receptors that are activated by neurotransmitters and this causes the opening of ion channel causing depolarization of the cell.
2. Responsible for establishing resting membrane potential.
3. Activated by binding of ligands to the channel receptor.
4. It is a receptor mediated ion channel.
5. Non specific ion channels
6. E.g., Acetylcholine receptor channel.

VOLTAGE GATED ION CHANNEL:

1. These channels are ion channels also present on the membrane but are activated by a change in voltage across the membrane and this will cause the ion channel to open and start depolarization of the cell membrane.
2. Responsible for shaping action potentials.
3. Activated by changes in voltage gradient across plasma membrane.
4. It transmits fast action potential from cell body to axon terminal in neurons.
5. These ion channel are specific.
6. E.g., K+ channel.

NOTES ON FOOD MICROBIOLOGY:

Food as substrate for microorganisms:

Microbial growth in food depends on following factors:

1. Intrinsic : These are the factors which are inherent to the food i.e., the physical and chemical properties of food. It includes pH, moisture content, oxidation, reduction potential, nutrient content, antimicrobial constituents and biological structure.
2. Extrinsic: Include environmental condition where the food is stored. (like temperature of storage, relative humidity of environment, presence and concentration of gases, presence and activity of other m/o)
3. Implicit: These are the physiological properties of micro-organisms (like hydrolytic activity).
4. Process factor: These factors will determine spoilage (vulnerability and type of m/o and preservation method)

INTRINSIC FACTORS:

pH: most m/o have an optimum range of pH for growth which is between 6.6-7.5.

• Growth range of m/o:  

1. BACTERIA : they do not grow below range pH 4.5. food which has pH more than 4.5 are more likely to be spoiled by bacteria. They were also grown in alkaline solution of egg white.
2.  FUNGI : There is no actual range. All food are prone to spoilage by fungi. they have to compete from bacteria for growth therefore mainly spoiled by acidic food.  

• pH range of foods depends on the acidity of food. They are classified as
• first low acid foods (above pH 5.3) - e.g., meat, fish, milk
• medium acidic food (pH 5.3 to 4.5) e.g., spinach, pumpkin.
• Acidic food (pH 4.5 to 3.7) e.g., tomato, pineapple.
• Highly acidic food (pH below 3.7) e.g., berries.

CATEGORIES: 

1. Fruits and Vegetables : Fruit (2-5 pH) and vegetables (pH- 4.2- 6). both have low pH.  However,  vegetables have higher pH compared to fruits. Therefore, fruits are liable to spoilage by fungi and yeast and vegetables are equally spoiled by bacteria.
2. Meat and fish : pH is above 5.6, can be spoiled by both bacteria and molds but bacterial spoilage is more dominant. The keeping quality of meat and fish :  Meat of fatigue animal spoil faster than rested animal. This is because a final pH attained upon completion of rigor mortis (stiffening of the joints and muscles a few hours after death). In case of fatigue animal, glycogen can utilized completely whereas in case of rested animal glycogen is 1% remain. It is converted to lactic acid. Therefore, pH of blood fluids drops to 5.6 from 7.4. This is responsible for increasing its keeping quality.
3. Milk and milk products : The pH of milk is around 6-6.5. Butter (6.1 to 6.4) Dahi (4.2-4.5).
4. Some foods have inherent buffering capacity. Meat is more highly buffered than vegetables as they have proteins. Milk is buffered due to the presence of casein. Buffers permit fermentation to go on longer with greater yield of products. Vegetables have low buffering capacity permitting decrease in pH by small amount of acid by lactic acid bacteria during early part of fermentation. E.g., in case of Sauerkraut fermentation (finely cut cabbage fermented by various lactic acid bacteria) - this results in suppression of competing organisms and rapid succession (due to low pH) .

MOISTURE CONTENT : 

m/o require moisture for growth. The water requirement of m/o is described in terms of water activity (aw) in the environment. Water activity = p/p0, where, p = vapor pressure of solution p0 = vapor pressure of solvent (water).

Water activity is defined by the ratio of water vapor pressure of food substrate to the vapor pressure of pure water at the same temperature. For pure water, aw = 1. For 1M solution of NaCl, aw = 0.98.

Relative humidity (RH) = 100 * aw 

Lowest  water activity values permitting growth of spoilage organism as follows:

 for most spoilage bacteria = 0.90,

 For most spoilage yeast = 0.88

For most spoilage mold = 0.80

For most spoilage halophilic = 0.75

Foe Xerophilic = 0.61

For Osmophilic yeast = 0.61

Yeast and fungi resists better aw values. Bacteria grow if high moisture content is available. For E.coli , aw is 0.96. Clostridium botulism = 0.93. Exception : Staphylococcus aureus and micrococcus are able to survive at low aw of 0.86.

If we are able to use freely available water by adding solutes like salt/sugar etc. It has drying effect, it can even take out water from the microbial cells. Thus, the oldest food preservation method i.e., drying and desiccation are based on lowering aw. Water can be made unavailable by :

• addition of solutes and ions that tie up water molecules out of food.
• hydrophilic colloids like gels etc. that make water unavailable. e.g., jelly has high conc. of gelatin.
• Water of crystallization i.e., ice is unavailable to m/o in a food as more ice is formed, the conc. of solutes in unfrozen water is increased lowering its aw.

OXIDATION - REDUCTION POTENTIAL O/R (Eh):

m/o display varying degrees of sensitivity to the O/R potential of their growth medium. The O/R potential of a substrate may be defined as " the ease with which the substrate losses or gains electrons." When electron are transferred from one compound to another, a potential difference is created between the two compounds. This difference can be measured in millivolts. More highly oxidized a substance the more positive will be its electrical potential, it will favor the growth of aerobic m/o. More reducing the substance, more negative will be its electrical potential, less O2 is available. Therefore, it favors the growth of anaerobic microbes. Hence, aerobic m/o requires +ve Eh for growth and anaerobic m/o requires -ve Eh.

The O/R potential of a food is determined by the :

• Characteristic potential
• Poising capacity : resistance to change in the potential of the food.
• O2 tension of atmosphere around the food.
• Access that the atmosphere has to the food.

Different m/o have different pH requirements, aerobes require oxidized environment, e.g., Bacillus. Anaerobic require reduced environment. e.g., Clostridium.. Microaerophiles (require air but a low O2 tension) slightly reduced environment (Lactobacillus). Facultative anaerobes can grow in any condition.
O/R potential of food: fruits and vegetables : Eh of -100mV; low O/R potential inside however, higher O/R in the outer layers. They have presence of reducing sugars, ascorbic acid. Therefore, O/R is low inside and resist a change in O/R. In case of fruit juice, Eh = +400mV because of free diffusion of O2. Clearing of fruit juices leads to removal of oxidizing and reducing substances. Therefore, juice present in pulp is less susceptible to growth of aerobic m/o than clear juices. In case of meat, intact big chunks of meat have an Eh of -100mV, interiors have low O/R potential because of presence of more proteins and sulfur-hydryl groups. Exterior have high O/R. Therefore, outer layer susceptible to spoilage by aerobic m/o whereas interiors may be undergoing Putrefaction. by anaerobic m/o. Minced meat has +ve Eh of 200mV because surface area increases. Now, they can be spoiled by aerobic m/o.
Techniques to control O/R potential of food:

• Vacuum packaging
• Skin tight packaging 
• Canning
• Addition of anti-oxidants (natural oxidant- ascorbic acid ; synthetic oxidant- propyl gallate (PG))

NUTRIENT CONTENT:

m/o require following : 1) Water (aw)   2) Source of energy which comes from sugar, aa etc.

3) Source of nitrogen (amino acid, protein )   4) Vitamins and growth factors

Nutrient requirement are least for molds followed by yeast, gram negative bacteria and gram positive bacteria. Fruits tend to be lower in vitamin B than meat and this fact  along with usual low pH and +Eh helps to explain the usual spoilage of fruits by molds rather than bacteria.

ANTIMICROBIAL CONSTITUENTS OF FOOD:

Eggs contain lysozyme in egg white and conalbumin which is responsible for binding albumin making it unavailable for m/o. Milk has lactoferrin which is able to bind iron making it unavailable for m/o. Lactoperoxidase   system which also has antimicrobial effect. Spices has essential oils that possess antimicrobial activity. e.g., Eugenol in cloves, allicin in garlic, turmeric, cinnamic aldehyde in cinnamon. 

Fruits and vegetables has organic acid derivative e.g., hydroxycinnamic acid derivative which has both anti bacterial and antifungal activities. m/o growing in food may produce inhibitory substances e.g., propionic acid produced by Propioni bacterium in Swiss cheese is inhibitory to molds. NISIN produced by Streptococcus lactis may be useful for inhibiting Clostridia in cheese.

Heating foods may result in the formation of inhibitory substances. e.g., heating lipids may increase auto-oxidation and make them inhibitory. Browning concentration of sugar syrups may result in production of furfurals which are inhibitory to fermentation organisms.

BIOLOGICAL/OUTER STRUCTURE:

The natural covering of some foods provide protection against the entry and damage by spoilage organisms. e.g., shells of eggs, peels of fruits, hides of animals, layer of fat over meat.

EXTRINSIC FACTOR:

TEMPERATURE OF STORAGE OF FOOD:

M/O grow over a wide range of temperature. Psychotropic optimize15-30C e.g., Pseudomonas, Alcaligenes Enterococcus. These m/o grow well at refrigerator temperature and cause spoilage of eggs, meat, fish etc. Thermophiles (55-65C)  e.g., Clostridium. the quality of food product must be taken into account in selecting storage temperature. e.g., banana are kept better if stored at 13-17C than 5-7C. Vegetables at 10C if stored at normal temperature they are susceptible to Mesophiles. In pasteurization, it is important to immediately chill the food.\

RELATIVE HUMIDITY OF ENVIRONMENT:

When food with low aw values are placed in an environment of high relative humidity, the food pick up moisture from environment which can lead to surface microbial growth. Foods with high aw, lose moisture when placed in an environment of low humidity.

Foods that undergo surface spoilage from molds, yeasts and certain bacteria should be stored under low relative humidity. In properly wrapped meats undergo surface spoilage in the refrigerator due to high relative humidity of the refrigerator.

PRESENCE AND CONC. OF GASES IN THE ENVIRONMENT:

This storage of food in atmosphere containing increased amount of CO2 up to 10% is referred to as a controlled atm or CA storage. It is done for fruits such as apples pears. CO2 retards fungal blotting it acts as competitive inhibitor of ethylene. Ethylene acts as an senescence factor in fruits and its inhibition has the effect of maintaining the fruit in a better state of natural resistance. Steaks (meat product) are stored 100% CO2 atm. fish in 80% CO2 atm. The inhibitory effect of CO2 increases with decrease in temperature due to increasing solubility of CO2 at lower temp. Therefore, they are more effective during cold storage.

Ozone also has preservative effects as it is a strong oxidizing agent. It has been used to retard surface spoilage of beef.

PRESENCE AND ACTIVITY OF OTHER M/O:

E.g., Bacteriocins, organic acids, antibiotics etc are produced by food-borne organisms that are lethal to other m/o.

Lactic antagonism- inhibition of food spoilage organism is due to NISIN produced by lactic acid bacteria, lowering of pH, production of organic acids, diacetyl etc.

REASONS FOR INTERFERENCE ARE :

• Competition for nutrients
• Rendering the environment unfavorable