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PRAKASH.S.P
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 Enzymes are biological polymers that catalyses the
biochemical reactions.
 The vast majority of enzymes are proteins
 They convert the substrates into different molecules,
called products.
1/32
1.Oxidoreductases:
catalyze oxidations and Reductions.
2.Transferases :
catalyze transfer of groups such as methyl or glycosyl
groups from a donor molecule to an acceptor molecule.
3. Hydrolases: catalyze the hydrolytic cleavage of C-C,
C-O, C-N, and acid ,anhydride bonds.
2/32
4.Lyases ; Removal of a group of a atom from the
substrate leaving double bond or add group to double
bond
5. Isomerases :catalyze geometric or structural changes
within a single molecule.
6. Ligases: catalyze the formation of covalent bond
between two substrate molecules with help of ATP,
which provide energy for the reaction
3/32
 Bacteria
 Yeast
 Molds
 Plants and animals
4/32
The enzymes are used to obtain
 efficient processes,
 leading to reduced use of raw materials,
 improved or consistent quality,
 replacement of chemical food additives
 avoidance of potential harmful by-products in the food.
 Use of specific enzyme instead of microorganism has
several advantage .
 A specific substrate can be converted into a specific
product by an enzyme through single step reaction.
5/32
Enzymes from these sources are used in the production
process of
 High-fructose corn syrup
 Extraction of juice from fruits and vegetables
 Enhancement of flavor in cheese
6/32
Enzyme Source Substrate Use
A- amylase Bacteria Starch Production of dextrins , brewing and
baking
Catalayse Molds Hydrogen
Peroxide
Removal of hydrogen peroxide;
milk,
D-Glucose
isomersase
Bacteria Glucose Glucose to fructose; high-fructose
corn syrup
7/32
Hydrolases (Hydrolyze)
Isomerases ( isomerization )
Oxidoreductase (oxygenation or hydrogenation)
8/32
Alpha-amylase On Bread Quality:
 Addition of alpha-amylase in the sponge and dough
bread can prolong shelf life for at least four days
longer compared to 0.5% powdered distilled
monoglycerides.
 The crumb softness of the loaf with maltogenic alpha-
amylase at day 7 was as soft as the loaf with 0.5%
distilled monoglycerides at day 3.
 The elasticity of the loaf with maltogenic alpha-
amylase at day 7 is even higher than the loaf with
distilled monoglycerides.
9/32
 Acid proteinases helps in Milk coagulation
 Neutral proteinases - accelerated cheese ripening
 Lipases - Accelerated cheese ripening
 Lacto peroxidase aids Cold sterilization of milk
10/32
 Some of the enzymes used in food waste treatments are
polysaccharidases
 Cellulase
 pectinase
 Hemicellulase
 chitinase, and amylase,
 lactases,
 proteinases.
 Treatment of fruits with cellulase and pectinase has
increased juice yield and improved separation of solids
from the juice. The solids can be used as animal feed.
11/32
 Chitinases are used to depolymerize the shells of
shellfish, and the product used to produce SCPs.
 Amylases are used to treat starch-containing
wastewater to produce glucose syrup for use in alcohol
production by yeasts.
 Proteases are used to treat wastewater from fish and
meat-processing operations. Some of these products are
used as fish food.
12/32
Pasteurized Milk:
 Heat-stable proteinases and lipases of
psychotropic bacteria are not inactivated by
pasteurization and can cause proteolysis of
casein and lipolysis of milk lipids to produce
flavor defects.
 Under normal short-term refrigerated storage,
these defects may not be enough to detect.
13/32
 In addition, psychrotropic bacteria contaminating
milk after pasteurization can also multiply during
storage and cause spoilage, especially when the milk
is either stored for a long time or temperature abused.

 In that event, it is difficult to differentiate the role of
bacterial growth and heat-stable enzymes in spoilage.
14/32
Ultrahigh Temperature (UHT)-Treated Milk
Products:
 UHT-treated milks, heated at135˚C for 1to2sec, are
considered commercially sterile products with a shelf life of
6months at Room temperature
 Spoilage of these products during storage at room
temperature has been observed in the form of bitter flavor,
sediments, and gel formation, due to the action of heat-
stable proteinases .
 Rancid flavor from the action of heat-stable lipases.
 The changes produced by proteinases are more predominant
than those associated with lipases.
10/22
15/32
Cheeses:
 Proteolytic activity by the extracellular proteinases of
psychotropic bacteria in raw milk was reported to reduce
cheese yield and increase the levels of nitrogenous
compounds in whey.
 Depending on the proteolysis of caseins, the loss in
cheese yield can be as high as 5%.
 The loss was directly related to the storage time of the
raw milk and psychotropic counts.
11/22
16/32
 In addition, the heat-stable proteolytic enzymes were
associated with increased proteolysis of cheeses
(especially soft cheeses such as cottage cheese), lower
flavor quality, and higher texture problems in
cheddar cheese.
 Lipases have also been implicated in the development
of off-flavor in cheese.
17/32
Cream and Butter:
 Cream and butter are more susceptible to spoilage by
heat-stable lipases than by proteinases.
 Extracellular lipases of psychotropic bacteria
preferentially partition with the cream phase of milk,
increasing their concentrations in cream.
 Lipases are responsible for off-flavor development in
cream. A cream that has undergone lipolysis foams
excessively and takes a longer time to churn during
processing of butter.
12/22
18/32
 The butter prepared from such cream is susceptible to
rancidity more quickly.
 Butter containing residual heat-stable bacterial lipases
undergoes rapid lipid hydrolysis even during storage at
–10˚C. Lipases that preferentially release short chain
fatty acids (C4 to C8), with and without long-chain
fatty acids, cause the most off-flavor in butter.
19/32
 Rich source of Protein – Molds, Yeast, Bacteria and Algae.
 Digestibility of these protein – 65-96%.
 Most preferred – Yeast ( Candida, Saccharomyces)
 Advantages - Microbial protein are a good sources of
Vitamin B and Carotene
 Can be produced under laboratory setting.
 Increase of microbial protein will reduce the use of grains as
animal feed, which then can be used as human food.
 Disadvantages- Poor in essential amino acids (methionine)
13/22
20/32
Amino Acids:
 Proteins of most cereal grains are deficient in the essential
amino acids, particularly lysine, and tryptophan. To improve the
biological values, cereals are supplemented with essential
amino acids.
 Supplementing vegetable proteins with essential amino acids
has been suggested to improve the protein quality for people
who either do not consume animal proteins (people on
vegetarian diets) or do not have enough animal proteins.
14/22
21/32
 To meet this demand as well as for use as nutrient
supplements, large amounts of several essential acids are being
produced. At present, because of economic reasons, they are
mostly produced from the hydrolysis of animal proteins
followed by purification.
 Now we have bacterial strains that have been isolated, some of
which are lactic acid bacteria that produce and excrete large
amounts of lysine in the environment.
 Isolating high-producing strains of other amino acids, and
developing strains by genetic and metabolic engineering that
will produce these amino acids in large amounts, can be
important for economical production of essential amino acids.
22/32
The different parameters or factors that are used in
combination include
 intrinsic factors (e.g., aw, pH, Eh, and natural
inhibitors),
 processing factors (e.g., heating, drying, fermentation,
and preservatives)
 extrinsic factors (e.g., temperature and aerobic or
anaerobic environment).
Example : Preservation of jams and jellies - high heat,
low pH (of fruits), low water activity (sugar in fruits and
added), and anaerobic packaging are used to reduce
microbial numbers as well as the growth of survivors.
15/22
23/32
 The greatest challenge will be the long-term preservation
of meat, dairy, and fish products at refrigerated temperature
or even at ambient temperature, where refrigeration is not
always available (military operation)
 The effectiveness of combining different factors to control
target microorganisms in a food has to be studied.
 In conducting such studies, one should also recognize that
the factors could have additive, synergistic, or even adverse
(neutralizing) effects when used in combination.
24/32
Low-Heat Processing:
 Low heat (100ºC) does not kill many pathogenic and
spoilage bacterial spores.
 They can be heat activated, which leads to germination and
outgrowth.
 However, if the pH of the food is reduced to 4.5 or NO2
and NaCl are added, the heat-shock spores will not
germinate.
16/22
25/32
 Clostridium botulinum grows at 35ºC at an aw of 0.95.
 If the storage temperature is reduced to 20ºC, it does
not grow unless the aw is increased to 0.97.
 Similarly, L.monocytogenes grows at 25ºC in a broth
containing 6.5% NaCl in 3d, but fails to grow under
similar conditions at 14ºC.
26/32
Low pH:
 Clostridium botulinum grows at pH 7.0, 37ºC, and an aw of
0.95
 pH is dropped to 5.3, no growth is observed, even at an aw
of 0.99. .
 Clostridium botulinum produces a toxin during incubation
at 16ºC in 28 d at pH 5.5, but at pH 5.2 under the same
conditions, no toxin is produced.
 In using acid to reduce pH, it is important to recognize that
organic acids, such as acetic, propionic, and lactic acids, are
more effective than HCl and phosphoric acid.
17/22
27/32
Preservatives:
 Preservatives such as NaCl and BHA act synergistically
to increase the antimicrobial action of sorbates.
 Organic acids are also effective at low pH because of
higher concentrations of undissociated molecules.
 The bactericidal effect of bacteriocins can be enhanced
when used with acids and EDTA.
18/22
28/32
Conclusion :
 Sublethal injury to microbial cells and spores and increased
susceptibility of injured cells and spores to one or more
preservation methods can play important roles in controlling
microorganisms by a combination of factors.
 The hurdle concept is based on the scientific concept that when
two or more methods are used together the combinations are
more effective, even at a much lower treatment level, because of
the large number of possibilities.
 Sublethal injury occurs due to the exposure of bacterial cells to
unfavorable physical and chemical environments (beyond the
growth range but not in the lethal range) that cause reversible
alterations in the functional and structural organizations of the
cells.
20/22
29/32
 Handbook of Food Enzymology - CRC Press
Book. ... 1st Edition. John R. Whitaker,
Alphons G. J. Voragen,
 Food Microbiology. MJP Publishers, 2007
K. Vijaya Ramesh.
Effect of enzyme ,protein and other combination system n food microbiology

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Effect of enzyme ,protein and other combination system n food microbiology

  • 2.  Enzymes are biological polymers that catalyses the biochemical reactions.  The vast majority of enzymes are proteins  They convert the substrates into different molecules, called products. 1/32
  • 3. 1.Oxidoreductases: catalyze oxidations and Reductions. 2.Transferases : catalyze transfer of groups such as methyl or glycosyl groups from a donor molecule to an acceptor molecule. 3. Hydrolases: catalyze the hydrolytic cleavage of C-C, C-O, C-N, and acid ,anhydride bonds. 2/32
  • 4. 4.Lyases ; Removal of a group of a atom from the substrate leaving double bond or add group to double bond 5. Isomerases :catalyze geometric or structural changes within a single molecule. 6. Ligases: catalyze the formation of covalent bond between two substrate molecules with help of ATP, which provide energy for the reaction 3/32
  • 5.  Bacteria  Yeast  Molds  Plants and animals 4/32
  • 6. The enzymes are used to obtain  efficient processes,  leading to reduced use of raw materials,  improved or consistent quality,  replacement of chemical food additives  avoidance of potential harmful by-products in the food.  Use of specific enzyme instead of microorganism has several advantage .  A specific substrate can be converted into a specific product by an enzyme through single step reaction. 5/32
  • 7. Enzymes from these sources are used in the production process of  High-fructose corn syrup  Extraction of juice from fruits and vegetables  Enhancement of flavor in cheese 6/32
  • 8. Enzyme Source Substrate Use A- amylase Bacteria Starch Production of dextrins , brewing and baking Catalayse Molds Hydrogen Peroxide Removal of hydrogen peroxide; milk, D-Glucose isomersase Bacteria Glucose Glucose to fructose; high-fructose corn syrup 7/32 Hydrolases (Hydrolyze) Isomerases ( isomerization ) Oxidoreductase (oxygenation or hydrogenation)
  • 10. Alpha-amylase On Bread Quality:  Addition of alpha-amylase in the sponge and dough bread can prolong shelf life for at least four days longer compared to 0.5% powdered distilled monoglycerides.  The crumb softness of the loaf with maltogenic alpha- amylase at day 7 was as soft as the loaf with 0.5% distilled monoglycerides at day 3.  The elasticity of the loaf with maltogenic alpha- amylase at day 7 is even higher than the loaf with distilled monoglycerides. 9/32
  • 11.  Acid proteinases helps in Milk coagulation  Neutral proteinases - accelerated cheese ripening  Lipases - Accelerated cheese ripening  Lacto peroxidase aids Cold sterilization of milk 10/32
  • 12.  Some of the enzymes used in food waste treatments are polysaccharidases  Cellulase  pectinase  Hemicellulase  chitinase, and amylase,  lactases,  proteinases.  Treatment of fruits with cellulase and pectinase has increased juice yield and improved separation of solids from the juice. The solids can be used as animal feed. 11/32
  • 13.  Chitinases are used to depolymerize the shells of shellfish, and the product used to produce SCPs.  Amylases are used to treat starch-containing wastewater to produce glucose syrup for use in alcohol production by yeasts.  Proteases are used to treat wastewater from fish and meat-processing operations. Some of these products are used as fish food. 12/32
  • 14. Pasteurized Milk:  Heat-stable proteinases and lipases of psychotropic bacteria are not inactivated by pasteurization and can cause proteolysis of casein and lipolysis of milk lipids to produce flavor defects.  Under normal short-term refrigerated storage, these defects may not be enough to detect. 13/32
  • 15.  In addition, psychrotropic bacteria contaminating milk after pasteurization can also multiply during storage and cause spoilage, especially when the milk is either stored for a long time or temperature abused.   In that event, it is difficult to differentiate the role of bacterial growth and heat-stable enzymes in spoilage. 14/32
  • 16. Ultrahigh Temperature (UHT)-Treated Milk Products:  UHT-treated milks, heated at135˚C for 1to2sec, are considered commercially sterile products with a shelf life of 6months at Room temperature  Spoilage of these products during storage at room temperature has been observed in the form of bitter flavor, sediments, and gel formation, due to the action of heat- stable proteinases .  Rancid flavor from the action of heat-stable lipases.  The changes produced by proteinases are more predominant than those associated with lipases. 10/22 15/32
  • 17. Cheeses:  Proteolytic activity by the extracellular proteinases of psychotropic bacteria in raw milk was reported to reduce cheese yield and increase the levels of nitrogenous compounds in whey.  Depending on the proteolysis of caseins, the loss in cheese yield can be as high as 5%.  The loss was directly related to the storage time of the raw milk and psychotropic counts. 11/22 16/32
  • 18.  In addition, the heat-stable proteolytic enzymes were associated with increased proteolysis of cheeses (especially soft cheeses such as cottage cheese), lower flavor quality, and higher texture problems in cheddar cheese.  Lipases have also been implicated in the development of off-flavor in cheese. 17/32
  • 19. Cream and Butter:  Cream and butter are more susceptible to spoilage by heat-stable lipases than by proteinases.  Extracellular lipases of psychotropic bacteria preferentially partition with the cream phase of milk, increasing their concentrations in cream.  Lipases are responsible for off-flavor development in cream. A cream that has undergone lipolysis foams excessively and takes a longer time to churn during processing of butter. 12/22 18/32
  • 20.  The butter prepared from such cream is susceptible to rancidity more quickly.  Butter containing residual heat-stable bacterial lipases undergoes rapid lipid hydrolysis even during storage at –10˚C. Lipases that preferentially release short chain fatty acids (C4 to C8), with and without long-chain fatty acids, cause the most off-flavor in butter. 19/32
  • 21.  Rich source of Protein – Molds, Yeast, Bacteria and Algae.  Digestibility of these protein – 65-96%.  Most preferred – Yeast ( Candida, Saccharomyces)  Advantages - Microbial protein are a good sources of Vitamin B and Carotene  Can be produced under laboratory setting.  Increase of microbial protein will reduce the use of grains as animal feed, which then can be used as human food.  Disadvantages- Poor in essential amino acids (methionine) 13/22 20/32
  • 22. Amino Acids:  Proteins of most cereal grains are deficient in the essential amino acids, particularly lysine, and tryptophan. To improve the biological values, cereals are supplemented with essential amino acids.  Supplementing vegetable proteins with essential amino acids has been suggested to improve the protein quality for people who either do not consume animal proteins (people on vegetarian diets) or do not have enough animal proteins. 14/22 21/32
  • 23.  To meet this demand as well as for use as nutrient supplements, large amounts of several essential acids are being produced. At present, because of economic reasons, they are mostly produced from the hydrolysis of animal proteins followed by purification.  Now we have bacterial strains that have been isolated, some of which are lactic acid bacteria that produce and excrete large amounts of lysine in the environment.  Isolating high-producing strains of other amino acids, and developing strains by genetic and metabolic engineering that will produce these amino acids in large amounts, can be important for economical production of essential amino acids. 22/32
  • 24. The different parameters or factors that are used in combination include  intrinsic factors (e.g., aw, pH, Eh, and natural inhibitors),  processing factors (e.g., heating, drying, fermentation, and preservatives)  extrinsic factors (e.g., temperature and aerobic or anaerobic environment). Example : Preservation of jams and jellies - high heat, low pH (of fruits), low water activity (sugar in fruits and added), and anaerobic packaging are used to reduce microbial numbers as well as the growth of survivors. 15/22 23/32
  • 25.  The greatest challenge will be the long-term preservation of meat, dairy, and fish products at refrigerated temperature or even at ambient temperature, where refrigeration is not always available (military operation)  The effectiveness of combining different factors to control target microorganisms in a food has to be studied.  In conducting such studies, one should also recognize that the factors could have additive, synergistic, or even adverse (neutralizing) effects when used in combination. 24/32
  • 26. Low-Heat Processing:  Low heat (100ºC) does not kill many pathogenic and spoilage bacterial spores.  They can be heat activated, which leads to germination and outgrowth.  However, if the pH of the food is reduced to 4.5 or NO2 and NaCl are added, the heat-shock spores will not germinate. 16/22 25/32
  • 27.  Clostridium botulinum grows at 35ºC at an aw of 0.95.  If the storage temperature is reduced to 20ºC, it does not grow unless the aw is increased to 0.97.  Similarly, L.monocytogenes grows at 25ºC in a broth containing 6.5% NaCl in 3d, but fails to grow under similar conditions at 14ºC. 26/32
  • 28. Low pH:  Clostridium botulinum grows at pH 7.0, 37ºC, and an aw of 0.95  pH is dropped to 5.3, no growth is observed, even at an aw of 0.99. .  Clostridium botulinum produces a toxin during incubation at 16ºC in 28 d at pH 5.5, but at pH 5.2 under the same conditions, no toxin is produced.  In using acid to reduce pH, it is important to recognize that organic acids, such as acetic, propionic, and lactic acids, are more effective than HCl and phosphoric acid. 17/22 27/32
  • 29. Preservatives:  Preservatives such as NaCl and BHA act synergistically to increase the antimicrobial action of sorbates.  Organic acids are also effective at low pH because of higher concentrations of undissociated molecules.  The bactericidal effect of bacteriocins can be enhanced when used with acids and EDTA. 18/22 28/32
  • 30. Conclusion :  Sublethal injury to microbial cells and spores and increased susceptibility of injured cells and spores to one or more preservation methods can play important roles in controlling microorganisms by a combination of factors.  The hurdle concept is based on the scientific concept that when two or more methods are used together the combinations are more effective, even at a much lower treatment level, because of the large number of possibilities.  Sublethal injury occurs due to the exposure of bacterial cells to unfavorable physical and chemical environments (beyond the growth range but not in the lethal range) that cause reversible alterations in the functional and structural organizations of the cells. 20/22 29/32
  • 31.  Handbook of Food Enzymology - CRC Press Book. ... 1st Edition. John R. Whitaker, Alphons G. J. Voragen,  Food Microbiology. MJP Publishers, 2007 K. Vijaya Ramesh.