• Various techniques can be used for determination of microorganisms in food.
• Some of them give total count (viable+ non-viable) while others give only viable count.

1. Standard plate count (SPC):

• Standard plate count gives viable count of organism present in food.
• Procedure of performing standard plate count is given above in figure.
• The number of organisms in original food is determined by counting the colony on agar plate.
• Two major assumptions of SPC are:
  • Microorganisms in suspension are separated as single cell so that each colony is developed from single cell.
  • All viable cells placed on medium will multiply and produce a colony
• Incubation time and temperature for different microorganisms:
  • Psychrophilesà 7^oC, 7 days
  • Mesophilesà 35^oC, 24-48hrs
  • Thermophilesà 55^oC, 48hrs
• Advantages:
  • It gives viable count.
  • It is extremely sensitive i.e. extremely low and high microbial population can be counted.
• Disadvantages:
  • If the suspension is not homogenous and contain aggregate of cells, the colony count will be lower than the actual number of microorganisms.
  • If the suspension contains different types of microorganisms, all of them cannot grow in the same medium and under the same condition.

Types of Standard plate count (SPC) method:

1. Pour plate technique:
   • In this method, food is firstly serially diluted in appropriate diluent.
   • Then, measured volume of sample from diluted tube is placed in petriplate.
   • Melted agar at 44-45^oC is mixed with it.
   • After homogenous mixing of sample with melted agar, it is kept for solidification.
   • Then the petri plates are incubated at appropriate time and temperature.
   • Plate containing colonies between 30-300 is selected and number of colonies are counted.
   • Now, number of organisms in original food sample is calculated by the following formula:
     • Colony forming units (CFU/ml) = (Number of colonies/volume of sample ) x dilution factor
   • Since psychrophiles cannot survive temperature of melted agar, this technique is not suitable for them.
   • In this method, both surface and subsurface colonies are developed.
   • Subsurface colonies are difficult to be isolated.
   • Spread plate technique:
   • In this method, appropriately diluted sample is placed on the surface of solidified agar.
   • Then the drop of sample is spread over agar surface using bent glass rod.
   • Plate is incubated for sufficient time and temperature, then number of colonies are counted.
   • Calculation of number or organisms is done similarly as in pour plate technique.
   • This method is suitable for psychrophile also and only surface colonies are developed.
2. Streak plate technique:
   1. In this technique, a transfer loop is used to spread the specific volume of specimen over a surface of solidified agar.
   2. The transfer is done by calibrated loop of specific volume.
   3. Sometimes, selective and differential media can be used to select growth of specific organism.

2. Membrane filter technique:

• This technique is particularly important to analyze microorganisms in liquid food in which microbial content is too low.
• In this methods, major measured volume of liquid filtered through membrane filter of specific porosity.
• Then filter pad is removed and placed on the surface of agar plate and then incubated.
• Microorganisms grow on surface of membrane filter to form colony.
• Then total number of organisms in original sample is calculated.
• Nutrient or selective agar media can be used for microbial growth.

3. Most probable number (MPB) method:

• It is statistical technique to determine number of organisms in sample.
• It gives most probable number but not the actual number.
• Turbidity, gas production and acid production are observed to determine microorganisms.
• This method is based on 3 steps:
  • Presumptive test
  • Confirmed or confirmatory test
  • Completed test

4. Direct microscopic count (DMC):

• In this method, there is no difference between dead and viable cells.
• Total cells are counted.
• In this method, the result is obtained faster than most other methods because incubation period is not required.
• Procedure of Direct Microscopic Count is given in above chart.
• In case of liquid food, direct smear is made.
• For solid food, it must be first divided up to 10^-1.
• Fatty foods must be defatted in xylene or acetone for preparation of smear.
• The xylene/acetone is then removed by dipping it in ethanol.
• In this method, number of microorganisms in microscopic field are counted directly.
• This technique is widely used to assess the quality of raw milk and other dairy product.
• Breed count method:
  • It is an example of Direct Microscopic Count.
  • This method was initially developed by R.S Breed.
  • In this method, 0.01ml sample is spread over 1cm square area on slide.
  • If sample is fatty, it should be defatted with xylene or acetone.
  • Excess xylene or acetone is then removed by dipping it into ethanol.
  • Then slide is dried, fixed and stained with appropriate dye and observed under microscope.
  • Average number of microorganisms per field is counted.
  • Then area of microscopic field is determined from which number of microorganisms in original sample is calculated.
  • It is not practical to count entire field.
  • So, only few microscopic fields are counted to determine average number of organisms in sample.

• Advantages of DMC:
  • It is simple and rapid technique.
  • Morphology as well as gram reaction of microorganism spore production etc. can be observed in microscope.
  • Very small amount of sample is needed.
  • The prepared slides can be stored and maintained as permanent record.
• Disadvantages of DMC:
  • DMC cannot distinguish viable and non-viable cells.
  • Food particles are not always distinguishable from microorganism’s cell.
  • Some microorganisms do not take stain and may not be counted.
  • It is very difficult to count microorganisms when the initial load is very high.

5. Electronic counter:

• In this method, standard volume of suitable dilution of suspension is placed in electronic counter.
• The machine has small aperture through which microorganisms can pass.
• The passage of microorganisms through aperture causes alteration in electric resistance across it which is recorded as on impulses.
• These impulses are counted by suitable circuit in the counter.
• Number of impulses from fixed volume of sample is used to calculate number of organisms in original food sample.

6. Dye reduction test:

• Two dyes are commonly employed in dye reduction test to estimate viable number of organisms.
• Methylene blue reduction test:
  • Methylene blue reduction test is commonly used to determine number of viable organisms in raw milk.
  • In this method, methylene blue is mixed with raw milk or incubated.
  • Microorganisms present in milk reduce methylene blue to form leucomethylene blue so that milk becomes blue to colorless.
  • The time of decolorization of milk is indicative of number of viable organisms.
  • If number of organisms are higher it is decolorized in shorter time and vice-versa.
  • In this method microbial quality of milk assessed by reduction time.
• Resazurin reduction test (rapid test):
  • It is an example of rapid dye reduction test use to determine number of viable organism in food such as raw milk.
  • In this test, resazurin dye is mixed with raw milk. Microorganism present in milk reduce resazurin such that its color changed from stale blue to pink or colorless.
  • If the number of microorganism is higher, dye is reduced in shorter time and vieversa
  • Therefore, microbial load of milk can be predicted by reduction time of resazurin. In this method, result is obtained within 10 mins.
• Advantages of dye reduction test:
  • It is simple, easier, and inexpensive test.
  • Only viable cells actively reduce the dye. So, that number of viable organism can be predicted.
• Disadvantages of dye reduction test:
  • Not all microorganisms reduce the dye equally.
  • They are not applicable for food that contain reducing substances such as reducing enzymes unless special steps are employed.

Detection of Microorganisms in foods: methods and techniques

The post Detection of Microorganisms in foods: methods and techniques appeared first on Online Biology Notes.

• Contamination of milk occurs at two levels:
• On farm:
  • Freshly drawn milk contains relatively few bacteria however Micrococcus and Streptococcus are usually found in aseptically drawn fresh milk.
  • During normal milking process, milk is subjected to contamination from udder of animal and adjacent areas.
  • Bacteria found in manure, soil and water contaminate are udder of animal from where they enter into the milk.
  • Other possible source of contamination is hand and finger of milker or other dairy workers.
  • Contamination also occurs from dairy utensils.
• During transport and at processing plant:
  • During transport and manufacturing, contamination occur through tanker, transfer pipes, sampling utensils and other equipment.
  • Sometimes, pathogen may contaminate the milk from hand and finger of milk handler.

Microbial Spoilage of milk and mik products:

• Milk is an excellent culture media for growth of many microorganisms.
• Therefore, different types of microorganisms grow in it and cause spoilage.

i. Spoilage of Milk and cream:

• Souring:
  • Evidence of souring of milk are sour flavor and then coagulation of milk to form solid like curd.
  • Many lactic acid bacteria, coliform and other bacteria ferment sugar of milk and produce acid.
  • At temperature of 10-37^oC, Streptococcus lactis is most likely to cause souring with possible growth of Coliform, Enterococci, Lactobacillus and Micrococcus.
  • At higher temperature, 37-50^oC, Streptococcus thermophilus and Streptococcus faecalis may produce 1% acid and it may be followed by Lactobacillus which produces more acid.
  • Little souring occurs in milk held at refrigeration temperature.
  • Pasteurization of milk kills more active acid forming bacteria but permit survival of thermoduric lactic acid bacteria such as Enterococcus, Streptococcus thermophilus, Lactobacillus, etc.
  • Bacteria other than lactic acid bacteria produce acid specially if conditions are unfavorable for lactic acid bacteria.
  • For example: coliform produce acetic acid, formic acid, ethanol, CO₂, H₂ etc.
  • Similarly, Clostridium produce butyric acid.
• Gas production (Strong fermentation of milk):
  • Sugar fermenting organism produce gas together with acid.
  • Main gas formers, Coliform, Clostridium, Heterofermentative lactic, Propianics bacillus, etc.
  • Coliform, Clostridium, and Bacillus produce both H₂ and CO₂, while others produce only CO₂.
  • Gas production in milk is evidenced by foam at top of liquid milk by gas bubble trapped in curd, by formation of curd.
  • Excessive gas production causes cracking or breakdown of curd causing so called stormy fermentation of milk.
  • Clostridium perfringens mainly causes stormy fermentation.
• Proteolysis:
  • Proteolysis is facilitated by storage at lower temperature by destruction of lactic acid bacteria or by distribution of already produced acid by mold and yeast.
  • Changed cause by proteolytic organism include:
  • Acid proteolysis in which acid production and proteolysis occur simultaneously.
  • Proteolysis with little acidity or even alkalinity.
  • Sweet curdling which is caused by renin like enzyme of microorganisms.
  • Slow proteolysis by intracellular enzyme of bacteria after their autolysis.
  • Residual proteolytic activity of some heat stable proteinase.
  • Acid proteolysis is caused by Micrococcus , Streptococcus faecalis var liquefaciens and some lactose fermenting proteolytic Bacillus species.
  • Sweet curdling is caused by Bacillus cereus.
• Ropiness/ sliminess:
  • Ropiness of milk occur both by bacterial and non-bacterial causes non-bacterial ropiness occurs due to thickness of cream or due to film of cousin or Lactalbumin during cooling.
  • Bacterial ropiness is caused by slimy capsular material of bacteria which usually develop at low storage temperature.
  • Bacteria producing ropiness in milk are Alcaligenes viscolactis, micrococcus freudenreichii, Enterobacter aerogenes, Klebsiella oxytoca, E. coli.
• Change in milk fat:
  • Various bacteria, yeast and mold hydrolyses fat of milk and cause rancidity.
  • Species of Proteus, Pseudomonas fragi, Staphylococcus, Bacillus, Micrococcus, Clostridium, etc. are lipolytic.
  • Pseudomonas fragi and Staphylococcus aureus produce fairly heat resistant lipase.
• Alkali production:
  • Pseudomonas fluorescence and Alcaligene viscolactis produce alkali.
  • Alkali production is due to formation of ammonia from urea and formation of carbonate from organic acid.
• Flavor defect:
  • Acid flavor: Acid flavor may be aromatic or sharp. Sharp flavor is caused by production of acetic acid formic acid, butyric acid etc. by Coliform and Clostridium. It is undesirable. Aromatic flavor is caused by Streptococcus lactic and Leuconostoc when they grow together. It is desirable.
  • Caramel or burnt flavor: It is caused by Streptococcus lactic var. maltigens.
  • Bitter flavor: It is caused by proteolytic organism.
  • Other flavor: They include earthy flavor by Actinomycetes, fruity flavor by Pseudomonas fragi, soapiness by Pseudomonas sapolactic etc.
• Color defect: Growth of pigmented bacteria and other organism give undesirable color. Some examples include:
  • Blue milk: It is caused by Pseudomonas syncyaneum
  • Yellow milk: caused by Pseudomonas synxantha and also by flavobacterium.
  • Red milk: caused by Serratia marcescencs and Micrococcus roseus.
  • Brown milk: caused by Pseudomonas putrefaciens and by enzymatic oxidation of tyrosin by Pseudomonas fluorescence.

ii. Spoilage of Butter:

• Many spoilage microorganisms come in butter from cream or milk from which it is prepared.
• Color defect:
  • Some color defect of butter is non-microbial. They include pink color caused by sulphur-dioxide refrigerant, surface darkening caused by evaporation of water from surface.
  • Discoloration caused by microorganisms depend on type of organism. For example, Stemphylium give black spots, Penicillium give green spot, Alternaria or Phoma give brown spots, Pseudomonas nigrificans give reddish brown spot etc.
• Flavor defect:
  • Cream and butter have capacity to absorb moisture from surrounding.
  • Butter may gain such flavor from absorption of flavor is developed in butter during microbial growth.
  • Some odors in butter caused by growth of organisms include:
  • Fishiness caused by Aeromonas hydrophila.
  • Ester like flavor caused by Pseudomonas fragi.
  • Rancid odor caused by lipase producing organism.
  • Yeasty flavor caused by yeast etc.

iii. Spoilage of Cheese:

• Spoilage of cheese occurs either by mechanical damage or by microorganisms.
• Microbial spoilage of cheese occurs during following three stages:
• Spoilage during manufacturing:
  • During manufacture of most cheese lactic starter culture is added to carry out lactic acid fermentation.
  • If these lactic starters are not effective or when contamination is heavy, many contaminating organisms grow in it and bring undesirable changes in cheese. For.eg. if starter culture is not effective, Clostridium and Bacillus grow and produce holes and other changes.
  • Acid proteolytic bacteria may produce bitter flavor.
  • Leuconostoc may produce holes in cheese.
  • Various organism cause proteolysis, gas production, sliminess and off flavor that damage the quality of cheese.
  • Cheese with too low acidity because of failure of starter culture or because of addition of cream is often made slimy by alcaligenes, melalcaligenes and Pseudomonas fragi.
• Spoilage during ripening:
  • During ripening, spoilage occurs by enzyme released from autolyzed bacteria or by growth of microorganisms during ripening.
  • Main type of spoilage differs with type of cheese.
  • In most of cases, like gas production by Clostridium, Heterofermentative lactis, Propionibacterium, Yeast etc. cause eye formation or cracking of cheese.
  • Clostridium also produce undesirable flavor by production of butyric acid.
  • Certain lactic streptococci give bitter flavor.
  • Some bacteria and yeast give sweet, fruity and yeasty flavor.
  • In cheese with insufficient acidity, putrefaction is caused by anaerobic Clostridium.
  • Microorganisms also caused discoloration on surface of cheese.
  • Bluegreen or black discoloration are produced by reaction of H₂S produced by Microorganisms with metal or metallic salt.
  • Oxidation of tyrosine by bacteria give reddish brown to greyish brown color.
  • Propionibacterium grow as yellow, pink or brown colored complex.
• Spoilage of finished cheese:
  • Soft cheese is most perishable and hard cheese such as cheddar and swiss cheese are most stable.
  • Most common spoilage organism of finished cheese are molds.
  • They grow on surface or into holes or cracks and cause discoloration.
  • Sometimes off flavor is also produced.
• Some molds causing spoilage of cheese are:
  • Cladosporium: It grows on surface and gives black discoloration.
  • Oospora (Geotrichum): Oospora lactis (called dairy mold) grow on surface of soft cheese. In this case, curd gradually becomes liquified under the growth. Oospora crustacea give red spots.
  • Penicillium: Penicillium puberulum and other green spored species grow on surface or into holes and give green coloration.
  • Monilia: Monilia nigra grow on surface of hard cheese and give black discs.

Preservation of milk and milk products from microbial spoilage

1) Reducing contamination:

• Keeping quality of milk is improved when contamination of milk is reduced.
• Udder and adjacent areas should be washed with water and if possible, by germicidal solution before milking.
• Hand and finger of milker should be clean.
• Dairy equipment and other milk processing equipment should be sanitized properly.
• Packaging of milk and milk products in can or packages also avoids contamination.

2) Removal of microorganisms:

• Microorganisms from milk are difficult to remove.
• High speed centrifugation (called bactofugation at 10,000 rpm removes about 99% spores and more than 50% of vegetative cell plus some protein.
• However, bactofugation is not done commercially for removing bacteria from milk.
• Mold can be removed physically from surface of cheese by scrapping or periodic washing.

3) Use of heat:

• For milk, pasteurization is used for preservation.
• Cream can be heated by injecting steam or by combination of steam injection and evacuation in a process called vacreation.
• Heat is also applied for cheese.
• Cooking at 65.6^oC or higher in melting of cheese during manufacture reduces many spoilage microorganisms.
• Evaporated milk is canned and then heated by steam under pressure.

4) Use of low temperature:

• Refrigeration temperature is recommended for milk or similar product during storage in plant during transportation and during storage in home until consumption.
• Fermenter milk and cheese are chilled after their manufacture and kept chilled until they reach the consumption.
• Chilling storage is most commonly applied technique of preservation of milk and milk product in home for short term storage.

5) Drying:

• Many types of milk product are prepared by drying or removing moisture from them.
• There are two types of such product, one is condensed product and the other is dry product.
• One condensed product is evaporated milk, in which 60% moisture is removed, so that remaining high lactose concentration in solution is inhibitory to micro-organisms.
• Bulk condensed milk, sweetened condensed milk, semisolid butter are other examples of condensed product.
• Milk product prepared in dry form include dry milk, ice-cream mix etc.
• Milk can be dried by drum drying or spray drying.

6) Use of chemical preservative:

• There are two types of chemical preservatives i.e. developed and added preservative important in milk product.
• Developed acidity in many fermented milk product retards growth of many microorganisms.
• Propionic acid develop in swiss cheese is inhibitory to mold.
• Many chemical preservatives are added from outside in many milk products.
• Sorbic and propionic acid added in cheese and yoghurt prevent surface spoilage by mold.
• Sugar is added in sweeten condensed milk to reduce A_w.
• NaCl used in cheese during manufacturing gives flavor and also acts as preservative.
• The addition of H₂O₂ combine with mild heat treatment has been used for pasteurization of milk for certain kind of cheese. Some cheese is also smoked.

Microbial spoilage of milk and milk products

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• Heated canned foods are microbiologically more stable than most other foods.
• Better keeping quality of heated canned food is due to:
  • Heat treatment applied during canning kills most of the microorganisms present in original food.
  • Recontamination of food from outside is prevented by sealed can.

Spoilage of heated canned food:

• Spoilage of heated canned foods occurs by two ways:
  • Survival of spores of some thermophilic bacteria during heating of canned foods.
  • Recontamination of microorganisms from outside through leak in container.

Various types of spoilage of heated canned food are:

i. Spoilage of canned food by spore forming thermophilic bacteria:

• Spores of thermophilic bacteria are not killed easily by heat treatment applied during canning.
• Later, spore germinates in food and cause spoilage.
• Three important types of spoilage caused by thermophilic spore formers are:
• Flat sour spoilage:
  • This type of spoilage occurs mainly in low acid foods and is caused by species of Bacillus, such as Bacillus coagulase and Bacillus stearothermophilus.
  • Later, spore germinate when canned food is kept hot for sometimes as in case of slow cooling of can.
  • They produce acid without gas in food.
  • Therefore, the can remains flat during spoilage and hence spoilage cannot be detected by examination of can from outside.
• Thermophile anaerobe spoilage:
  • A spoilage is caused by Clostridium thermosaccharolyticum, which is a thermophilic anaerobe.
  • It produces acid and gas in food. Gas is a mixture of CO₂ and H₂.
  • Due to gas production, can swells and finally burst if it is kept for long time.
  • The spoiled food has usually sour odor or taste.
• Sulfides or sulfur stinker spoilage:
  • It is caused by Desulfotomaculum nigrificans.
  • Spoilage usually occur in low acid food.
  • Spore of these organisms are usually less heat resistant than those of flat sour and thermophile anaerobes bacteria.
  • Therefore, spoilage by this organism indicates inadequate heat treatment during canning.
  • The organism is also obligate thermophile. Therefore, spoilage by this organism occur in case of poor cooling or hot storage of can.
  • The organism produces H₂S that react with tin of can to form black spots of FeS in food and on inner wall of can.
  • H₂S produced give putrid odor which is widened when can is opened.

ii. Spoilage of canned foods by spore forming mesophilic bacteria:

• Spoilage by mesophilic spore formers result from inadequate heat treatment during canning.
• Spoilage by mesophilic Clostridium species:
  • Several mesophilic Clostridium causes spoilage of canned food.
  • Sugar fermenting species such as Clostridium butyrium and C. pasteurianum produced butyric acid by fermentation of sugar.
  • They also produce hydrogen and carbon-dioxide that sweet the can other species such as Clostridium botulinum, C. sporogens and C. putrifaciens are proteolytic or putrefactive.
  • They decompose protein to produce H₂S, endole, skatable and mercaptans that give bad odor to the food.
• Spoilage by mesophilic Bacillus species:
  • Many species of Bacillus are aerobic and therefore cannot grow in well evacuated can.
  • They cause spoilage especially in poorly evacuated can.
  • Bacillus subtilis, B. mesentericus and B. polymyxa cause spoilage of many canned foods such as corn, tomato etc.
  • Since, spores of these organisms are less heat resistant, they usually spoiled food by contaminating through leakage container.

iii. Spoilage of canned foods by non-spore forming bacteria:

• Non spore forming bacteria cause spoilage of canned food if mild heat treatment is applied during canning or, when they enter into the can through leakage during cooling by contaminated water.
• Many thermoduric bacteria such as Enterococcus, Streptococcus thermophilus, Micrococcus, Lactobacillus and Leuconostoc species can survive pasteurization temperature and spoil the canned food.
• However, in most of the cases, spoilage by non-spore formers indicates leakage of container.
• Type of spoilage depends on type of microorganisms. For example: Coliforms and heterofermentative lactic acid bacteria swell the can by production of gas.
• Non gas forming bacteria such as Pseudomonas, Alcaligenes, Micrococcus, Proteus etc. also spoiled canned food.

iv. Spoilage of canned foods by yeast and mold:

• When yeast grow in canned food, they cause swelling of can by production of CO₂.
• Spoilage by yeast indicates recontamination through leakage or lack of heat processing plus poor evacuation.
• Spoilage by mold:
  • Some species of mold such as Sclerotia and Byssochlamys fulva are somewhat heat resistant and they survive mild heat treatment.
  • Other molds enter through leakage in can.
  • Mold cause spoilage of high acid and high sugar foods such as jam strain of Aspergillus penicillium and Citromyces are found commonly in canned food.

Microbiological spoilage of Canned foods

The post Microbiological spoilage of Canned foods appeared first on Online Biology Notes.

• The healthy inner flesh of the meat contains few or no micro-organisms although they have been found in lymph node and bone marrow.
• Upon, death of animal, invasion of tissue by contaminating microorganisms takes place.
• In meat, microorganisms come from external source or from meat animal itself.
• Factors that influence invasion of meat by contaminating microorganisms are:
  • The load of organism in intestine. The greater the load, the greater will be the invasion of meat tissue. Therefore, starvation of animal for 24hrs before slaughter has been recommended.
  • The physiological condition of the animal immediately before slaughter:
  • The method of killing or bleeding: The better and more sanitary bleeding method contributes less contamination.
  • The rate of cooling: The rapid cooling will reduce the rate of invasion of meat by contaminants.
• Microorganisms are spread in the meat through the blood and lymph vessel, connective tissue spaces and by grinding in ground meat.

Microbial Spoilage of meat:

• Common type of microbial spoilage of meat can be classified on the basis of whether they occur under aerobic or anaerobic condition and whether they are caused by bacteria, yeast, or mold.

1. Spoilage of meat under aerobic conditions:

i. Bacterial spoilage of meat:

• Surface spoilage:
  • It is caused by Pseudomonas, Acenatobacter, Streptococcus, Leuconostoc, Bacillus and Micrococcus.
  • Temperature and available moisture influence type of microorganisms causing slime.
• Change in color of meat:
  • Red color of meat may be changed into green brown or grey due to production of oxidising agent, H₂S, etc. by microorganisms. For example, Lactobacillus and Leuconostoc cause greening of sausage.
• Change in fat:
  • Fat of meat may become rancid due to lipase producing microorganisms such as Pseudomonas and Achromobacter.
• Surface color due to pigmented bacteria:
  • Serratia marcescens give red spots.
  • Pseudomonas syncyanea give blue color, Chromobacterium lividum gives greenish blue to brownish black color, Flavobacterium give yellow color.
• Phosphorescence:
  • It is caused by luminous bacteria e.g. Photobacterium growing on surface of meat.
• Off odors and off taste:
  •  Undesirable odor and taste called taint are caused by many bacteria due to production of volatile acids such as formic acid, acetic acid, butyric acid etc.
  • Actinomycetes give musty or earthy flavor.

ii. Fungal spoilage of meat:

• Stickness:
  •  Many molds grow on surface of meat and make it sticky to touch.
  • Whiskers: when meat is kept at temperature near freezing, mold grow slowly without sporulation on surface producing while cottony growth.
  • It may be caused by Thamnidium, Mucor mucedo, Mucor racemosus etc.
• Black spot: It is caused by Cladosporium herbarum.
• White spot: It is caused by Sporotrichum carnis.
• Green spot: It is caused by Penicillium species.
• Change in fat: Many molds produce lipase and cause hydrolytic rancidity of fat.
• Off odor and off taste:
  • Many molds give musty flavor to meat in the vicinity of their growth.
• By yeast:
  • Under aerobic condition, yeast grow on surface of meat causing sliminess, rancidity of fat, off odor and taste and discolorations like white, pink, brown spots.

2. Spoilage of meat under anaerobic conditions:

• In anaerobic condition, anaerobic or facultative anaerobic bacteria spoil the meat.
• Souring:
  • It is caused by formic acid, acetic acid, butyric acid, propionic acid, higher fatty acids and other organic acids. E.g. lactic acid produced by bacteria.
  • Souring may also be caused by foods own enzyme.
• Putrefaction:
  • It refers to the anaerobic decomposition of protein with production of offensive smelling compounds such as H₂S, mercapatans, indole, skatole etc.
  • It is usually caused by Clostridium species but species of Pseudomonas proteus and Alkaligens may cause putrefaction.
• Taint:
  • It refers to any undesirable odor or taste.

Spoilage of different types of meat products:

Spoilage of fresh meat:

• Fresh beef:
  • Change in hemoglobin and myoglobin so as to cause loss of bloom and production of reddish-brown methemoglobin and metmyoglobin.
  • White, green, yellow, greenish blue, black spots due to pigmented micro-organism’s phosphorescence.
  • Sliminess on the surface due to slime forming organism, stickiness due to mold, whiskers due to mold etc.
  • Souring and putrefaction.
• Fresh pork sausage:
  • Souring is the most common type of spoilage of refrigerated sausage.
  • It may be caused mainly by Lactobacillus and Leuconostoc.
  • Enclosed sausage undergoes slime formation by mold on long term storage.
  • Colored spots also appear on surface due to pigmented organism.

Spoilage of cured meat:

• Curing salts make meat more susceptible to gram +ve bacteria and mold than to gram -ve bacteria.
• Sausage:
  • If moisture is available micrococcus and yeast form slime in the surface.
  • With less moisture, mold may give cottony growth and colored spots.
  • Microorganisms that produce peroxide cause greening of sausage. E.g. Lactobacillus, Leuconostoc and other catalase -ve bacteria.

Spoilage of Refrigerator packaged meat:

• Packaging film permitting good penetration of oxygen and CO₂ favor more aerobic bacteria such as Pseudomonas, Acenatobacter and Moraxella.
• They cause spoilage like off flavor slime and sometimes putrefaction.
• Film with poor gas penetration encourage lactic acid bacteria causing sourness and slime.
• Curing solution or pickles:
  • Spoilage of curing solution is usually putrefactive and is caused by Vibrio, Alcoligens and Spirillum.
• Souring can be caused by Lactobacillus and Micrococcus.
• Slime is formed by Leuconostoc and Micrococcus.

Preservation of meat from spoilage:

i. Asepsis:

• Asepsis prevents external microorganisms from entering into the meat.
• Water spraying before slaughter is recommended to remove dirt and microorganisms from hair and hide and foot bath may be used to remove dirts from hoofs.
• Other method of asepsis includes sanitization of knife, used for killing and personal hygiene of food handler.
• Films may be used to keep out bacteria and other microorganisms.

ii. Use of heat:

• Canning is a specialized technique of preservation for meat.
• Canned meats are divided into two groups on the basis of heat treatment employed for canning:
• Self-stable canned meat:
  • The processing temperature is 98^oC and the size of container is usually less than 1lb.
  • They are made sterile or at least commercially sterilized.
• Non-self stable canned meat:
  • The processing temperature is about 85^oC and the size of container is upto 22 lb.
  • They kill only part of spoilage organisms and the meat must be kept frozen to prevent spoilage.

iii. Use of low temperature:

• Chilling:
  • Chilling temperature vary from -1.4^oC to 2.2^oC with the lower temperature preferred.
  • Meat are chilled rapidly because there is opportunity for growth of mesophiles if cooled suddenly.
  • The time limit for chilling storage depends on meat. For.eg. 30 days for beef, 1-2 weeks for mutton.
• Freezing:
  • Freezing is increasingly effective as the temperature drops from -12.2^oC towards -28.9^oC.
  • Meats are frozen quickly in package.
  • Freezing process kills about half of the bacteria and their number decrease slowly during storage.

iv. Preservation by radiation

v. Preservation by chemical preservative:

• Chemical preservatives are added in meat as a component of wood smoke and curing solution.
• Smoking:
  • Smoking of food usually have two main purposes i.e. adding desired flavor and preservation.
  • Other desirable effect include improvement in color and flavor and tenderization of meat.
  • Commonly smoke is obtained by burning hard wood, and temperature of smoking generally vary from 48^oC- 72^oC.
  • Smoking period lasts from a few hours to several days.
  • Preservative action of smoking is due to three factors i.e. chemical preservatives, drying action and heating.
  • Wood smoke contains large number of volatile compounds with bacteriostatic and bactericidal activity.
  • Some of which are formaldehyde, phenol, cresol, aliphatic acids, ketones, aldehyde etc.
  • Composition of anti-microbials depends on type of wood used.
  • Wood smoke also contains large number of volatile coloring and flavoring agent.
• Curing:
  • It is a common preservation method for meat.
  • Both solid cuts and ground meat are subjected to curing.
  • Curing agents permitted in meat are NaCl, sugar, sodium nitrate and vinegar, but only the first three are commonly used.
  • There are few methods of introducing curing agents in meat:
  • The dry cure in which the dry ingredients are rubbed into the meat.
  • The pickles cure in which meat is immersed in the solution of curing agent.
  • The injection cure in which concentrated solution of agent is injected into the meat by needle.
  • The direct addition method in which curing agents are added directly in the finely ground meat.
  • The curing temperature (especially with pickle cure) usually is about 2.2^oC – 3.8^oC and the time of curing vary with the meat and the method used.
    • Function of ingredients used in curing are:
    • NaCl: It lowers A_w and gives flavor.
    • Sugar: It lowers A_w and gives flavor.
    • Vinegar: It is preservative.
    • Sodium nitrite: It is a coloring agent. It decomposes into nitric oxide that reacts with myoglobin of meat to form nitroso-myoglobin that gives red color to the meat. Sodium nitrate is used as a source of sodium nitrite, but it is not a real coloring agent.

v. Spices and other condiments:

• Spices used in meat are not in concentration high enough to be preservative but they help other preservative methods.

vi. Antibiotics:

• Antibiotic most commonly recommended in meat are chloramphenicol, oxyltetracycline, chlortetracycline, nisin etc.
• Antibiotics may be applied in meat by various ways.
• It may be fed to the animal for long period.
• It may be fed in high dose for a short period before slaughter.
• It may be injected into flesh.
• It may be applied or mixed directly to the meat

Microbial spoilage of meat and methods of preservation

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• Food is a good culture medium for growth of many microorganisms.
• The presence of microorganisms in food causes decomposition and spoilage of food.
• Therefore, microbial analysis of food is important in quality assurance of food.
• Microbial analysis of food gives idea about:
• The quality of food:
  • By determining type and number of microorganisms in food and by comparing this standard value, we can evaluate quality of food
• The sanitary condition of food:
  • Microbial analysis helps to predict possible contamination of food.
• The storage condition:
  • By microbial analysis we can detect whether food is stored in proper condition or not.
• The types of microorganisms in food:
  • By microbial analysis, we can detect pathogen or their toxic product if present in food.
• The quality of raw material:
  • It gives quality of raw materials used in food.
• Evaluation of effectiveness:
  • Microbial analysis also evaluates the effectiveness of preservative methods employed.

Steps in microbial analysis of food:

1. Sampling:

• Sampling is an important step in microbial analysis.
• Sample of food can be obtained from processing line from storage house or from market.
• Sample:
  • The sample should be representative of whole lot of food.
  • Sample should be collected randomly from the lot.
  • The principle is to avoid bias and to draw sufficient number of sample units.
• Sampling device:
  • Sterile spoon pipette, stirrer, swab etc. are used for taking samples.
  • Sterile scissors, knife, can opener etc. are used for opening food packages.
  • Liquid and small particles are sampled with sampling tube, spoon, droppers etc.
  • Large food materials (e.g. Cheese) are cut by knife.
  • For surface samples, knife, swabbing or sticky cello phone tape is used.
  • Sticky cello phone tape is pressed on the surface of solid agar to transfer microorganisms.
• Number of samples:
  • If food is examined for Salmonella, usually co-samples are collected 5 samples are needed for other organisms.

2. Techniques for sample collection:

• When the product is in big container: Steps in sample collection are:
  • Outer surface of container is cleaned by washing or by 70% ethanol.
  • Container is opened with the help of sterile cutting instrument.
  • If the food is in bulk, the sample are collected from various places within container.
  • In case of liquid sample, it is first agitated to form homogenous mixture and sample is taken.
• When the food is in small container:
  • When the sample is in small container, they should be taken directly to the lab for analysis.

3. Preparation of sample:

• For solid food sample: Solid food is mixed with sterile diluent in mechanical blender to obtain homogenous suspension. For example, when 10gm food is added to 100ml diluent, 10^-1 dilution is obtained.
• For liquid food sample: Liquid food is serially diluted to obtain 10^-1, 10^-2, 10^-3 dilution.

4. Transportation of sample:

• Multiplication and death of sample (microorganisms) should be prevented during transport of sample.
• The sample should be delivered to lab immediately.
• Perishable and unfrozen sample should be cooled to 0-5^oC and transported in insulated container.
• Frozen product must be maintained in frozen condition until analysis.
• Sampling reports should be prepared and sent with sample.

5. Sampling report:

• The report should provide the following information:
• Name and address of person collecting sample.
• Date, time, place and purpose of sample collection.
• Nature of food
• Name of manufacturer, importer, seller, batch number, manufacture data, expiry date etc.
• Method of sampling, size and no. of samples, temperature of product at the time of sampling.
• Suggested test can also be given.

6. Sampling plan:

• Choice of sampling plan depends upon the organism present in food, the nature of food and whether the food is raw or processed or eaten directly, type of consumer i.e. normal, infant, old person or ill person.
• Sampling plan is basically a statement of criteria of acceptance based upon examination of required number of sample units by specific method.
• Sampling plan are of two types:
• Two class plan:
  • It consists of three specification ‘n’, ‘c’, and ‘m’.
  • This plan is simplest and is used to ‘accept’ or ‘reject’ food product.
  • E.g. Two class of certain food for coliforms is:
  • N=5, c=2 and m=10²
  • It means 5 sample must be tested.
  • In order for that to be acceptable not more than two samples must contain greater than 100 coliforms per grams.
  • The food will be rejected, if 8 or more sample units exceed 100 organism per grams.
  • e.g. n=5, c=0, m=0 (Salmonella). It means food will be rejected if any one sample out of five contained organisms.
  •  Where, n= number of sample unit to be tested.
  • m= the maximum number of organisms per gram. Value above this is non-acceptable.
  • c= the maximum no. of allowable samples that may extend the microbiological criteria ‘m’.
  • when this number is exceeded, the lot is rejected.
• Three class plan:
  • It consists of four specifications i.e. n, c, m, and M.
  • M- the quantity is used to separate marginally accepted quality from unacceptable quality.
  • Number or value above M in any sample are unacceptable.
  • e.g. n=5, c=2, m=100, M=150 e.g. food A. coliform
  • It means 5 samples must be tested. If not more than two sample.
  • 2 sample exceed equal or more than can organism, it is acceptable if any one sample.
  • Sample contain equal or greater than M, than sample is non-acceptable.

I. Sample procedure for dairy product:

• Milk and milk product:
• No. of samples:
  • Fluid milk= 5 field samples
  • Dried milk and ice cream = 10 field samples
  • Cheese = 5 field samples
  • Butter = 5 field sample
• Methods of analysis:
  • Enumeration of mesophilic aerobic bacteria
  • Enumeration of coliforms
  • Enumeration of Staphylococcus aureus (cheese)
  • Detection of Salmonella
  • For butter, enumeration of yeast and mold as well as coliforms.

i. Microbial analysis of Milk:

• In general market milk is sampled in the container in which it is sold.
• Sterile plunger is used for sampling and 10ml sample is taken. The collecting sample is put in sterile screw capped tube and immediately send to lab.
• Before performing test, the sample is mixed by inverting up and down for more than 10 times.
• For milk bottle, the top is flamed, cap removed with flame forceps.
• For plastic packets, the top and corner of packet is swabbed with spirit.
• After allowing to air dry, the swabbed area is cut with sterile scissors.
• The collected milk is tested by methylene blue test resazurin  test, MPN test.
• Three routine tests prescribed for milk are:
  • Plate count at 32^oC: serial dilution is made and pour plating is done from tubes of  10^-2– 10^-8 dilution.
  • For raw milk, DMC is usually done.
  • Coliform test: It is done by MPN
• Mesophilic aerobic bacteria in Milk:
  • Raw milk should not exceed total plate count = 2x 10⁵/ml
  • Pasteurized milk should not exceed total plate count = 2x 10⁴/ml
  • At 37^oC, coliform = 0 (best)
  • At 42^oC, fecal coliform = 0 (best)
• For dried milk,
  • Mesophilic aerophilic bacteria = n=5, c=2, m= 5X 10⁴, M = 5×10⁵
  • Coliform= n=5, c=2, m=2, M=102
  • Staphylococcus aureus = n=5, c=1, m=10, M=102
  • Salmonella = n=10, c=0, m=0

ii. Microbial analysis of Cheese:

• Cone sample is taken with the help of borer.
• The collected sample is made into finer pieces with a sterile grater. A grater is a device with a rough surface for grating food.
• The holes remaining after borings are sealed with paraffin wax.
• 10gm of grated sample is mixed with 90ml of warm diluent and homogenized in a blender.
• Serial dilution is performed.
• Plate count is performed on yeast extract milk agar. Similarly, coliforms can also be counted.
• For direct microscopic examination, thin film of slices of cheese are made. They are defatted with xylol. Xylol is washed and smear is stained and examined.
• For cheese,
  • Staphylococcus aureus, n=5, c=1, m= 10³, M=10⁴.

iii. Microbial analysis of Ice cream:

• If the ice cream is in packet of small size, it should be taken to lab in frozen condition (dry ice can be used).
• Melting ice cream should not be taken from the field.
• The wrapper is removed aseptically and content are put in sterile screw called tubes.
• The collected sample is allowed to melt in the lab below 20^o C.
• 10ml sample is weighed in a screw capped jar and serial dilution is performed in 0.1% peptone water.
• Plate count is performed from each dilution.
  • Mesophilic aerobic bacteria, n=5, c=2, m=2.5X10⁵.
  • Coliforms: n=5, c=2, m= 10², M=10³
  • S. aureus: n=5, c=1, m=10, M=10²
  • Salmonella: n=10, c=0, m=0

iv. Microbial analysis of Butter:

• The sampling of butter is done by using sterile knife.
• 10g of butter is emulsified in 90ml of warm water (40-45^oC) diluent and plate count is performed.
• Cultures are made on agar free nutrient medium.

v. Microbial analysis of Yoghurt:

• Yoghurt is sampled from the container in which it is fermented using spoon or spatula.
• Double dilution of yoghurt is prepared to 0.1% peptone water.
• 5ml of dilution is mixed with 5ml of melted LS differential medium.
• It is poured in plates, cooled, to solidify and incubated around 43^oC for 48hrs.

II. Sampling procedure for meat and meat products:

• No. of field samples=5
• Method of analysis:
• Enumeration of mesophilic aerobic bacteria
• Detection of Salmonella

i. Microbial analysis of Carcass sampling (raw meat):

• Sterile large swab is taken and they are moistened in 0.1% peptone water.
• The carcass is wiped with the swab.
• The carcass is again cleaned with dry pad.
• The pads are taken and 250ml of diluent is added in the pad to disperse the microorganisms from the trap.
• Serial dilution is performed.
• Plate count is made ta 20^oC.
• For detection of Salmonella, plating is done in selective medium.

ii. Microbial analysis of Chopped meat (raw meat):

• It may be fresh, minced or chopped meat.
• Several random samples are taken so as to detect universally distributed microorganisms.
• The sample is mixed with diluent and mixed well.
• Sample is serially diluted.
• Plate count is performed from dilutions 10^-4 to 10^-8.

iii. Microbial analysis of Poultry:

• Swab with lengthy rod is taken and it is sterilized.
• For each chicken, 2 swabs are taken. One for swabbing outside the body and other for swabbing body cavity of chicken.
• The swabs are rinsed in 100ml peptone water.
• Sample is serially diluted.
• Plate count is performed for total count and coliform count using selective media.
• To test for Salmonella, the dilution should be enriched in selective or tetrathionate broth and sample is further processed.

iv. Microbial analysis of Fresh fish:

• The whole surface of fish is either swabbed or washed with sterile 0.1% peptone water +1% NaCl in sterile plastic bag.
• Appropriate dilutions are prepared and total count is performed in marine agar which is incubated at 20^oC for 5 days.
• Cultures can also be done in glucose tryptone media, macconkey agar is or media containing 5-10% NaCl for halophilic bacteria.

v. Microbial analysis of Eggs:

• Outer surface of egg shell is swabbed with sterile swab. Sterile swab must be moistened in peptone water prior to swabbing.
• Then appropriate dilution is made from the swab.
• Inner portion of egg can be taken as sample and diluted.
• No. of field samples:
  • Salmonella = 10 field samples
  • Mesophilic bacteria = 5 field samples
  • Coliforms = 5 field samples
• Methods of analysis:
  • Enumeration of mesophilic aerobic bacteria (TPC)
  • Enumeration of coliforms
  • Detection of salmonella
  • For TPC= n=5, c=2, m=5x 10⁴, M=10⁶
  • For coliforms = n=5, c=2, m=10, M=10³
  • For Salmonella = n=10, c=0, n=0

Food sampling and preparation for microbial analysis

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• Food spoilage refers to the damage or destruction of food so as to make it undesirable for human consumption.
• Such spoilage may be due to:
  • Physical damage caused by cutting, bursting, freezing etc.
  • Mechanical damage caused by insect, bird, animal etc.
  • Activity of food enzyme itself
  • Growth of spoilage micro-organisms in food.
  • Improper environmental condition during harvesting transport, storage and marketing.
• The spoiled food may transfer the spoilage organism to the fresh food and result in spread of spoilage.
• Spoilage increase the amount of wastages.

Spoilage of eggs:

1. Contamination of eggs:

• Freshly laid egg is sterile but the egg shell soon becomes contaminated by fecal matter of hen by nest, by washing water, by handling and by other material in which it is stored.
• If a total number of micro-organisms per shell of hen’s egg has been reported to range from 10²-10⁷ with average of 10⁵.
• Salmonella spp. may be found on shell or inside egg.

2. Non-microbial spoilage of eggs:

• These include loss of moisture and hence loss of weight during long term storage.
• Change in physical state of egg contents also occur during long term storage.
• They include thinning of egg white and breaking of yolk membrane.
• As the yolk membrane weaken and break, yolk becomes flat and homogenously mixed in egg white.

3. Microbial spoilage of eggs:

• In order to cause spoilage of shell of egg, microorganisms must contaminate the shell, penetrate through the pores in shell and inner membrane, reach the eggwhite and yolk and grow there.
• Some microorganisms cannot grow in egg white but can grow rapidly in egg yolk.
• Change in storage temperature facilitates penetration of organism through shell and hence facilitates microbial spoilage.

1. Bacterial spoilage of egg:

• Bacteria are more common spoilage organism than mold.
• Bacteria cause rots in egg.
• When bacteria grow within the egg, they decompose the content and form byproduct.
• This result in characteristic odor, appearance or color from which various microorganisms acquire their name:
• Green rot:
  • It is caused by Pseudomonas fluorescence.
  • Green egg white shows fluorescence when exposed to UV light.
  • In later stage of spoilage, egg yolk disintegrates and mask green color of egg white.
  • Odor is lacking or fruity or sweetish.
• Colorless rot:
  • It may be caused by Pseudomonas, Acetobacter, Acinatobacter and coliform.
  • In later stage of spoilage, egg yolk disintegrates or at least have incrustations.
• Black rot:
  • It is caused by Proteus and sometimes Pseudomonas and aeromonas.
  • Egg yolk blackens and then breakdown to give whole egg content muddy brown color.
  • Odor is putrified due to H₂S.
• Pink rot:
  • It is caused by Pseudomonas usually at the later stage of green rot.
  • They are similar to colorless rot except that pink coloration occurs in yolk and white.
• Red rot:
  • It is caused by Serrotia marcesceus.
  • These eggs are distinguished by a rod dissociation of egg white and the surface of the yolk in ammonical i.e. putrified odor.
• Custard rot:
  • In this rot, yolk is incrusted with custard like material and occasionally have green to olive pigment.
  • The albumin become thin with orange coloration.
  • This type of spoilage is caused by Citrobacter and Proteus vulgaris.

2. Fungal spoilage of egg:

• Fungal spoilage goes through following stages:
• Pin spot molding:
  • In this case, small compact colonies of mold appear on the shell and usually just inside the shell.
  • The color of pin spots varies with the type of mold. For example: Cladosporium give black spot and Sporotrichum give pink spot.
• Superficial fungal spoilage:
  • This occurs if eggs are stored in atmosphere of high humidity.
  •  In this case, molds grow on shell in the form of whiskers.
• Fungal rotting:
  • It is the final stage of spoilage by mold.
  • In this case, mycelium of the mold grows through the pores and cracks in the shell.
  • Jellying  of egg white may occur and colored spots may be produced.
  • Hypha of mold grows through the yolk membrane and rupture it, so that yolk mixes with the white.
• Molds causing spoilage of egg include Penicillium, Sporotrichum, Mucor, Botrytis, Alternaria, Thamnidium etc.

Preservation of eggs from spoilage:

• Eggs have several ways of protecting itself from microbial spoilage.
• Shell and underlying membrane serve as first line of defense to prevent entry of microorganisms.
• In addition to physical barrier, egg albumin is not suitable growth medium and discourage growth of many microorganisms.
• Characteristics of egg albumin that discourage microbial growth include pH of 9-10, low level of simple nitrogenous compound, apoprotein that binds riboflavin, avidin that binds biotin, ovotransferrin that chelate iron, and lysozyme that hydrolyze peptidoglycan of bacteria.
• Despite physical barrier and other anti-microbial factors, many microorganisms can invade and cause spoilage of egg.
• Therefore, following methods are employed for its preservation:

i. Asepsis:

• Great care should be taken to reduce contamination by fecal matter by dust and nest.
• When eggs are broken for freezing or drying, spoiled egg should be discarded and contamination from equipment can be reduced by sanitizing it.

ii. Removal of microorganism:

• Various methods can be employed to remove dirt and faecal matter from egg shell.
• Dry cleaning by sand blasting removes dirt and bloom.
• Washing with warm water removes dirt, bloom and apart of microorganisms but encourage penetration of bacteria into egg through pores in shell.
• Use of disinfectant in washing water reduce number of microorganisms.

iii. Use of heat:

• Heat treatment suggested include heating whole egg in oil for 10 minutes at 60⁰C or in water at 54.4^oC for 80 min, Immersion of egg in boiling water for few second, immersion of egg in hot detergent, Sanitizer solution at 43.3^oC to 54.4^oC.
• Pasteurization is required for most egg product.
• Because of heat coagulability of egg, stabilization is required before pasteurization.
• This includes addition of aluminum salt and adjustment of pH.

iv. Preservation by low temperature:

• Chilling:
  • Eggs are commercially stored for six month or longer, at a temperature of -1.7^oC to – 0.55^oC and relative humidity of 70-80%.
  • If temperature is greater than -1.67, there is more rapid penetration of microorganisms and growth into egg and more physical and chemical changes.
  • Special treatment like impregnation of egg shell with colorless and odorless mineral oil keeps out moisture, slows dessication and air penetration during chilling.
• Freezing:
  • Eggs are first washed with 200-500ppm chloride solution and broken.
  • Egg yolk and egg white are separated and they are frozen separately at -17.8^oC to -20.5^oC.

v. Preservation by drying:

• Eggs are first washed with chloride solution, broken and then yolk and white are separated.
• Liquid egg is then dried by drum drying or spray drying method.
• Glucose should be removed from egg before drying because it causes browning of egg.

vi. Preservation by chemical preservative:

• Preservatives may be used on shells of egg in the atmosphere around them and on containers for egg.
• Some of them are:
  • Waxing and oiling of shell keep the shell dry, reduce penetration of oxygen into the egg and reduce passage of carbon dioxide and moisture out.
  • Immersion of whole egg in solution of sodium silicate is also a good preservative.
  • Use of CO₂ in ozone in storage atmosphere improves quality of egg.

Microbial spoilage of eggs and methods of preservation

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2. E. coli gastroenteritis:

• E. coli is generally regarded as normal flora of gastrointestinal tract of animal but several strain of E. coli cause diarrheal disease which are usually foodborne.
• Several outbreaks of E. coli gastroenteritis have been reported so far including recently occurring gastroenteritis outbreak in some European countries.

Etiological agent:

• Natural habitat of E. coli is gastrointestinal tract of animal and it is excreted in stool.
• Therefore, presence of sufficient number of E. coli in food indicates stool contamination E. coli strain causing gastroenteritis are divided into 5 groups:
  • Enteropathogenic E. coli (EPEC)
  • Enterotoxigenic E. coli (ETEC)
  • Enterohaemorrhagic E. coli (EIEC)
  • Facultative enteropathogenic E. coli (FEPEC)

Food involved in E. coli gastroenteritis::

• Outbreak of E. coli gastroenteritis usually associated with cheese and vegetable product but other food like creamed fish are also found associated.

Pathogenesis and symptoms of E. coli gastroenteritis::

• E. coli gastroenteritis is caused by ingestion of about (10⁶-10¹⁰) viable bacterial cells.
• After ingestion, they attach to epithelium of intestine by pili or fimbriae.
• Gastroenteritis caused by E. coli is characterized by non bloody diarrhea.
• Diarrhea is watery and is similar to that caused by Vibrio cholera.
• Pathogenesis and detailed symptoms differ with type of strain as given below:
• EPEC strain attach to the surface of intestinal epithelium by colonization factor antigen (CFA) type of pili and produce two types of toxin i.e. heat labile toxin and heat stable toxin.
• Heat labile toxin enter into intestinal cell and increase the level of CAMP.
• Similarly, heat stable toxin enter into intestinal cell and increase the level of CGMP.
• Increased level of CAMP and CGMP inhibit absorption of Na⁺ ion by epithelial from the intestinal lumen and at the same time it causes secretion of more water and Cl^– ion from the epithelial cell into intestinal lumen.
• Therefore, ETEC strain also causes watery diarrhea. ETEC strain is associated with travellers diarrhea.
• ETEC strain is represented by E. coli O₁₅₇H₇.
• They produce two types of toxin i.e. shiga like toxin 1^st (SLT-I) or (verotoxin or verocytotoxin) and shiga like toxin 2^nd (SLT-II).
• SLT -I and SLT-II are cytotoxin and kill the cell by inhibiting protein synthesis.
• EHEC causes hemorrhagic colitis which is a serious type of diarrhea.
• It also causes hemolytic uremic syndrome (HUS) which is characterized by renal failure.
• EIEC strain causes diarrhea by invading through the intestinal epithelium, the mechanism is similar to the Shigelosis.

Treatment of E. coli gastroenteritis::

• Treatment of diarrhea caused by E. coli is giving salt solution to counteract loss of water and electrolyte during diarrhea.

Prevention and control E. coli gastroenteritis:

• Main source of E. coli is stool of human beings and other animals.
• Therefore, sanitary practice is important to prevent contamination of food by stool.

3. Gastroenteritis caused by Vibrio spp.

• The genus vibrio consist of at least 28 species and four species are often associated with food borne gastroenteritis.
• They include:
  • Vibrio parahaemolyticus
  • Vibrio vulnificus
  • Vibrio alginolyticus
  • Vibrio cholerae
• Of them, Vibrio parahaemolyticus is most commonly associated with food borne gastroenteritis.

Vibrio parahaemolyticus gastroenteritis:

• Vibrio parahaemolyticus is halophilic bacteria and causes acute gastroenteritis after ingestion of sea food like raw fish and shell fish.
• It is the most common type of food borne gastroenteritis caused by vibrio species.
• Most other bacterial food poisoning are associated with wide variety of food, but this food poisoning is associated only with sea food.
• If any other foods are found associated it indicates direct mixing of sea food with that food.
• Etiological agent:
  • Vibrio parahaemolyticus is halophilic and grow in the range of 1-8% NaCl, but maximum growth occurs at 2-4% NaCl.
  • Range of temperature for growth is 4-44^oC with optimum pH being 7.6-8.6.
  • It is heat sensitive.
  • Most strain produce thermostable direct hemolysis (TDH) and some produce heat labile hemolysis and other produce both.
  • Some strain also produces thermostable related hemolysis (TRH), which is an important virulence factor.
  • This bacteria attach to intestinal epithelium by hemagglutinin. Pilli also helps in attachment.
• Food involved :
  • Natural habitat of this bacteria is sea water.
  • Therefore, food poisoning by this bacteria is mainly associated with sea food like fish, shell fish etc.
  • In rare cases, other foods are also found associated.
  • If this occurs, it indicates direct-mixing of sea food with other food.
• Disease symptoms:
  • Incubation period ranges from 2-48hrs.
  • Abdominal pain and diarrhea are common symptoms.
  • During diarrhea, watery stool with blood and mucus appear.
  • Nausea, vomiting, mild fever and headache also occurs.
  • The disease is self-limiting and recovery occurs within 2-5 days.
• Treatment:
  • The disease is self-limiting and requires no specific treatment.
  • In extreme cases salt solution is given to counteract loss of water and electrolyte during diarrhea.
• Prevention and control:
  • Natural habitat of this bacteria is sea water.
  • Therefore, sea water should not be used for processing of food, that prevent contamination.
  • Microorganisms present in contaminated food can be prevented from growing by sufficiently refrigerating during storage.
  • Finally, bacteria present in food can be killed by heating the food just before eating.

Vibrio cholerae gastroenteritis:

• Vibrio cholerae causes epidemic cholera which is a serious type of gastroenteritis.
• Although cholera is usually waterborne disease, some outbreaks are also found associated with ingestion of contaminated food.
• It is associated with ingestion of sea food.
• Usually Vibrio cholerae strain O₁ cause food borne gastroenteritis but several outbreaks are also found associated with non O₁ strain.
• Food involved:
  • Cholera is usually waterborne, sometimes is caused by ingestion of contaminated sea food.
• Etiological agent:
  • Usually Vibrio cholerae strain O₁ causes food borne cholera but some outbreaks are found to be associated with non O₁ strain.
• Pathogenesis:
  • It is a non-invasive type of infection and microorganisms do not penetrate through intestinal epithelium.
  • After ingestion, Vibrio cholerae multiply in intestine and produce enterotoxin (cholera toxin).
  • When vehicle is water, person with normal activity becomes infected only by ingestion 10¹⁰ or more bacterial cell.
  • But if vehicle is food, just 10²-10⁴ bacterial cells are sufficient to cause cholera.
  • It is because buffering capacity of food protect the ingested bacteria from stomach acidity.
  • After production, cholera toxin enters into the intestinal epithelial cell and increases the level of CAMP in cell.
  • Increased level of CAMP inhibits absorption of Na⁺ and Cl^– ion from intestinal lumen by intestinal epithelial cell.
  • At the same time, it causes hypersecretion of water and ions from intestinal epithelial cell into intestinal lumen.
  • This causes loss of water and electrolyte and produces diarrhea.
• Symptoms:
  • Incubation period is usually 1-4 days depending on amount of organism ingested.
  • Nausea, vomiting, diarrhea and abdominal pain occur very suddenly.
  • Stool resemble rice water in appearance which contains mucus epithelial cells and large number of Vibrio cholerae.
• Treatment:
  • Give salt solution to counteract loss of water and electrolyte during diarrhea.Give tetracycline orally, that inhibits Vibrio spp. in intestine.

other Vibrio species

1. Vibrio vulnificus:
   • This organism is found in sea water and sea food.
   • It is highly invasive and causes septicemia. It also produces hemolysin and cytotoxin.
2. Vibrio alginolyticus:
   • Its natural habitat is sea water. In human being, it causes soft tissue and ear infection.

Food borne bacterial gastroenteritis caused by Salmonella, E. coli and Vibrio spp

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• Clostridium perfringens causes food borne gastroenteritis which is due to enterotoxin production.
• Clostridium perfringeus multiply and produce enterotoxin in food.
• After ingestion, it further multiplies in intestinal tract and produce further enterotoxin.
• Therefore, gastroenteritis caused by Clostridium perfringens is food infection rather than intoxication.

Etiological agent:

• Clostridium perfringens is gram +ve, non-motile, anaerobic, spore forming rod shaped bacteria.
• However, Clostridium perfringens occasionally survive in presence of O₂ , minimum, optimum and maximum temperature for growth are 15^oC, 43-47^oC and 55^oC respectively.
• pH range is 5-9. It is inhibited by 5% NaCl (minimum A_w 0.97).
• They produce 5 types of antigenically different enterotoxins A, B, C, D, and E.
• Clostridium perfringens are also classified into 5 groups on the basis of type of enterotoxin they produce.
• Main food poisoning strain is type A. However, food poisoning by type C strain are also reported in some countries.
• Enterotoxin of Clostridium perfringens is a structural component of spore. Therefore, toxin is produced during spore formation.
• Clostridium perfringens is found in soil, water, dust and intestinal tract of human beings.
• Therefore, it is excreted in stool of healthy individual, from where it contaminates the foods.

Food involved:

• Meat and meat products are mainly associated with food poisoning by Clostridium perfringens.
• It is because it is a normal flora of intestinal tract from where it directly enters into meat.
• In case of other food, it occurs by contamination of bacterial spore from stool, soil, dust, etc.
• Insufficient heating or warming of food stimulates spores for germination.
• Therefore, food which are insufficiently heated and then stored unfrozen are highly susceptible to spoilage.

Pathogenesis of Clostridium perfringens gastroenteritis:

• When contaminated food is ingested, pre-formed enterotoxin as well as viable bacterial cell enter into intestinal tract.
• In intestine Clostridium perfringens multiply and produce further enterotoxin during sporulation.
• Then enterotoxin bind to the cell of intestinal epithelium and enters into intestinal epithelial cell.
• This enterotoxin causes secretion of water and electrolyte from intestinal cell into intestinal lumen and causes diarrhea.
• This enterotoxin is also cytotoxic and kills intestinal epithelial cells.

Disease symptoms of Clostridium perfringens gastroenteritis:

• Incubation period is 8-24hrs.
• Abdominal pain and diarrhea are two typical symptoms.
• In contrast to staphylococcal food poisoning, vomiting is rare.
• The disease is of short duration and usually remains for a day or less.
• Mortality rate is very low and requires high dose of bacteria to be fatal.

Treatment of Clostridium perfringens gastroenteritis:

• Treatment involves giving self-solution to counteract loss of water and electrolyte during diarrhea.

Prevention and control of Clostridium perfringens gastroenteritis:

• Food poisoning by Clostridium perfringens can be prevented by:
• Preventing contamination:
  • For this purpose, sanitary practice is important to prevent contamination of spore of bacteria into food.
• Preventing germination of spore and growth in food:
  • Various methods can be used to prevent germination of spore and multiplication of bacteria in food.
  • One simple method is to freeze food adequately during storage.
• Killing organism before eating food:
  • By heating vegetative cells are easily kill but spores are resistant.
  • γ radiation can be used to kill spore in food just before eating.

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• Botulism is a neuro paralytic intoxication caused by ingestion of preformed enterotoxin in food produced by Clostridium botulinum.
• Etiological agent for Botulism:
  • Clostridium botulinum is gram +ve, anaerobic, rod shaped, spore forming bacteria.
  • They produce 8-types of neurotoxins (A, B, C₁, C₂, D, E, F and G) in food.
  • Therefore, Clostridium botulinum strains are classified into 8-group on the basis of type of neurotoxin they produce.
  • Type A, B, E and F cause botulism in human being and type C and D cause botulism in other animals.
  • It usually produces gas in food. It may or may not be proteolytic.
  • Proteolytic strain produces various bad smelling compounds like H₂S in food.
  • But, in case of non-proteolytic strain, spoilage cannot be judged by smell of food.
  • Similarly, bad odor is not developed in low protein or non-protein food.
  • Various factors affect spore germination growth and toxin production by C. botulinum.
  • Moisture below 30% inhibit toxin production. Similarly, salt concentration of 8% or more prevent neurotoxin production in food.
  • Similarly, neurotoxin is not produced if pH of food is 4.5 or below.
  • Composition of food determine type of toxin produced. For.eg. food containing milk or casein give more toxic type of type A toxin.

Properties of botulinum toxin (Botox):

• Toxin is produced in food during growth and autolysis of bacteria.
• Proteolytic strain produce toxin in active form but some non-proteolytic strain produce toxin in inactive form.
• This inactive toxin become active in stomach by acid hydrolysis.
• Unlike enterotoxin of S. aureus, this neurotoxin is damaged by heating at 80^oC for 10 minutes.
• Botulinum toxin is most toxic chemical found in nature.

Pathogenesis of botulism:

• Botulism occur by ingestion of pre-formed neurotoxin on food.
• After ingestion it is absorbed by cell of upper intestinal tract, then enters into lymph then into blood.
• From blood, it reaches neuromuscular junction and binds to nerve ending. After binding it inhibit release of acetylcholine neurotransmitter that gives paralysis of muscle.
• If this paralysis extends to breathing muscle, death occurs by respiratory failure.

Infant botulism:

• In adult botulism is intoxication i.e. it is caused by ingestion of preformed neurotoxin of Clostridium botulinum in food.
• In adult, ingested Clostridium botulinum cannot multiply in intestinal tract.
• It is because Clostridium botulinum is highly acid sensitive and it is killed by stomach acidity.
• Furthermore, this bacteria is very less competitive and cannot compete with normal flora of intestinal tract.
• So, in case of adult botulism occurs only by ingestion of pre-formed neurotoxin, but not by ingestion of bacterial cell but in infant stomach is not acidic and intestinal normal flora are not fully developed.
• Therefore, ingested Clostridium botulinum multiply and produce endotoxin in intestinal tract of infant.
• So, botulism in infant is an example of food infection.

Symptoms of Botulism:

• Incubation period is 12-36hrs, but it may be shorter or longer.
• Earliest symptom includes acute digestive disturbance followed by vomiting, sometime diarrhea, fatigue and headache.
• After above symptoms typical neurological symptoms appear. They include double vision, difficulty in speaking and swallowing, mouth becomes dry and tongue swells.
• Paralysis of involuntary muscle occurs. Paralysis of respiratory muscle results in death.

Treatment of Botulism:

• Treatment of botulism can be separated into three processes:
• First treatment is removal of unabsorbed neurotoxin from intestinal tract. For.eg. by inducing vomiting.
• Second treatment involves neutralization of neurotoxin by ingestion of antibody (anti-toxin). It is the only one successful treatment of botulism.
• Third treatment involves curing of symptoms. For.eg. by artificial breathing.

Prevention and control of botulism:

• Preventing contamination of food:
  • For this purpose, food should be kept away from soil and dust because its natural habitat is soil.
• Preventing growth and toxin production in food:
  • It can be done by making the food aerobic by adding chemicals, heat treatment etc.
• Damaging neurotoxin in food:
  • It can be done by heating the food at 80^oC for 10 minutes just before eating.

Botulism: Food poisoning caused by Clostridium botulinum

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• Food borne disease are divided into two main types i.e. poisoning and infection.
• Food poisoning:
  • If disease is caused by infection of toxic chemical present in food, it is called food poisoning.
  • Food poisoning is further divided into two types i.e. chemical poisoning and food intoxication.
  • If poisoning is caused by injection of poison which is accidently or purposely added in food, it is called chemical poisoning. E.g. disease caused by injection of rat poison in food.
  • If poisoning is caused by injection of toxin secreted by living being, it is called food intoxication.
  • Sometimes tissue of food is itself toxic and causes intoxication. E.g. mushroom poisoning.
  • Some microorganisms contaminate food and produce toxin in the food.
  • If intoxication is caused by injection of microbial toxin in food, it is called microbial food intoxication.
• Food infection:
  • If disease is caused by ingestion of viable microorganisms in food, it is called food infection.
  • In food infection microorganisms multiplies in intestinal tracts and cause disease.
  • There are two types of food infection i.e. invasive type and enterotoxigenic type.
  • In enterotoxigenic type of food infection, microorganisms produce enterotoxin in intestinal tract that cause disease. E.g. food infection caused by EPEC, vibrio cholera.
  • In invasive type of food infection microorganisms cause disease by invading through intestinal tract. E.g. food infection caused by Shigella, EIEC.
  • In some food borne disease, food simply serves as carrier of microorganisms and microorganisms do not multiply in food. For e.g. in case of vibrio cholera, Corynebacterium diphtheria, Mycobacterium bovis.
  • In other food borne diseases food serves as cultivation medium for growth of microorganisms and microorganisms multiply in food. E.g. EPEC, vibrio parahaemolyticus.

Difference between food infection and food intoxication:

Food poisoning caused by Staphylococcus aureus (Staphylococcal food intoxication):

• Disease caused by Staphylococcus aureus is intoxication caused by ingestion of pre-formed enterotoxin in food.
• It is one of the most commonly occurring food borne disease.

Etiological agent:

• Staphylococcus aureus is gram positive cocci that occur in grape like cluster.
• Intoxication is caused by enterotoxin producing strain of S. aureus.
• Most enterotoxin producer are coagulase +ve, however, not all coagulase +ve Staphylococcus aureus produce enterotoxin.
• It can tolerate high concentration of salt 10-20% of NaCl and sugar (50-60%).
• It is nitrate tolerant and can grow in curing solution of meat.
• They are proteolytic but usually do not produce bad smell in food.
• Therefore, food spoilage caused by S. aureus is difficult to judge by its smell and appearance.
• Temperature range for growth and toxin production is (14-46)^oC and minimum value of A_w for growth is 0.86.
• Maximum pH for growth in food is 8.
• Natural habitat of S. aureus is skin and upper respiratory tract of human being and other animal.
• From these sources, it contaminates in food.

Enterotoxin:

• Staphylococcus aureus produces six antigenic type of enterotoxins.
• They include A, B, C₁, C₂, D and E, of which, type A and D are more frequently associated with food intoxication.
• Condition that favor multiplication of S. aureus also favor toxin production.
• Toxin is produced at an appreciable rate at (15.6-46.1)^oC and production is best at 40^oC.
• Under best condition, sufficient amount of toxin appears in food within 4-6 hrs.
• At lower temperature, it takes very long time to produce enough enterotoxin in food.
• Therefore, poisoning by S. aureus can be prevented by adequate refrigeration during storage of food.
• An important characteristic of enterotoxin is that it is heat stable.
• Therefore, this enterotoxin is not easily damage by normal cooking of food.
• So, food poisoning by S. aureus, occur when food is not sufficiently heated just before eating.

Food involved:

• Creamed filled bakery product are more commonly associated with S. aureus food poisoning.
• However, many other food products like meat and meat products, milk and milk products, vegetable, egg containing products etc. are also found associated.

Pathogenesis of staphylococcal food intoxication

• Staphylococcal food poisoning is caused by ingestion of pre-formed toxin in food.
• Enterotoxin acts on intestine to induce vomiting and diarrhoea.
• Production of diarrhoea by enterotoxin is not clear.
• Enterotoxin binds to the wall of small-intestine, stomach and large intestine with high affinity and it causes inflammation and irritation of lining of stomach and intestine.
• Induction of vomiting by enterotoxin occurs due to stimulation of central nervous system.
• Enterotoxin binds to receptor of vomiting in intestine, which stimulates vomiting center in brain via vagus and sympathetic nerves.

Disease symptoms of staphylococcal food intoxication :

• Symptom of the disease vary with amount of enterotoxin ingested and the immunity of the host.
• Staphylococcal food poisoning is characterized by very short incubation period of 2 – 4 hrs.
• Most common symptoms include vomiting, diarrhea, retching (sound in stomach), abdominal cramp, nausea.
• In severe case blood and mucus may be seen in stool and vomits.
• Mortality rate is extremely low and the disease is usually self-limiting.

Treatment of staphylococcal food intoxication:

• Treatment of diarrhea include giving salt solution to counteract loss of water and electrolyte during diarrhea.

Prevention and control of staphylococcal food intoxication:

• Staphylococcal intoxication can be prevented by:
• Preventing contamination of food:
  • Good personal hygiene of food handler is very important to prevent contamination because S. aureus enters into food from skin and upper respiratory tract of human being.
  • Person who is carrier of S. aureus should not be allowed to handle food.
• Preventing growth and toxin production:
  • Growth of S. aureus in food and toxin production can be prevented by adequate refrigeration of food, by lowering A_w value of food below 0.86, by adjusting pH at very low level etc.
  • Enterotoxin of S. aureus is heat stable. Therefore, it can be damaged by sufficient heating the food before eating.

Food borne disease: food poisoning and food infection with example

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