Module 3. Microorganisms associated with milk

Lesson 14

EFFECT OF PROCESSING ON MICROORGANISMS IN MILK

14.1 Introduction

In Indian subcontinent milk production is regularly increasing during the last few decades and acquires the position of nutritious food. Different plans have been introduced in past to improve the production and quality of milk from farm to consumers. However, in India dairy sector is still dominated by the rural people, especially farmers and at farm the advanced facilities to protect the milk from spoilage are scanty but upcoming. Because of this, a major portion of total milk produced is spoiled by different factors during milking, transportation, handling and storage of milk. The final quality of milk or its products is directly related to the hygienic quality of raw milk produced. Therefore, milking production practices plays a major role in determining the hygienic quality of raw milk or milk products as discussed earlier.

Table 14.1 Hygienic parameters [pH and microbial counts (cfu/ml)] of freshly drawn milk

Parameter(s)	Minimum	Maximum
pH	6.33	7.0
Aerobic plate count	1.3x10^4	1.5x10^9
Total coliforms	<30	20.8x10^6
Fecal coliforms	<30	2035
Staphylococci	<30	10820

14.2  Effect of Cooling on Milk

Cooling of milk is a process of bringing the temperature of the milk below ambient temperature using a cooling medium. The purpose of the cooling is to reduce the spoilage of milk by preventing or retarding the growth of different group of microorganisms. Milk contains various microbes derived from different sources. The most important spoilage and pathogenic micro-organisms in milk are mesophiles with a growth temperature ranging between 20°C to 40°C. Cooling reduces the metabolic activity of micro-organisms by reducing the activity of the enzymes necessary for metabolism, thereby reducing the growth and reproduction of the bacteria, especially mesophiles.

Table 14.2 The effect of storage temperature of milk on the growth of microbes

Temperature of storage (°C)	Growth rate (µ) after 18 hr of storage
0	1.00
5	1.05
10	1.80
15	10.00
20	200.00

From the above data it is evident that the critical temperature is 10°C as beyond this the growth increased further. Hence in practice, milk is cooled to below 5°C to increase the shelf life of raw milk.

14.3 Bactofugation

Centrifugal force provides an efficient means of improving the bacteriological quality of milk. The principle is bacterial super centrifugation but when it is carried out under pasteurization, it is called ‘bactofugation’. However, there is a tendency to use the term irrespective of processing temperature. Bactofugation removes bacteria, both living and dead, from treated substances, whereas traditional heat treatment kills bacteria and leaves them in food. Bactofugation is important in food-stuffs infected with bacteria, especially those containing thermo-stable endotoxins.

The purposes of bactofugation are:

· To improve the hygienic quality

· To avoid heat resistant bacteria without resorting to excessive heating

· Where exceptionally high degree of bacteriological purity is sought

The process of bactofugation is based on Stoke’s laws

V = [r (d₁-d₂) g]/9ɳ

Where:

V = Velocity or solid or semi solid particle; d₁ = density of particle to be separated; d₂ = density of fluid; r = radius of the particle; g = centrifugal force; ɳ = viscosity of fluid

Since, bacterial dimension is nearly 1.2μm and density is 1.070 - 1.120, it requires high centrifugal force. Based on size and density certain species are removed more easily than others. Therefore, 90-95% of bacteria can be removed by the application of 8000-12000 g of gravitational field for 8 sec at 50˚C.

With two bactofuges in a series, 99% of bacteria can be removed at 72°C. This treatment has two fold effects. In this condition a cell count reduction of 99% and colony count reduction of 99.9% is obtained. Bactofugation is not a substitute for pasteurization or sterilization but used in conjunction with them to improve their efficiency.

14.3.1  Advantages of bactofugation

· In cheese:

§ Prevents swelling in certain cheeses by butyric acid bacteria which are heat resistant

§ Removal of bacteria without pasteurization which enables raw milk cheese production with more typical cheese flavor.

· In milk Powder: To reduce the count of microbes and significant removal of heat resistant bacteria.

· Sterilized milk: The severity of heat treatment can be reduced.

· Cream: Bittiness caused by heat resistant Bacillus cereus is avoided.

14.4 Thermization

Thermization is the least heat treatment followed in dairy industry. Heat treatment used is in the range of 63-65°C/15-20 s and cooling to below 6°C, optimum being 66-70°C/15 s.

Purpose of Thermization is:

· To destroy enzyme ‘lipase’ because milk produced on farm may not be taken daily to dairy

· To extend storage of raw milk during refrigeration

· To maintain daily production of products, when supply is reduced

It is designed to lower the number of psychrotrophs of raw milk to take care of lipolysis and proteolysis.

14.4.1  Factors affecting thermization process

· Raw milk - Initial load should be less than 5,00,000 cfu/ml. As load increases, the corresponding count after thermization in 3 days storage at 6°C also increases because of elimination of only of psychrotrophs

·  Heat treatment above 65°C is effective, but increasing the temperature at constant holding time promoted of shelf life than increasing holding time at constant temperature. As temperature increases more number of flora of enterobacteriaceae are destroyed

· Some bacteria may increase in number e.g. S. thermophilus (at 68°C/10 s)

14.5 Destruction of Microbes by Heat

Microbes are destroyed by heat, when microbial proteins are coagulated and enzymes required for their metabolism are inactivated. The death of microbes is also due the thermal denaturation of the secondary and tertiary structure of macromolecular cellular organizations i.e. DNA, proteins and membranes.

Decline of viable count in a given interval is proportional to the initial concentration of living cells. The effect of heat depends on the intensity of temperature and duration of exposure. The effect varies with the type of micro-organism, state of organism and environment during heating. Following term involving are used to denote heat destruction:

· Decimal reduction time (D value)

· Z-value

· Q₁₀ value

· F₀ value

D-Value is defined as the time in minutes taken to destroy 90% of the viable micro-organisms at specific temperature. D-value is the index of the time-temperature needed to reduce microbial numbers in a system by one log cycle e.g. one million microorganisms – reduced to 100 by heating the milk for the time equivalent to four D values.

Z-Value designates the slope of a thermal death curve. It is the number of degree of temperature required for a specific death curve to reduce the counts by 1 log cycle. In other words, it is the temperature difference that results in ten folds change in D value. Z value for spore formers is 18 and for non-spore formers is 10-14.

Q₁₀ Value

It is defined as a factor by which reaction rate is increased by increasing the temperature by 10°C.

Q₁₀ Value = Rate of reaction (T + 10°C) divided by rate of reaction at T°C

Q₁₀ value for chemical reactions is 2 to 4; for microbial inactivation it is 10 to 30 and for spore destruction it is 8 to 12.

10 Z= log Q₁₀

F₀-Value is a total integrated lethal effect and it is used to measure microbial severity of a thermal process. It is expressed as minutes at a specified reference temperature of 121°C when Z equaled 18. F₀ value indicates F value when Z equaled 18 e.g. F₀ value is 10 minutes at 121°C which is equivalent to 1 minute at 131°C.

Clostridium botulinum cook

The minimum heat treatment recommended for low acid products (>4.5 pH) is 121°C for 3 min or equivalent. This results in 12 D reductions for Clostridium botulinum. It is selected because, it is most heat resistant pathogen and hence the minimum heat treatment recommended to achieve 12 D reductions is known as minimum Clostridium botulinum cook.

14.6  Pasteurization of Milk

Louis Pasteur (1860-64), a French scientist, heated wine to 50-55°C to increase the keeping quality and this method known as pasteurization has been frequently used in industry with some modification. As per the definition, pasteurization is: LTLT= 63°C/30 min or HTST= 72°C/15 s and cooled to 5°C or below.

14.6.1  Objectives of pasteurization

• Destroy all the pathogenic organisms present in milk.

• Reduce the load of non-pathogenic organisms and increase shelf life.

The most important heat resistant pathogen is Mycobacterium tuberculosis. Thus, the minimum heat treatment was established to guarantee the inactivation of M. tuberculosis. The pasteurization deals effectively with almost all pathogens satisfactorily except for Bacillus cereus.

Milk contains alkaline phosphatase enzyme that is inactivated at a temperature-time combination similar to that of pasteurization. Alkaline phosphatase can be measured by a simple chemical test known as phosphatase test. Some of the conditions of pasteurization are placed below:

Table 14.3 Time-temperature combinations

Microbes	Duration of exposure
	30 min (°C)	15 s (°C)
M. tuberculosis	58.9	70
Phosphatase	61.1	71.1
Pasteurization	61.7	71.7
Cream line reduced	62.2	72.2

14.6.2  Effect of pasteurization on Raw Milk Microflora

Raw milk contains a mixed microflora arising from several sources. Holding temperature prior to pasteurization is quite important. Above 10°C, all the micro-organisms in raw milk multiply actively. This includes heat resistant non-spore formers and spore formers. If this number of heat resistant micro-organisms increases the total count of milk after Pasteurization will also increase.

<10°C – Growth of contaminating strains rapidly falls.

<5°C – Gram negative rods grow significantly like Pseudomonas that are capable of producing heat resistant enzymes. B. cereus may grow and produce toxins.

Theoretically it is not possible to destroy all the microbes in milk by heat treatment but the purpose of Pasteurization is to reduce probability of pathogens surviving the process to such a level that the public health risk from drinking such milk is negligible.

Table 14.4 Thermal resistance of some microorganisms

Percent survivors	Heat treatment
	63°C/30 min	80°C/10 min
Bacillus	54	61
Coryneforms	46	37
Gram-positive	0	2
Gram-negative	0	0

14.6.3  Effect of pasteurization on thermodurics in milk

A. Source of thermodurics from milk:

· Gram-positive organisms: Only Alcaligenes tolerans survives pasteurization

· Gram-positive cocci

a) Micrococci – large number in raw milk but outgrown by others at 7°C. These are unable to grow due to lactenin, a natural inhibitor in milk. These also occur on dairy equipments.

b) S. thermophilus, E. faecalis and S. uberis: A number of strains of these are thermoduric but grow slowly at refrigeration temperature between pasteurization and consumption. Milk containing high thermodurics also contains appreciable number of haemolytic streptococci.

c) Anaerobic spore formers – unable to grow because of high redox potential of milk but sometimes isolated from milk.

d) Aerobic spore formers – survive pasteurization

Mesophilic – B. licheniformis most important followed by B. pumilus, B. subtilis

Thermophilic – B. stearothermophilus

Psychrotrophic – B. coagulans, B. circulans, B. mycoides

e) Coryneform groups – Survive and form substantial portion of microflora of pasteurized milk but do not grow at low temperature.

B. Inadequately cleaned utensils.

C. Accumulated in pasteurization plant due to improper cleaning methods.

D. Re-pasteurization of returned milk: because of growth of thermodurics on storage after first pasteurization.

E. Use of pasteurized skim milk or cream for standardization.

F. Summer months due to poor cooling.

14.6.4 Effect of pasteurization on psychrotrophs in milk

• Pseudomonas, Flavobacterium, Acaligenes, Achromobacter are destroyed by pasteurization.

• Part of normal flora of milk and grow fast as temperature increases up to 25°C.

• Pasteurization destroys all psychrotrophs to the extent that survivors do not deteriorate flavor deterioration even over extended storage. But post pasteurization contamination is a major problem.

• Psychrotrophs can cause the problems in pasteurized milk like unclean flavor, putrid, fruity, rancid, sour, ropy, greenish yellow discolorations.

14.6.5  Effect of pasteurization on thermophilic organisms in market milk

These are non-pathogenic and usually associated with high acidity and off-flavors. Facultative thermophiles grow at 37°C and at 55°C however obligate thermophiles do not grow at 37°C but grow at 55°C and even up to 70°C. There are some factors that affect the thermophile count:

i) Raw milk – contains few thermophiles but these may increase on storage at higher temperature and gain access via soil beddings, feeds etc.

ii) Pasteurizer – inside surface and holder tubes, pre heaters, filter cloths etc. HTST – no problem of thermophiles because of too short residence time of milk (total 70-80 s), but filter cloths used are problematic.

iii) Re-pasteurization and returned milk:

a) addition of returned milk

b) dripping from bottle fillers to raw milk contain very high thermophiles

c) skim milk or cream from returned milk for standardization.

iv) milk foam left in equipment is an excellent source

v) dead ends where hot milk is allowed to stand for any appreciable time.

14.6.6  Effect of pasteurization on coliforms

Coliforms are undesirable in pasteurized milk. Placed below there are some factors that effects the counts:

a) Raw milk – improper sanitation of production i.e. fecal contamination through water, exterior of animal and improperly cleaned utensils.

b) Pasteurizing plants:

i. Improperly cleaned pipelines, pumps, fillers, bottles etc.

ii. Condensate drippings at various places

iii. Personnel unsanitary practices

iv. Defective or worn-out equipment having pits and pockets that favor accumulation of milk solids.

v. Defects - gassiness, ropiness, unclean flavor, medicinal, bitter flavor

14.6.7 Effect of pasteurization on pathogens

Pathogen presence is due to improper pasteurization and, post processing contamination both. No recorded cases of pathogens, if pasteurization is done as per IDF standards. Incidents of Staphylococcus, Salmonella, Campylobacter, and Yersinia are reported but B. cereus was not reported though it can survive pasteurization.

The possible sources of post-processing contamination:

(a) Human carriers

(b) Thermophiles in final regeneration are shed into pasteurized milk

(c) Holes in plate heat exchanger of regeneration section allow raw milk mixes with pasteurized milk that can be avoided using high pressure on processed milk

14.6.8 Effect of pasteurization on keeping quality

Keeping quality of pasteurized milk is 5-7 days at refrigeration temperature. Spoilage depends on type and number of micro-organisms and also on temperature of storage. Unclean and bitter flavors are due to Gram-negative rods and bitter off-flavors are due to B. cereus at 10⁶ cfu/ml. Acid production, coagulation, protein destabilization are brought by acid producers like Lactococcus lactis ssp lactis.

During Pasteurization, Psychrotrophs are destroyed but enzymes produced by these are ‘thermostable’. Proteases, lipases and phospholipase have less time to react at low temperature storage because of keeping quality of 5-7 days.

14.7 UHT Processing of Milk

The purpose of UHT treatment of milk is to produce sterilized milk that is meant to:

· Keep without deterioration i.e. remain stable and of good commercial value for a sufficient period

· Be free of microorganisms and toxins harmful to the health of consumers

· Be free of microorganisms liable to proliferate during storage

IDF recommended a temperature 135-150°C/1-4s for UHT treatment of milk.

14.7.1  Microbes in milk and their response to UHT

Class I: Microbes killed by conventional pasteurization temperature of 71-72°C for 15-30 s. This eliminates most vegetative cells of bacteria like S. aureus, haemotylic streptococci, Gram-negative enterococci (E. coli, Salmonella sp.), Pseudomonas, B. abortus, M. tuberculosis, all yeasts and moulds.

Class II: Resistant to HTST, but sensitive to UHT i.e. 135-150°C/1-4s HTST is tolerated by some thermoduric vegetative cocci like enterococci, some micrococci, microbacteria, thermophilic bacilli (L. bulgaricus, Lactobacillus lactis), S. thermophilus, thermoduric aerobic and anaerobic spores.

Class III: Obligate thermophilic soil bacterium B. stearothermophilus are known to withstand UHT treatment of milk. Some spores of mesophilic bacilli and clostridia may survive, if milk is heavily contaminated.

Table 14.5 Heat resistance of spore formers (in sterilized milks)

Microorganisms	Decimal reduction time (s) at 121°C
Clostridium botulinum	3
B. cereus	2-4
B. coagulase	18
B. subtilis	3-20
B. stearothermophilus	200-500

B. stearothermophilus is most resistant and constitute greatest hazard in spoilage of sterilized milk product.

14.7.2  Spoilage of UHT milk

It is caused due to

(a) Enzymes those are heat resistant

(b) Post processing contamination either from packaging operation or improper   cleaning of system.

Spoilage is characterized by bitter off flavor, gelation and coagulation of milk proteins. Psychrotrophs and aerobic spore formers do produce proteases and lipases at a storage temperature of as low as 4°C. Some of these enzymes are most heat resistant. Enough enzymes are produced at 5°C by as few as 10³-10⁴ pseudomonas/ml to cause significant loss of native milk proteins and bitter flavor. Most of UHT spoilage can be traced to packaging failures. Aseptic filling of UHT milk is of utmost importance. Since contamination with one viable bacterium is able to reproduce in milk will inevitably spoil the product during storage within few days. Contamination risk with sterility of packaging material and contamination is mainly by class I and II micro-organisms, is mainly due to

· During change of paper rolls

Worn out gaskets in sterile portions of equipment that harbor microbes

· Condensed water at the filling pipes

· Faulty sealing of packages.

If contamination by insufficient sterility of packaging material is due to faulty sealing or corroded plates of plate heat exchanger, then the contamination micro-flora is most variable and varies between package to package. If contamination is due to gaskets or condensed water, then all contaminated packages contain uniform microflora.

Microbes that will enter the product more probably are those found in water stagnant on dairy floors – Pseudomonas, Micrococcus, Enterococcus Bacillus and some yeasts. But these do modify milk visibly (i.e. coagulation, proteolysis, staining and flocculation) or produce off flavors or a measurable property (i.e. pH, acidity, redox potential) but UHT milk cannot attain cell concentration of >10⁵/ml so these must be detected by direct counts on culture medium.

14.7.3  Quality control of UHT milk

Satisfactory process shows spoilage level not higher than one per 1000 containers.

Packs test, 50 to 100 units’ daily/ line, if the line gives 4000 units/ hour. This amounts to 0.1 – 0.2% of the whole production.

· Pre incubation of packs for sufficient period is done. The temperature should be between 25-30°C/7-9 days and incubated at 55°C for thermophilic spores. The appearance, pH, taste and blowing of packages are recorded.

· The defective packages are tested by streaking on agar plate and incubated at 30°C/2 days or 55°C/4days. Streaking is done to know the nature of micro-organism by going morphological, oxidase and catalase tests.

· Interpretation should be:

Thermophilic spores – insufficient heating of the product

Mesophilic spores - survived heat treatment or recontamination.

14.7.4  Methods to improve the quality of UHT milk

· Use raw milk of low SPC and psychrotrophs

· Milk collected from hygienic milk shed areas

· Immediately after collection, milk is subjected to pasteurization (or) thermization

· Test sample of milk in sealed ampoule in oil bath at 130 – 140°C

· Better use recombined or reconstituted milk with very low spore count

· LP system preservation of farm milk

· Bactofugation with 1000 l/h flow rate

· Nisin – active against spore formers (aerobic/anaerobic) but that is not approved by PFA.

14.8  Sterilization of Milk

Sterilization means exposing the milk to 118 - 120°C for 15 to 20 min. Sterilized milk should contain neither bacteria nor bacterial spores.

14.8.1  Bacterial spores of raw milk

Raw milk contains a considerable number of bacterial spores although this number may be low in comparison with total bacterial count.

Bacterial spores gain entry into milk mainly by contaminate with:

- Dust particles

- Soil and

- Manures

Thus, the density of spores in the milk is higher, when the animal is milked in stable than in open air.

More than half of spores in raw milk belong to the species of Bacillus licheniformis. Other spores of considerable importance are B. pumillus, B. subtilis. But lesser number of spores of B. cereus, B. circulance, B. megatherium, B. stearothermophiulus (thermophilic) and Clostridium spp. are also encountered. Among the mesophilic spores present in raw milk those of B. subtilis are most thermoresistant.

14.8.2 Survival of bacteria in sterilized milks

Because of deleterious effect of sterilization upon certain physic-chemical properties of milk, there is often tendency to use the minimum amount of heat treatment, with the result occasionally bacteria are developed in sterilized product. When B. subtilis is present, it gives extremely ‘bitter taste’ and ‘proteolysis’ occurs. B. circulans presence in sterilized milk produces ‘carbolic taint’. When sterilized milk is incubated at higher temperature, it is possible that more species may be found. The presence of B. coagulans and of facultative thermophilic bacilli can be demonstrated by tests at 37°C. Test at 55°C can demonstrate the ‘presence of bacteria of B. coagulans and groups of obligate and facultative thermophilic bacilli. Presence of B. cereus and B. subtilis indicates the lower intensity of heat treatment. The spores of B. cereus are less thermo-resistant than those of B. subtitis. Hence the presence of B. cereus in greater in number than B. subtitis indicates insufficient degree of heat treatment.

The presence of B. circulans that produces carbolic taint is often associated with improperly washed bottles. These bacteria grow even at rather low temperature quickly and abundantly form spores in milk from returned bottles contaminate the bottle washing machine. When the temperature and alkalinity of detergent solutions are not sufficiently efficient, large number of B. circulans spores may survive the treatment and infect clean bottles. The in-bottle-treatment fails to kill the spores; hence, more infected bottles are returned and contaminate the bottle washing machine. Thus, a vicious circle is set up which is fatal to the product unless measures are taken to improve the standards for bottle sterilization.

The presence of B. coagulans and thermophilic bacilli is usually associated with the filler. For reasons of economy and quality milk is mostly bottled at higher temperature. These bacteria develop in filler, forms spores and contaminate milk during bottling, often surviving later in bottle treatment. Development of B. coagulans group can be easily controlled by using filling temperature and their growth is retarded at 60°C and prevented at 65°C. The development of thermophilic bacilli can be controlled only thorough frequent cleaning and disinfection of filler. At a maximum bottling temperature of 80°C, thermophiles develop much lesser than at frequently used bottling temperature of 65-70°C.

Anaerobic spore formers are practically never present unless milk has been strongly under sterilized, as anaerobic spore forms are less resistant then aerobic spores.