Module 4. Microbiological methods of milk testing

Lesson 20

METHODS OF ENUMERATION OF OTHER GROUPS OF BACTERIA

20.1 Introduction

Milk is a very good growth medium for different groups of microorganisms because of its high water content, near neutral pH, and variety of available nutrients. The spoilage of milk and milk products is due to the presence of proteolylic and lipolytic bacteria in milk. These microorganisms mostly produce extracellular enzymes that attack fat and proteins and thus, producing abnormal flavours/ odours, as well as bitter taste in dairy products. However, in case of heat treated milks, the major spoilage microorganisms are spore formers.

20.2 Proteolytic Counts

Most proteolytic bacteria belong to psychrotrophs that may grow at 7°C although their optimum temperature is bit higher. Rapid cooling and cold storage of raw milk favour the growth of psychrotrophs in milk. These become dominant microflora during cold storage of milk and their extracellular enzymes, particularly proteases contribute to the spoilage of milk. The rate of cooling to a required temperature and the length of storage are other important factors. Pseudomonas fluorescens and other psychrotrophs that may be present in raw milk generally produce protease during growth. Spoilage of pasteurized or raw milk by proteolytic bacteria can occur on prolonged storage at below 7°C. Different methods have been introduced for isolation and identification of proteolytic microorganisms in milk.

· Proteolytic count can be made oncasein agar plates on incubation for 6 days and the number of colonies counted after flooding the plates with dilute acid. However, the drawback of the method is that microorganisms cannot be picked up as pure culture.

· Milk agar plates are prepared by adding about 10 percent of sterile milk to nutrient agar. Microorganisms are usually considered proteolytic, if there is a clear zone around colony. The clear zone around colony may be produced by solvent action of dilute lactic or other acid. Method is unreliable in case of acid-forming bacteria. If sufficient acid is produced, there will be a zone of precipitation immediately around the colony, surrounded by a clear zone; but if little, or weak acid is formed, there may be a clear zone without any precipitate. It is therefore difficult to determine whether or not an acid-forming microorganism is weakly proteolytic.

· An improved medium for the detection of proteolytic microorganisms have been developed by adding caseinate (1% w/v), citrate (0·015 m) and Ca²⁺ (0·02 m) to agar. Its greater sensitivity compared with existing milk agar media is related to its ability to detect the first stage of casein breakdown as shown by the formation of a white zone of precipitation. The extent of proteolysis exhibited by amicroorganism is reflected both by the size and type of precipitation zone forming round colony. This medium can thus be used for simultaneous determination of proteolytic and total bacterial counts.

20.3 Lipolytic Counts

Lipolytic activities, supported by psychrotrophs are not significant, if count does not exceed 10⁶ CFU/ml. However, 2.7 × 10⁴ CFU/ml is a sufficient count of psychrotrophs microorganisms for the initiation of lipolysis. Unlike milk lipases, microbial lipases are thermo-resistant; remain active even after warming up. Extracellular enzymes of psychrotrophic microorganisms may resist pasteurization (72°C for 15 s) and Ultra high temperature (UHT) treatment of milk (138°C for 2 s or 149°C for 10 s).

Lipolysis results in the development of a high level offree fatty acids that cause a bitter taste of dairy products, making them hardly acceptable. Enzymes present in cooled raw milk are either endogenous or they come from growing psychrotrophs. Changes in the level of free fatty acids in milk can serve as an indirect indicator of lipolytic activity. The normal content of free fatty acids in milk fat is between 0.5 and 1.2 mmol/100 g. The permissible maximum content of free fatty acids is 13.0 mmol per kg for a churning method or 32.0 mmol/kg for an extraction-titration method. The free fatty acids content of 49 mmol/kg determined by the extraction-titration method as the limit value of a hazard of lipolytic changes in the taste characteristics of milk.

· Tributyrin agar can be used for the estimation of psychrotrophic lipolytic count by incubating at 6.5 °C ± 0.5 ° C for 10 days.

· Colonies appears with clear lytic zone.

· Lipolytic activity can also be tested in tributyrin agar by incubating at 37 °C for 3 days and screening the plates forclear zone of hydrolysis.

20.4 Spore Count

Spore formers constitute a group of undesirable bacteria that are heat resistant and are capable of withstanding pasteurization of milk i.e. 63°C for 30 min or 72 °C for 15 s. Some microbes can also withstand UHT heat treatment and cause problems in UHT milk. These microbes belong to Bacillus spp and Clostridium spp. Spoilage by spore-formers can occur in low-acid fluid milk products that are preserved by sub-sterilization heat treatments and packaged with little chance for re-contamination with vegetative cells. Products in this category include aseptically packaged milk, cream and sweetened and unsweetened concentrated canned milks. Under favourable conditions these microorganisms multiply rapidly and bring about different types of spoilages such as peptonisation and production of off flavours in milk.   Spore-forming bacteria that spoil milk products usually originate in the raw milk. Spore-forming bacteria from raw milk are predominantly Bacillus spp., with Bacillus licheniformis, Bacillus cereus Bacillus subtilis, and Bacillus megaterium most commonly isolated.

· Samples are transferred to water bath maintained at 80°C and milk is allowed to attain 80°C.

· After 0 min at 80°C, remove samples and allow cooling to room temperature slowly. During slow cooling, the spores will germinate and converts to a vegetative form.

· Prepare dilutions and transfer appropriate dilution to sterile Petri dish.

· Pour melted Standard plate count agar, mix contents and allow it to solidify.

· Incubate the plates for enumeration of mesophilic spore forming bacteria at 37°C for 48 h. and for thermophilic spore forming bacteria at 55°C for 48 h.

· Count the colonies after incubation period.

20.5 Thermophilic Count

The thermophilic microorganisms are capable of growing at 45°C and above. Majority of these microorganisms are spore formers such as Bacillus spp and Clostridium spp. These cause problems particularly in processing plants of milk. The standard plate count agar is used. The plates are incubated at 45°C or 55°C for 48 h and colonies are counted. Excessive number of thermophiles is problematic, if the milk is meant for heat processing.

20.6 Thermoduric Count

Thermodurics constitute a major group of undesirable bacteria that are heat resistant and they withstand the pasteurization temperature but not grow at 63°C for 30 min or 72°C for 15 s with an optimum growth temperature of 37°C. They are basically mesophilic microorganisms possessing a greater ability of withstanding the higher temperature. These belong to the genera Micrococcus, Microbacterium, Bacillus and Coryneform. These bacteria under favourable conditions multiply rapidly and bring about different types of spoilage like acid or rennet coagulation, peptonisation and production of off-flavours in milk.

20.6.1 Procedure

Place the milk samples in a water bath maintained at 63°C. As soon as the temperature of the milk reaches 63°C, note down the time and keep the samples at this temperature for a period of 30 min. Remove the samples and chill the milk by immersing in ice cold water to bring the temperature down to 5°C. Prepare dilutions and transfer 1 ml of appropriate dilution to sterile Petri dish. Pour melted standard plate count agar and allow it to solidify. Incubate the plates for enumeration of thermodurics at 37°C for 48 h. Count the colonies after incubation.

Table 20.1 Grading of milk based on thermoduric counts

Thermodurics (cfu/ml)	Grade of milk
<10,000	Good
10,000 to 30,000	Fair
>30,000	Poor

20.7 Psychrotrophic Count

Psychrotrophs are capable of growing at refrigeration temperature (7-10°C). There is often sufficient time between milk collection and consumption for psychrotrophs to grow. Pasteurized milk is expected to have a shelf life of 14 to 20 days at refrigerated temperature. The contamination of a container with even one rapidly growing psychrotrophs can lead to spoilage. Psychrotrophs in milk that spoil raw and pasteurized milk are primarily aerobic Gram-negative rods of family Pseudomonadaceae, with occasional representatives from Neisseriaceae and Flavobacterium spp. and Alcaligenes spp. Generally 65 to 70 per cent of psychrotrophs from raw milk are Pseudomonas. Their presence in milk will result in undesirable flavours during storage under refrigerated conditions due to the production of proteolysis and lipolysis.

The standard plate count agar is used after supplementation with 1 to 2.5 IU of penicillin to inhibit the growth of Gram positive bacteria, because the predominating psychrotrophs belong to Gram-negative. The plates for psychrotrophic count are incubated in a refrigerator (7-10°C) for 7-10 days or kept in an incubator at 15°C for 3 days and colonies are counted.

Suggested standards for psychrotrophs in raw milk

Less than 10⁴ cfu per ml - Satisfactory

Above 10⁴cfuper ml – Unsatisfactory