Module 5. Microbes in spoilage of milk

Lesson 24

ABNORMAL MILK FERMENTATIONS

24.1 Introduction

Milk and milk products though prepared using lactic acid bacteria can be spoiled by the growth of ‘wild’ strains that produce unwanted gas, off-flavours, or appearance defects. Non-spore-forming bacteria responsible for fermentative spoilage of dairy products are mostly in either the lactic acid-producing or coliforms. Genera of lactic acid bacteria involved in spoilage of milk and fermented products include Lactococcus, Lactobacillus, Leuconostoc, Enterococcus, Pediococcus, and Streptococcus. In addition to these micro-organisms, there are a number of non lacticgroups of micro-organisms that can produce enzymes, which can attack major milk constituents like fat, protein and carbohydrates causing abnormal textural defects or odour or colour defects.

24.2 Abnormal Fermentations

24.2.1 Gas production

The production of CO₂ by certain micro-organisms in dairy products is responsible for ‘gassiness’. Gassiness is associated with acid production causing‘frothiness’ due to the associative action of acid producing bacteria with yeasts. The production of gas in canned dairy products causes bulging of cans is called ‘blowing’ is highly undesirable. Gassy cream is rejected for butter making. However, in Swiss cheese a controlled production of gas gives the desirable texture by eye formation. In cheddar cheese, gas production due to coliforms is undesirable, as it is accompanied with off-flavors that may be carried to the final product. In canned dairy products, the production of gas may lead to early gas production or late blowing of cans. The early blowing condition is due to coliforms, whereas late blowing is due to Clostridium spp.

24.2.1.1 Causative organisms

· Coliforms: E. coli, E.aerogenes ferment lactose of milk or cream into gas and acid. These are called ‘early gas producer’that lead to blowing condition.

· Anaerobic spore forming bacteria e.g. Clostridium butyricum, Clostridium sporogenes produce only gas under anaerobic conditions, mostly prevailing in canned dairy products like processed cheese, concentrated milk. These are called‘late gas producers’ and produce late blowing condition.

· Lactose fermenting yeast produce CO₂ and small amounts of ethyl alcohol in milk, cream and whey at or below 37 °C(for example Candida pseudotropicalis).

24.2.1.2 Precaution and control

· Avoid contamination of causative micro-organisms

· Holding of milk and cream at ambient temperature should be avoided

· Adequate heat treatment of milk should be done

24.2.2 Ropiness/ Sliminess

Ropy fermentation is brought about by the growth of bacteria leading to change in consistency of the product that forms threads of viscous masses, when poured. Ropiness develops only on storage and milk is drawn out as fine threads and may appear gel like consistency. Sometimes the change is so prominent that the milk can be drawn into a long thread.

Ropiness has particularly been observed in milk held at low temperature as many causative microbes show active growth in milk at low temperature. At higher temperatures these ropy bacteria are over-grown by lactic acid bacteria. However, ropiness causing bacteria are not harmful to the consumers. Ropiness is developed before detectable acid development and it decreases as the acidity increases.

24.2.2.1 Causative organisms

I. Gram-negative rods: Alcaligenes viscosus

II. Coli-aerogenes group. Consists of ropy strains belonging to enterobacter, citrobacter and related genera:

a. Enterobacter aerogenes

b. Enterobacter  cloacae

c. Citrobacter freundii

d. Serratia marcescens

III. Aerobic spore formers have also been infrequently involved in ropiness.

a. Bacillus cereus

b. Bacillus subtilis

c. Bacillus circulans

IV. Lactic acid bacteria:

a. Lactococcus lactis ssp. lactis biovar hollandicus

b. Lactobacillus casei

c. Lactobacillus bulgaricus

24.2.2.2 Source

Bacteria causing ropiness are generally not present in the aseptically drawn milk, but come from water, dairy equipment, dust laden air, coat of cows, cow feed etc and enter the milk drawn.

24.2.2.3 Mechanism of ropiness

Materials that are capable of producing ropiness are:

· True gums or gum like substances: polysaccharides. Gums are derived from lactose that is fermented by Coliforms, Lactococcuslactis ssp. Lactis biovar hollandicus, Streptococci, Leuconostoc, Lactobacilli.

· Mains: Nitrogenous mucus like substances. Mucins are combination of proteins with a carbohydrate radical may be produced by peptonizing bacteria (Micrococci, Gram-negative bacteria and aerobic spore formers)

· Exopolysaccharides: produced as capsules associated with cell or as slimeunattached to the cell. Alcaligenes viscosus produces capsular material.

24.2.2.4 Significance of ropiness

Ropiness causing bacteria are not harmful to consumers. However, this defect affects the acceptability of products that occurs less frequently in pasteurized milk. Ropiness has particularly been observed in milk held at low temperature. At higher temperature,lactic acid bacteria dominate ropy bacteria. Ropiness is observed higher in summer, more prevalent in spring and autumn.

24.2.2.5 Prevention and control

a. Clean milk production and effective heat treatment

b. Post-pasteurization contamination should be avoided.

24.2.3 Sweet curdling

Curdling without pronounced acid production is sweet curdling. This defect is due to the production of an extracellular enzyme similar to rennin by bacteria that precipitate casein before the development of sufficient acidity. This defect is commonly observed in milk and cream particularly that are moderately heat treated particularly when held at higher temperatures during summer months. Rennin like is produced generally by aerobic spore formers.

The activity of this enzymeis known to increase at higher temperature. Little or no acid production is observed during sweet curdling. Under Indian conditions, it is observed that boiled milk, if allowed to cool and stand in same container for long time the product will undergo sweet curdling. This defect sometimes, becomes apparent on re-heating the product. This defect may also occur in pasteurized milk, if stored for longer times under refrigerated conditions. Under farm conditions milk contains microbes but the defect does not appear, as the commonly encountered lactic acid bacteria curdle the product by acid production.

24.2.3.1 Causative microorganisms

· Cocci - Streptococcus liquefaciens

· Aerobic sporeformers – Bacillus cereus, Bacillus subtilis

· Psychrophilic spore formers- Bacillus cereus, Bacillus licheniformis and certain Microbacterium spp.

· Non-spore forming rods: Proteus spp. and E. coli.

24.2.3.2 Significance

Sweet curdling is prevalent in heat treated products, particularly in summer. However, it is also observed inrefrigerated and boiled milk stored for longer durations.

24.2.3.3 Factors affecting sweet curdling

· High temperature : More prevalent in summer

· Age of milk: Milk held at ambient temperature for more than 24 h due toproduction of more rennin by Bacillus mycoides. Itdoes not growrapidly, as it may be inhibited by lactic acid bacteria.

· Pasteurization:It kills most of the bacteria, especially lactic acid bacteria, hence, no competition. The spore formers geminate and grow in these circumstances and cause sweet curdling.

24.2.3.4  Precaution and control

· Avoid contamination

· Control storage temperature

· Pasteurization at a temperature higher than normal

24.2.4  Proteolysis

It is a process in which casein or some insoluble casein derivatives are broken down to water soluble compounds through the action of micro-organisms or their enzymes. Milk contains different proteolytic enzymes that may bring about alterations of milk constituents. Some of these are naturally present in milk, while others are elaborated by micro organisms.Since, milk contains only small amounts of non-protein nitrogenous substances to sustain prolonged growth or survival, micro-organisms depend on enzymes that hydrolyze milk proteins. Changes in milk proteins, as a result of psychrotrophs growth or enzymatic action, are important in keeping quality of milk and milk products. Release of various nitrogenous compounds or degradation of individual protein fractions is observed while studying proteolysis caused by psychrotrophic enzymes. Raw milk supplies contain heat stable proteases or microbes that are able to produce these proteases can attack caseins and whey proteins leading to coagulation of milk and development of bitter taste.

Proteolytic flavours are attributed to the breakdown of proteins and amino acids and thus proteolysis is often accompanied by development of bitternessdue to the presence of peptides that are bitter in taste. Psychrotrophic proteinases primarily act during refrigerated storage of raw milk before heat treatment. Ultra high treated sterilized milk coagulated in the presence heat resistance pseudomonas proteases.

24.2.4.1  Causative microorganisms

Psychrotrophs are active proteolytic and grow at 7°C or less, especially Pseudomonas fluorescens, Pseudomonas fragi and Alteromonas putrefaciens. Thermodurics are especially Micrococcus caseolyticus, Bacillus stearothermophilus Bacillus cereus and Bacillus subtilis.

24.2.5  Lipolysis

It is hydrolysis of milk fat and subsequent production of off-flavours in dairy products. Lipolysis is carried out by lipase resulting into the accumulation of free fatty acids. The lower chain fatty acids particularly butyric and caproic are responsible for lipolytic off-flavours. The onset of lipolysis in milk at any stage from milking, storing and processing should be avoided, since rancid milk or cream will not yield products of high flavour quality.

The intrinsic milk lipase is present in sufficient quantities to cause hydrolysis of milk fat. However, fat globule membrane protects the milk triglycerides from attack by lipolytic enzymes, whose activity is further impaired by their association with casein and possibly by the presence of lipolytic inhibitors. The intrinsic milk lipase is well partitioned from the triglyceride substrate; hence little or no lipolysis occurs in milk.

Lipolytic microorganisms and their enzymes produced in bulk cooled milk/ cream by psychrotrophs that survives HTST pasteurization and UHT sterilization, causes lipolysis during subsequent storage of heat processed milk even at low temperature. However, in some instances lipolysis appears to have a favorable influence on the flavor of cheese. The hydrolysis of cheese constituents is essential for the development of mold ripened cheese. Not only the lipolytic activity of molds but also further metabolism of fatty acids especially their oxidation to methyl ketones. The addition of lipase of Candida lipolytica improve the quality of blue veined cheese made from raw or pasteurized milk and yeasts of Torulopsis had a desirable influence on the formation of methyl ketones.

24.2.5.1  Causes of lipolysis

1. Intrinsic milk lipase: Present in sufficient quantities to cause hydrolysis of milk fat. However, this fat globule membrane protects the milk triglycerides from attack by lipase and hence, little or no lipolysis occurs.

2. Lipolytic microbes or enzymes:

a) Psychrotrophs: Pseudomonas spp. mainly Pseudomonas fragi, Pseudomonas fluorescens and Achromobacter lipolyticum

b) Other types: Micrococcus freudenreichii, Bacillus subtilis and Bacillus coagulans

c) Yeast and moulds: Candida lipolytica, Geotricum candidum, Penicillium spp. and Aspergillus spp.

24.2.5.2  Precaution and control

· Clean milk production practices

· Cooling of milk as per recommendation

· Processing of milk prior to microbial growth reaching log phase.

24.2.6  Bitty cream defect

Bitty cream or broken cream is a well known example of the effect of spore formers on milk. This defect, which occurs in pasteurized and raw cream, is characterized by the appearance of flakes in the cream layer that do not mix even when milk is shaken. If such milk is used in tea, the flakes float on the surface making it unacceptable to consumers.

The flakes are of two types, one is of mechanical/ physical origin and there flakes occur when fat globule membrane is partly disrupted andglobule stick together, and when this condition is pronounced, it occurs as cream plug. The second type is of bacterial origin and is produced partly by lecithinase enzyme of Bacillus cereus, Bacillus cereus var mycoides that attacks phospholipid part of fat globule membrane and partly from the coagulation of casein associated with membrane. The bacterial flakes do not change materially, when heated to 100°C, whereas mechanical flakes change at 40–45°C

Bitty cream is amajor spoilage problem of pasteurized milk.The main reasons for this are failure of refrigeration, seasonal variation, prolonged storage etc.

24.2.6.1  Precautions and control

Clean milk production to avoid spore contamination is the best way.

24.2.7  Fermentation based discolouration

A number of different abnormal colours are developed depending on the growth of micro-organism as detailed below:

· Yellow: Pseudomonas synxantha

· Blue: Pseudomonas cyanogens

· Black: Pseudomonas nigrifaciens

· Red: Serratia marcescens

· Green: Pseudomonas fluorescens

24.2.8  Abnormal flavours

Most of the off-flavours are microbiologically originated. Microbial off-flavours are invariably encountered in raw milk through occasional neglect or failure in refrigeration. Improvement in processing operations has resulted in lower bacterial count and hence, most microbial spoilage and associated off-flavours can be traced to post-pasteurization contamination with psychrotrophs.

· Fruity Flavour: It is due to ethyl ester formation usually catalyzed by esterases produced by psychrotrophs or lactic acid bacteria. Ester formation by Pseudomonas fragi involves liberation of butyric and caproic acids from one and three positions of milk triglycerides and are esterified with ethanol. Predominating esters are Ethyl butyrate, Ethyl hexanoate.

· Malty flavour: caused by malty strains of Lactococcus lactis ssp. lactis var maltigenes

· Bitty flavour: caused by proteolytic microorganisms especially Bacillus spp. and Pseudomonas spp.

· Fishy flavour: caused by Pseudomonas icthyosmius, due to conversion of lecithin to trimethylamine.

· Potato flavour: caused by Pseudomonas mucidolens and Pseudomonas graveolens

· Phenolic flavour: caused by Bacillus circulans

· Musty flavour: caused by Actinomyces and certain yeast

· Burnt of caramel flavour: caused by Malty strains of Lactococcus lactis ssp. lactis

· Unclean flavour: E. coli