Module 2. Classification of dairy microorganisms

Lesson 5

CHARACTERISTICS OF IMPORTANT MICROORGANISMS – I

5.1 Introduction

Microorganisms play a significant role in manufacturing fermented milk and milk products. These groups of useful microbes are called as lactic acid bacteria. The pleasant flavour of some of the dairy products like butter and fermented milks, and the desired texture of many cheeses are mainly due to the activities of certain microbes during production and ripening. In this lesson, we shall be discussing the characteristics of some of the dairy microbes.

5.2 Streptococcus

Streptococcus is spherical, Gram-positive belonging to phylum Firmicutes and the lactic acid bacterial group. Cellular division occurs along a single axis in streptococcus and hence, these grow either in chains or pairs. The name from Greek streptos, means easily bent or twisted, like a chain (twisted chain) is given to these microbes. In contrast to this, staphylococci, divide along multiple axes and generate grape-like clusters of cells. Most streptococci are oxidase and catalase negative, and many are facultative anaerobes. In 1984, many micro-organisms formerly considered Streptococcus were separated out into two different genera Enterococcus and Lactococcus. Sherman divided streptococci into four groups based on the presence of different cell wall antigens, physiological properties and growth temperature requirements (Table 5.1). This genus comprised 27 species and the four groups are:

· Pyogenic streptococci (ABCEFGH antigen)

· Viridans streptococci (no group specific antigen)

· Faecal streptococci or Enterococci (D antigen)

· Lactic streptococci (N antigen)

Table 5.1 Sherman’s classification of group of streptococcus

Characteristics	Pyogenic	Viridans	Enterococcus	Lactococcus
Group antigen	A, B, C, D, E, F, G, H	Non specific	D	N
Growth at 10°C	-	-	+	+
Growth at 45°C	-	+	+	-
NaCl (6.5%)	-	-	+	-
pH 9.6	-	-	+	-
Methylene blue (0.1%)	-	-	+	+
NH₃ from Arginine	+	-	+	+/-
Litmus reduction before clotting	-	-	-	+
Resistance to 60°C/ 30 min	-	+/-	+	-
Haemolysis	Β	α, γ	α, β, γ	γ
Representative species	S. pyogenes (A) S. agalactiae (B) S. disgalactiae (C)	S. bovis (D) S. uberis S. thermophilus	S. durans, E. faecalis	L. lactis ssp lactis L. lactis ssp cremoris

5.2.1 Pyogenic group

Streptococcus pyogenes also known as Group A Streptococcus is the causative agent in Group A streptococcal infections, including streptococcal pharyngitis, acute rheumatic fever and scarlet fever. Streptococcus pyogenes is one of the other major causes of streptococcal infection in humans, after pneumococcus. However, rather than being directly invasive and inflammatory, it seems to cause local infection, but then its other actions are via toxins: whether affecting kidneys, heart valves in rheumatic fever or scarlet skin of scarlet fever. Group A Streptococcus infection is generally diagnosed with a Rapid Strep Test or by culture. Rheumatic fever, a disease that affects the joints, kidneys and heart valves, is a consequence of untreated Streptococcus A infection caused not by the bacterium itself. Rheumatic fever is caused by the antibodies created by the immune system to fight off the infection cross-reacting with other proteins in the body. S. pyogenes causes acute mastitis in ruminants.

Streptococcus agalactiae or Group B Streptococcus causes pneumonia and meningitis in neonates and elderly, with occasional systemic bacteraemia. These can also colonize the intestines and the female reproductive tract, increasing the risk for premature rupture of membranes and transmission to the infant. Women who test positive should be given prophylactic antibiotics during labour that will usually prevent its transmission to the infant. Is similar to S. pyogenes and causes mastitis in animals and is a natural inhabitant of udder of ruminants. Streptococcus dysgalactiae is also a member of Group C, β-haemolytic streptococci that can cause pharyngitis and other pyogenic infections. Similar to Group A streptococci, it also causes bovine mastitis.

5.2.2 Viridans group

5.2.2.1 Viridans

Streptococcus is a pseudo-taxonomic non-Linnaean term for a large group of commensals that are either α-haemolytic, producing a green colouration on blood agar plates (‘viridans’, from Latin, means green), or nonhemolytic. They possess no Lancefield antigens and in general, pathogenicity is low.

5.2.2.2 Streptococcus uberis

At dairy farms, mastitis due to environmental Streptococcus, especially S. uberis, is a major cause of concern. It is known that many of these infections develop into clinical cases that may persist up to months. There is also a tendency for these infections to develop during the dry period and the early part of lactation. Typically, it is classed as an environmental pathogen and focus has to be on preventing environment to cow transfer.

There are instances, however where S. uberis infections appear to behave somewhat as a contagious pathogen and may transfer from animal to animal. In typing strains of the micro-organisms it can be found that several animals may be infected with the same strain. This raises a question, as to whether the same strain went from the environment into several animals or did it transfer from animal to animal.

There is no doubt that the micro-organism are found in bedding, manure and general environment of the animal and so environment to animal transfer is a major factor in disease transmission. Under certain conditions, it appears that animal to animal transfer may also be responsible for within herd spread.

Streptococcus thermophilus is a Gram-positive bacteria and homofermentative facultative anaerobe of the viridans group. It tests negative for cytochrome, oxidase and catalase, and positive for alpha-haemolytic activity. It is non-motile and does not form endospores.

It is also classified as lactic acid bacteria (LAB). S. thermophilus is found in fermented milk products. It is not a probiotic (it does not survive the stomach in healthy humans) and is generally used in the production of yogurt, alongside Lactobacillus bulgaricus. The two species are synergistic, and S. thermophilus probably provides L. bulgaricus with folic acid and formic acid which it uses for purine synthesis.

5.3 Lactococcus

Lactococcus is a genus of lactic acid bacteria that were formerly included in the genus Streptococcus Group N1. These are known as homofermenters meaning that they produce a single product, lactic acid in this case, as the major or only product of glucose fermentation. Their homofermentative character can be altered by adjusting cultural conditions like pH, glucose concentration, and nutrient limitation. They are gram-positive, catalase-negative, non-motile cocci that are found singly, in pairs, or in chains.

Five species of Lactococcus are currently recognized along with three subspecies. They are:

· L. lactis (L. lactis subsp. Lactis, L. lactis subsp cremoris and L. lactis subsp. lactis biovar diacetylactis)

· L. garvieae

· L. plantarum

· L. raffinolactis

· L. piscium

These micro-organisms are commonly used in dairy industry in producing fermented dairy products. These can be used in single-strain, or in mixed-strain starter cultures with other lactic cultures i.e. Lactobacillus and Streptococcus. Special interest is taken in studying L. lactis subsp. lactis and L. lactis subsp. cremoris, as these are the strains used as starters in industrial fermentations. Their main purpose is rapid acidification of milk; this causes a drop in the pH of the fermented product that prevents the growth of spoilage causing microbes. The bacteria also play a role in flavour production in final product. Lactococci are currently being used in the biotech based industries, as these are easily grown on cheaper whey-based media. As food-grade bacteria, these are used in the production of foreign proteins applied in the food industry.

Lactococcus lactis ssp lactis is classified informally as lactic acid bacteria, because it ferments lactose to lactic acid. These are typically spherical or ovoid cells, about 1.2µm by 1.5µm, occurring in pairs and short chains. These are Gram-positive, non motile, and do not form spores. Lactococci are found associated with plant material, mainly grasses, from which these are easily inoculated into milk. Hence, these are found normally in milk and may cause natural souring. L. lactis has two subspecies such as, lactis and cremoris, both of which are essential in producing varieties of cheese and other fermented milks.

L. lactis ssp lactis is related to other lactic acid bacteria such as Lactobacillus acidophilus in our intestinal tract and Streptococcus salivarius in mouth. However, Lactococcus normally does not colonize human tissues and differs from many other lactic acid bacteria in its pH, salt, and temperature tolerances limits for growth, that are important characteristics relevant to its use as a starter culture.

Lactococcus lactis ssp lactis is vital for manufacturing cheeses, as well as other dairy products like cultured butter, buttermilk, sour cream and Kefir. It may also be used for vegetable fermentations (i.e. cucumber pickles and sauerkraut). This can be used in single strain, or in mixed strain cultures with other lactic acid bacteria such as Lactobacillus and Streptococcus species.

When L. lactis ssp lactis is added to milk, it uses enzymes to produce energy molecules, called ATP, from lactose. The by product of ATP production is lactic acid. The lactic acid curdles the milk that then separates to form curds that are used to produce cheese and whey. But curdling the milk is not its only role in cheese production. The lactic acid produced by it lowers the pH of the product and preserves it, by preventing the growth of unwanted microflora while other metabolic products and enzymes produced by L. lactis ssp lactis contribute to the more subtle aromas and flavours that distinguish different cheeses.

Lactococcus lactis ssp lactis biovar diacetylactis is quite similar to L. lactis ssp lactis. It utilizes citrate and produces carbon dioxide, diacetyl, volatile fatty acids and acetoin. The citrate utilization is a plasmid linked trait and is quite unstable. The acetic acid produced is inhibitory to a number of microbes - Pseudomonas, coliforms and Salmonella.

Lactococcus lactis ssp cremoris

The importance of lactococci, specifically L. lactis subsp. cremoris, is demonstrated by its continual use in food fermentations. L. lactis subsp. cremoris strains are preferred over L. lactis subsp. lactis strains because of their superior contribution to product flavor via unique metabolic mechanisms.

5.4 Bifidobacterium

Bifidobacterium is a genus of Gram-positive, non-motile and often branched anaerobe. These are ubiquitous, endosymbiotic inhabitants of the gastrointestinal tract, vagina and mouth (B. dentium). Bifidobacteria are one of the major genera that make up the colon flora.

Prior to the 1960s, Bifidobacterium species were collectively referred as ‘Lactobacillus bifidus’. Some Bifidobacterial strains are considered as probiotics, as these may exert a range of beneficial health effects, including regulation of intestinal microbial balance, inhibition of pathogens and harmful bacteria, modulation of immune responses, repression of procarcinogenic enzymatic activities, production of vitamins, and bioconversion of dietary compounds into bioactive molecules. Bifidobacterium spp are known to discourage the growth of Gram-negative pathogens in infants. Bifidobacterium possesses a unique fructose-6-phosphate phosphoketolase pathway employed to ferment carbohydrates.

Species: B. adolescentis, B. animalis, B. bifidum, B. breve, B. infantis, B. longum, B. ruminantium