Module 2. Classification of dairy microorganisms

Lesson 6

CHARACACTERISTICS OF IMPORTANT MICROORGANISMS – II

6.1 Introduction

As already said in lesson 5, 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 flavor 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. Here also we present different groups of microbe associated with dairy.

6.2 Lactobacillus

Lactobacillus is Gram-positive facultative anaerobic or microaerophilic, rod-shaped bacteria. These are a major part of lactic acid bacteria, because most of its members convert lactose to lactic acid. In humans these are present in vagina and gastrointestinal tract asymbionts. These are usually benign, except in mouth, where these have been associated with dental caries. Many species are prominent in decaying plant material. The production of lactic acid makes its environment acidic that inhibits the growth of harmful bacteria. Overall it is a quite heterogeneous group of Gram-positive, asporogenous rods, homo/ heterofermentative, aerobic/ facultative anaerobe; nonmotile, microaerophilic, aciduric, catalase negative and requires a complex medium (i.e. MRS medium/ Rogosa acetate agar) for growth.

Orla-Jensen divided the genus of lactobacilli into three (i.e. Thermobacterium, Streptobacterium and Betabacterium) groups based on their optimum growth temperature and fermentation patterns (Fig. 6.1 and Table 6.1). There are 64 species reported in this genus.

Table 6.1 Orla-jensen division of genus Lactobacillus

Characteristics	Thermobacterium	Streptobacterium	Betabacterium
Growth at 15°C	-	+	-
Growth at 45°C	+	-	+/-
Voges-Proskauer	+	+	-
Fermentation types	Homofermentation	Homo/ heterofermentation	Heterofermentation
Representative species	L. bulgaricus L. acidophilus L. helveticus	L. casei L. plantarum	L. brevis L. fermentum
	Hexoses fermented to lactic acid  by EMP, but pentoses not fermented	Hexoses fermented to lactic acid   by EMP. Some species also produce acetic, formic and ethanol under glucose limitation. Pentoses fermented to lactic acid   and Acetic acid  involving phosphoketolase	Hexoses fermented to lactic acid   and acetic acid involving phosphoketolase pathway

· Lactobacillus bulgaricus is one of the cultures used for production of yogurt and that also found in other naturally fermented products. It utilizes lactose to produce lactic acid, which is used to preserve milk. It is Gram-positive rod, non-motile, and does not form spores. This bacterium is regarded as aciduric or acidophilic, since it requires a low pH (around 5.4-4.6) to grow effectively. The bacterium has complex nutritional requirements, including the inability to ferment any sugar except lactose. It is a thermophilic microbe whose optimum temperature is 40˚C.

Lactobacillus bulgaricus is commonly used with Streptococcus salivarius subsp thermophilus as a starter in making yogurt. The two species work in synergy, with Lactobacillus bulgaricus producing amino acids from milk proteins that are used by S. thermophilus. Both species produce lactic acid that gives yogurt its tart flavour and acts as a preservative. The resulting decrease in pH also partially coagulates the milk proteins, such as casein, resulting in thickness of yogurt. While fermenting milk, L. bulgaricus produces acetaldehyde, one of the main yogurt aroma components. Some strains of L. bulgaricus also produce bacteriocins named ‘bulgaricin’ (stable at 100˚C/hr), that kill undesired bacteria.

· Lactobacillus acidophilus (Latin: acid-loving milk-bacterium) is a homofermentative species, fermenting sugars into lactic acid, and grows readily at rather low pH values (below pH 5.0) having an optimum growth temperature of around 37°C. L. acidophilus occurs naturally in human and animal gastrointestinal tract, mouth, and vagina. Some strains of L. acidophilus have probiotic characteristics and are used in many dairy products, sometimes are associated with S. salivarius ssp. thermophilus and Lactobacillus bulgaricus for the production of yogurt.

Some strains of L. Acidophilus are extensively studied for health effects able to survive gastrointestinal transit, resistant to bile, low pH, and digestive enzymes. These may then be able to adhere to human epithelial cell lines and human intestinal mucus.

· Lactobacillus casei is found in the human intestine and mouth. This is documented to have a wider pH and temperature ranges, and supports the growth of L. acidophilus, the producer of amylase. L. casei is typically, a dominant species of nonstarter lactic cultures present during ripening of Cheddar cheese. L. casei is also the dominant species in naturally fermented Sicilian green olives. It is mesophilic in nature.

· Lactobacillus plantarum is commonly found in number of fermented food products and is also present in saliva from where it was first isolated. It has the ability to liquefy gelatine and has one of the largest genomes known among the lactic acid bacteria. It is a very flexible and versatile species.

L. plantarum is Gram-positive, aerotolerant that grows at 15°C but not at 45°C, and produces both D/L isomers of lactic acid. This species and related lactobacilli are unusual in that these can respire oxygen but have no respiratory chain or cytochromes, the consumed oxygen ultimately produce H₂O₂ that excludes components from the food. In place of protective enzyme superoxide dismutase, present in almost all other oxygen-tolerant cells, L. plantarum accumulates millimolar quantities of manganese polyphosphate that is also used by L. plantarum in a pseudo-catalase to lower reactive oxygen levels. Lactobacillus plantarum, like many lactobacillus species, can be cultured using MRS media.

· Lactobacillus brevis is found in many different types of environment and fermented foods such as sauerkraut and pickles. It is also one of the most common causes of beer spoilage. Its ingestion by consumers improves immune function. L. brevis is one of the major Lactobacillus species found in kefir grains, and has been responsible for the production of the polysaccharide (dextran) that forms the grains. Major metabolites of L. brevis include lactic acid and ethanol. Strains of L. brevis and L. hilgardii have been found to produce the biogenic amines tyramine and phenylethylamine.

6.3 Leuconostoc

‘Leucos’ means colorless and ‘Nostoc’ means encapsulated blue green algae. The leuconostoc species are Gram-positive cocci in pairs or short chains, microaerophilic/ aerobic/ facultative anaerobic. There are seven recognized species of Leuconostoc: L. citrovorum, L. dextranicum, and L. mesenteroides.

Leuconostoc is placed within family Leuconostocaceae. These are generally ovoid cocci often forming chains. Leuconostoc spp are intrinsically resistant to vancomycin and are catalase-negative that which distinguishes these from staphylococci. All species of Leuconostoc are heterofermentative and are able to produce dextran from sucrose. These are generally slime-forming and flavour producing in the presence of lactic acid.

Blamed for causing 'stink' when creating a sourdough starter, some species are also capable of causing human infections.

Leuconostoc is, along with other lactic acid bacteria such as Pediococcus and Lactobacillus, responsible for the fermentation of cabbage, making it sauerkraut. In this process the sugars in fresh cabbage are transformed to lactic acids that give it a characteristic sour flavour and improved shelf life.

6.4 Enterococcus

Enterococcus is a genus of lactic acid bacteria of phylum firmicutes. These are Gram-positive diplococci or short chains, and are difficult to distinguish from streptococci on physical characteristics alone. Two species E. faecalis (90-95%) and E. faecium (5-10%) are common commensal in the intestine of humans. Rare clusters of infections occur with other species, including E. casseliflavus, E. gallinarum, and E. raffinosus. There is a question whether enterococci should be regarded as starter culture for dairy because these are mainly of faecal origin and do not have GRAS status, as some of them are pathogenic in nature. These are included in lactic acid bacteria group because these are mainly found in artisanal cultures and have a positive effect on the development of flavour and texture.

Enterococcus differ from other lactic acid bacteria in a sense that these are more resistant to processing conditions and these may grow at temperatures i.e. 10 and 45°C; tolerate pH 9.6 and NaCl at a level of 6.5%.  The species are distinguished based on the arginine/ hippurate hydrolysis and lysis against micrococi as a typical characteristic of each species.

Enterococcus faecalis, earlier classified as a part of Group D Streptococcus, is Gram-positive, commensal inhabiting the gastrointestinal tracts of humans. It is among the main constituents of some probiotic food supplements. Like other species Enterococcus, E. faecalis can cause life-threatening infections, especially in nosocomial environment, where naturally high levels of antibiotic resistance contribute to its pathogenicity. E. faecalis has been frequently found in root canal-treated teeth in prevalence ranging from 30 to 90% of cases.

6.5 Pediococcus

Pediococcus is Gram-positive lactic acid bacteria, placed within the family of Lactobacillaceae. These usually occur in pairs or tetrads, and divide along two planes of symmetry, as do other lactococci. These are purely homofermentative. Pediococcus dextrinicus has been reassigned to the genus Lactobacillus. Pediococcus, along with other lactic acid bacteria (i.e. Leuconostoc and Lactobacillus), is responsible for the fermentation of cabbage, making it sauerkraut. During this process, sugars in fresh cabbage are fermented to lactic acid that gives sauerkraut a characteristic sour flavour and good shelf life. Pediococcus are usually considered the contaminants of beer and wine, although their presence is sometimes desired, like inlambic beer. Certain Pediococcus produce diacetyl that gives a buttery or butterscotch aroma to some wines (i.e. Chardonnay) and a few types of beer. Pediococcus are often used as silage inoculants, probiotics, and usually added as beneficial microbes in making of some cheeses and yogurts.

Pediococcus are the only lactic acid bacteria that form tetrad by dividing typically in two different planes. Typically, these microbes are homofermentative and produce L, D, and DL lactic acid as the major end product. These comprise of six different species but only two (i.e. P. pentosaceus and P. acidilactici) are mainly used in dairy. These are significant to food industry in negative and positive sense. As P. pentosaceus leads to spoilage, while also used as starter cultures for dairy as well as silage. The main characters to distinguish between species are type of lactic acid produced, arginine hydrolysis, and growth at different pH and in sugar fermentation pattern (Table 6.2).

Table 6.2  Biochemical and physiological characteristics of Pediococcus spp.

Characteristic(s)	P. pentosaceus	P. acidilactici
Growth at 10% NaCl	+/-	-
Arginine hydrolysis	+	+/-
Acid from: a) Maltose b) Trehalose c) Arabinose	+ + +	- V V