Clostridium tyrobutyricum

Clostridium tyrobutyricum is a spore-forming bacterium whose spores are heat-resistant. It comes in large numbers before the silage used for cows as feed. It belongs to the genus Clostridium, which includes numerous pathogens, but is not considered pathogenic.

Occurrence

Clostridium tyrobutyricum occurs in the ground, from where it is distributed by using the endospores and thus passes on plants, which are used for the production of silage. From this it can be isolated and was also detected in dairy products such as Emmental cheese, in which it had arrived on the cows' milk after receiving contaminated feed.

Features

As a typical representative of the clostridial bacteria to grow obligate anaerobes and thus are catalase - negative and oxidase - negative. They are gram- positive, showing a microscopic image of a rod-like shape and are motile by peritrichous flagella arranged.

Under adverse environmental conditions, they are able to form endospores. These are larger than the vegetative cells and lead to a bulge in the mother cell. Furthermore, the spores are characterized by thermal resistance: While most vegetative bacterial cells are killed by briefly heating to temperatures of about 80 ° C ( pasteurization), this heating does not damage the endospores, they remain viable and can germinate again.

The species name indicates that this species forms butyric acid by fermentation, as butyrate salts of butyric acid are known. The genome of Clostridium tyrobutyricum ( strain DSM 2637 ) is currently being sequenced.

The optimum temperature for the cultivation of C. tyrobutyricum is from 40.2 to 43.3 ° C, therefore, the bacterium is one of the more thermophilic organisms. At 10 ° C, it shows no growth, however, the cells survive the temperature, and begin at slightly higher temperatures (12-15 ° C), again with the increase. The pH value for the growth of most strains tested in the range of pH 5.5 to 7.5. C. tyrobutyricum able to tolerate moderate concentrations of sodium chloride, a content of 2.0 % does not prevent the growth of a content of 3.0 % allows at least some of the tested strains have multiplication and only starting from a sodium chloride content of 3, 5% is no more growth is possible. This yields an estimate as to the conditions under which to be reckoned with unwanted growth of Clostridium tyrobutyricum in cheese production.

Metabolism

Clostridium tyrobutyricum utilized carbohydrates by fermentation. First, a gradual reduction of monosaccharides occurs ( simple sugars ) such as D- glucose (dextrose ) in glycolysis to pyruvate. Under anaerobic conditions, the consumed here NAD ( nicotinamide adenine dinucleotide ) must be regenerated, this is done by butyric acid fermentation. From pyruvate ( butyric acid salt), acetate ( salt of acetic acid ), carbon dioxide (CO2) and elemental hydrogen arise as a result of fermentation by C. tyrobutyricum butyrate (H2).

Butyric acid fermentation proceeds in many Clostridia, as a special feature, however, C. tyrobutyricum use instead of glucose and lactate (salt of lactic acid) as a substrate. Lactic acid is produced by representatives of Lactobacteriaceae in lactic acid fermentation and lowers the pH of the environment. Most clostridial grow only in neutral or alkaline media, the presence of lactic acid bacteria to suppress their growth. Clostridium tyrobutyricum, however, tolerated a slightly acidic environment to gain ( to about pH 5.0), and is also capable of the degradation product of lactic acid bacteria themselves energy.

Evidence

The bacterium can be cultivated in a nutrient medium containing meat extract, yeast extract, peptone from casein, glucose and other constituents. It is important that the medium prior to inoculation is anoxic, because oxygen for the cells of C. tyrobutyricum toxic. This is achieved by boiling and cooling under a nitrogen atmosphere, thus the presence of oxygen is excluded from the air. Also in the inoculation and incubation must be paid to strict compliance with the Anaerobentechnik. Is incubated at a temperature of 37 ° C.

Include biochemical assays to identify, as described above, the catalase and oxidase test, and tests of a typical "Bunte series", inter alia, on the usability of various carbohydrates and other substrates is investigated. A work based on the rapid determination system in miniature (Analytical Profile Index ) for the determination of anaerobes is commercially available and also includes the detection of Clostridium species. These classic microbiological and biochemical detection methods are quite time consuming, so that there are now direct detection of Clostridium tyrobutyricum by using the multiplex PCR method. It ensures the differentiation from other Clostridium species.

Industrial importance

Clostridium tyrobutyricum is feared as the cause of a faulty fermentation in cheese production. The germ can be entered on silage as feed the cows in the animal and passes through the milk in the cheese. In the production of silage, the appropriate plant parts are crushed, compacted in the silo and is hermetically sealed. Now a fast lowering of the pH is desirable, as is the case with lactic acid fermentation, while maintaining the anaerobic atmosphere. The low pH value and the absence of oxygen prevent numerous harmful for the preparation of the silage micro-organisms from growing. This is also true for most of clostridia, but not C. tyrobutyricum, the tolerated slightly acidic pH values ​​and also can still metabolise lactic acid as a substrate.

The silage thus contains either still vegetative cells or spores of the bacterium. The spores pass through the lining in the body of the cow and from there directly into the milk, or this is done via contamination of the udder with sporenhaltigem material. Even with a pasteurization of the milk prior to cheese production, the spores are at least not killed, so they are included in the cheese and can germinate again inside the whole cheese. When now expiring butyric acid fermentation which is also present in the cheese lactic acid is broken down to butyric acid, carbon dioxide and hydrogen. The gases carbon dioxide and hydrogen cause the cheese to a strong flatulence ( late blowing, blowing referred to in English as late ) that makes itself as cracks and crevices in the Käsekrume noticeable and nothing with in some cheeses such as Emmental, desired holes has to do in the cheese. Butyric acid also causes an unpleasant odor. These cheeses are inedible, no longer usable and can lead to substantial financial losses in cheese production.

Milk from cows fed with silage is not suitable for cheese production because of possible risk of faulty fermentation. In Switzerland, will be omitted in cheese milk on the use of silage. In the EU, a product derived from chicken egg white enzyme as an antibacterial agent in the cheese industry is lysozyme, admitted. As a food additive with the number E 1105 it may only be used for ripened cheese to prevent damage caused by a faulty fermentation by Clostridium tyrobutyricum.