Instituto Valenciano de Microbiología

Masía El Romeral
Ctra. de Bétera a San Antonio Km. 0.3
46117 Bétera (Valencia)
Phone. 96 169 17 02
Fax 96 169 16 37
CIF B-96337217


Clostridium butyricum and others related (Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium tyrobutyricum, Clostridium sporogenes, ...): interest of their control in dairies industries. 

Information (04/14/11)

Clostridium butyricum is a strict anaerobic bacteria, producing spores, found in soil and in the intestinal tract of animals and people.

In dairies that produce cheese, may cause problems. The main alteration is caused swelling, followed by the possible explosion or burst (blowing), early or late, during cheesemaking. This alteration occurs more often in semi - hard cheeses or cured (Emental, Guda, ...) with little healing time and high pH. On the contrary, it is less common in dry - cured cheeses (Parmesan, Cheddar).

Alteration is increasing due to changes in breeding animals. It occurs as a result of inadequate or late development of lactic bacteria or by some errors, for the technical process of making cheese. The onset is usually delayed as fresh milk is not a favorable environment for germination of spores of Clostridium butyricum, while during chemical fermentation caused by lactic acid bacteria which induces spore germination accumulates.

That explosion, causes changes in the appearance and taste of cheese, butyric acid and the generated gas, mostly hydrogen and carbon dioxide. Gas production will induce an expansion balloon, and cambo consistency. This phenomenon has not only been implicated Clostridium butyricum, but also other species such as C. acetobutylicum, C. beijerinckii, C. tyrobutyricum andC. sporogenes.

Control of the presence of spores in milk

Contamination clostridia spores in milk can be controlled before and after milking. On the one hand spores they can accumulate in silos, and for this reason some producers of cheeses require animals from which comes the milk used for cheese making, not fed with products stored in silos. During the production of milk, animal behavior should be controlled to prevent throw in dirty areas with feces because this case, the udder is contaminated with feces that can be found spores, causing contamination of milk. After collecting milk at farms, and before use, follow some recommendations, given that pasteurization has no effect on spores, so other procedures must be followed. In many European countries the milk treatment with lysozyme or nisin is recommended. Lysozyme is a hydrolase acid produced by animal cells, and is present for example in egg white. This enzyme hydrolyzes the bacterial walls, and 0.5% concentration, will kill most vegetative cells of clostridia, but however, has no effect on the spores. This enzyme is admitted as food additive, including cheeses (Directive no. 95/2 / EC).

Nisin is a lantibiotic classified as antibacterial peptide, obtained from various species of Lactobacillus and kills bacteria by forming pores in the membrane joining the lipid II. Although it has effect on vegetative cells, no effect on spores.

Lacking these substances effects on spores, filtration techniques, or centrifugation, to remove bacteria and spores in milk are used. Centrifugation at high speed is able to remove 92% of the spores of aerobic bacteria and 91-97% of the spores of anaerobic bacteria.

Microfiltration with ceramic membranes microfiltration, retains the suspended particles, microorganisms and fats. Proteins, carbohydrates, minerals and cross, but retains 99% of the spores of aerobic bacteria and anaerobic bacteria. Both centrifugation and microfiltration, change the composition of milk to be marketed as such and should contain fats, which are mainly used for making cheese. Thus it is avoided that the milk used for cheese making, contain clostridia spores as Clostridium botulinum, which must be altered during manufacturing cheese.

Neurotoxigénicas strains of Clostridium butyricum

Clostridium butyri cum, is a species of Clostridium not proteolítca, like Clostridium botulinum. Some strains of Clostridium butyricum can produce a potent neurotoxin, antigenically identical to that produced by Clostridium botulinum type E, encoded by a gene present in a plasmid large, which can be transferred between strains. For this reason, the germination of Clostridium botulinum should be controlled, since during it, can produce the neurotoxin if the strain possesses the plasmid encoding it .

Tests in IVAMI:

1. Detection and identification of Clostridium butyricum and other related species (Clostridium  acetobutylicum, C. beijerinckii, C. tyrobutyricum y C. sporogenes), by culture in liquid medium under conditions of strict anaerobiosis, followed by seeding to isolate colonies solid media, and the identification of colonies compatible with this species, by sequencing of the 16S rRNA gene. Counting colony forming units (CFU) is not recommended because the distribution of their presence inside the cheese is not uniform.

2. Detection of Clostridium butyricum neurotoxin by enrichment culture sample and subsequently inoculated mice. If it is negative the test is not necessary to continue. If mice affected with paralytic symptoms, a neutralization test should be performed to confirm that it is similar to that of Clostridium botulinum neurotoxin type E.

3. Detection producer neurotoxin gene, similar to Clostridium botulinum, using Real-time PCR (polymerase chain reaction in real time).

Recommended sample:

It is recommended to send full cheese or pieces affected to process in the same laboratory as it is required to collect the contents of the look of the formed air cavities.

Preservation and shipment of sample:


Cost of the test: