Medium for the selective enrichment of lactate-fermenting Clostridia spp.
(Cl. tyrobutyricum) which are responsible for "late blowing" in brine-salted semi-hard and hard
cheeses and other dairy products.
The medium is used to enumerate the spores of lactic acid fermenting Clostridia spores in silage,
milk, and dairy products. During miling process low numbers of butyric acid fermenting bacteria (BAB)
originating from silage are introduced into the raw milk. When the contaminated milk is used for cheese
production, cheese brines become contaminated with heat resistant Clostridia spores. During
the ripening of salt brined, semi- and hard cheeses (for example, Gouda, Edammer, Emmentaler, Greyere, and
Parmesan) "late blowing" gasogenic Clostridia ferment lactate into butyric acid, acetic acid,
and gas (CO2 and H2). The gas swells the cheese and is responsible for a defect
termed "late blowing" or butyric swelling, resulting in bad taste. The main species causing this butyric
swelling defect is Cl. tyrobutyricum. Other Clostridia belonging to the butyric acid
fermenting bacteria (BAB) are Cl. butyricum or Cl. sporogenes. The causative Clostridia
spp. are anaerobic, Gram-positive microorganisms forming heat resistant endospores, which survive
pasteurization but not UHT or sterilization of milk.
Mode of Action
Vegetative cells are killed by a heat
treatment (75°C for 10 min). Resazurin is a redox indicator and monitors the oxygen level. The
nutrient composition of the basal medium, particularly the high quality of the peptones, creates the
conditions for a rapid growth of lactate fermenting Clostridia spp. Sodium acetate promotes the
spore germination which is activated by the heat treatment of the sample. Lactate is the substrate
for the Clostridia spp. to produce gas, visualized by a raised paraffin plug.
Preparation
Suspend 38 g in 1 liter of purified
water. Autoclave (15 min at 121°C). Cool to 45-50°C and dispense in bottles or tubes.
pH: 5.9 + 0.2 at 25°C.
The prepared non-boiled broth is pink and the boiled broth is colorless. A pink color indicates the
presence of oxygen.
Experimental Procedure and Evaluation
The MPN method is employed in the examination
on lactate fermenting Clostridia spp. Tubes of medium are boiled (100°C for 10 min) where
appropriate to regenerate anaerobiosis and cooled down to 25-30°C. Colorless tubes are inoculated with
sample or sample dilutions and overlaid with 2 cm of sterile (121°C for 20 min.) melted (58-60°C)
paraffin. The tubes are heated (75°C for 10 min) to kill vegetative microorganisms and cooled down
to 37°C to solidify the paraffin.
The inoculated medium is incubated at 37°C for up to 7 days. The tubes are evaluated every 48 hours.
Tubes with growth and gas formation indicated by a raised paraffin plug are considered positive. The MPN
index is used to calculate the number of Clostridia.
Further biochemical identification verifies the presence of CI. tyrobutyricum.
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consequences sur la qualite du lait et des produits laiters. Revue laiterie Francaise
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Bryant, M.P. & Burkey, L.A: 1953 Cultural Methods and some characteristics of some more numerous groups
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Cerf, O. & Bergere, K.L. 1968 La numeration des spores de Clostridium et son aplicatoin au lait
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