Isolating lactic acid bacteria from milk

The isolation of lactic acid bacteria from raw and pasteurized milk is discussed.

Isolation of lactic acid bacteria from raw milk

It can be challenging to isolate lactic acid bacteria (LAB) from raw milk that has been refrigerated without pre-incubation since the flora tends to be dominated by Gram-negative bacteria and the LAB are present in low numbers.

There is no single agar medium that is suitable for the selective isolation of strains all genera of LAB present in raw milk.  

While selective media can be used for some LAB including lactobacilli, some streptococci including faecal streptococci, leuconstocs and pediococci general purpose growth media and incubation conditions generally need to be adapted for particular projects. M17 (Terzaghi and Sandine, 1975) which is a good general purpose growth medium is generally not selective for LAB. While some strains of Lactobacillus delbrueckii subsp. bulgaricus  are inhibited by the β-glycerophosphate present in M17 (Table 1) caution should be used in using M17 to enumerate Streptococcus thermophilus in environments containing L. delbrueckii subsp. bulgaricus.

Table 1. Effect of β-glycerophosphate (1.9 %) on growth of L. delbrueckii subsp. bulgaricus strains in MRS broth. Incubation at 37°C for 48 h


 

MRS

MRS + GP

No growth

0

9

Poor growth

1

29

Medium growth

0

1

Good growth

57

13

*No of strains (58 tested). From: Shankar and Davies (1977)

Note that 23% of the L. delbrueckii subsp. bulgaricus strains tested grew well on M17 indicating the need for authors using this medium to enumerate Str. thermophilus in an environment containing L. delbrueckii subsp. bulgaricus  to confirm the absence of L. delbrueckii subsp. bulgaricus (Gram stain and catalase reaction as a minimum).

The simplest method for isolating LAB from raw milk is probably to use M17 or preferably PLGYG agar (Mullan et al., 1981) containing thallium acetate and adjusted to pH 5.5 with lactic acid. As discussed M17 is inhibitory to many strains of L. bulgaricus. The thallium acetate inhibits the growth of Gram-negative bacteria, the usual concentration is 1 part in 2000 parts of media (Harrigan and McCance, 1998). The lactic acid and the lower pH provide a more selective environment for LAB.

The lactic acid is added aseptically to the agar media after sterilisation and prior to use. The selective pressure can be increased further by e.g. lowering the pH below 5.5, addition of salts e.g. sodium chloride, sodium or calcium lactate, sodium acetate, replacing the glucose with sucrose or other sugars and using antibiotics (Billlie et al, 1992).

Incubation temperature can be varied (e.g. 30°C for mesophiles) as required as can gaseous environment.

Isolating thermoduric / thermophilic lactic acid bacteria from pasteurized milk

These bacteria can be isolated from pasteurised milk, either laboratory-pasteurized (60°C, 30 minutes) or in samples of milk from a pasteurizer. Expect low counts in good quality raw milk that has been subjected to laboratory pasteurisation. Much higher counts are usually obtained from pasteurizers in cheese factories especially those operated for an extended period.

These bacteria may belong to several genera including Lactobacillus and Streptococcus.

Streptococci are generally sensitive to the acetate used in media like Rogosa. So Rogosa agar cannot be used as the only isolation medium. While incubation temperature can be used to provide a selective pressure, workers should also be aware that on occasions that may be relatively high concentrations of aerobic sporeformers present particularly in milk samples from pasteurizers operated for extended periods. These can form spreading colonies obscuring other colonies on plates.

The isolation media and incubation conditions required to enumerate these bacteria are similar to those described for raw milk except that incubation temperatures ranging from 42°C-45°C are used.

Designating lactic acid bacteria to genus and species

Key distinguishing attributes of major current genera important in food fermentations are given in Table 2.

 Table 2. Characteristics of genera of lactic acid bacteria used as starter cultures

Genus

Cell Morphology*

Fermentation

Lactate isomer

DNA (mole % G+C)**

Growth on Rogosa agar

Lactococcus

Cocci in chains

Homo

L

33-37

-

Lactobacillus

Rods

Homo/hetero

D/L, D, L

32-53

±

Leuconostoc

Cocci

Hetero

D

38-41

±

Oenococcus

Cocci

Hetero

D

 

 ±, Most strains are positive

Streptococcus

Cocci in chains

Homo

L

40

-

Pediococcus

Cocci, tetrads

Homo

D/L

34-42

+

Tetragenococcus

Cocci, tetrads

Homo

D/L

 

+

*Distinguishing between a short rod and a coccus can be difficult.

From: Mullan (2014)

The differential characteristics listed in Table 2 are based on phenotypic properties and despite their limited validity in current microbial classification they are still used and provided the limitations are understood have some utility.

Molecular and chemotaxonomic methods are being used to assign genus and species designations. The extent of DNA–DNA and DNA–rRNA hybridization, similarity between profiles produced by restriction mapping of chromosomal DNA, and the nucleotide sequence of the 16S and 32S RNAs have been found to be particularly useful in the creation of the genus. Additional methods, including serology, also have provided further evidence for the validity of genus designation (e.g., antisera) against purified superoxide dismutase, which has been used to demonstrate a similarity between lactococci but not streptococci or enterococci (Mullan,2014).

Verifying that isolates are safe to use

 Many lactic acid bacteria can produce bioamines, such as histamine, putrescine, tyramine, and cadaverine. These are produced by the action of amino acid decarboxylases and can cause headaches and other physiological effects. Screening new strains for bioamine production before starter use is recommended.

There is also the possibility that new isolates may have antibiotic-resistant genes e.g. for glycopeptide antibiotics (vancomycin and teicoplanin) and new isolates should be screened to ensure that strains with antibiotic resistance genes are not used.

Genes for virulence traits associated with adherence to host tissue, invasion and abscess formation, modulation of host inflammatory responses, and secretion of toxic products (e.g.,bioamines) have been identified and should be screened for in new isolates (Mullan, 2014).

 Literature cited

Bille, P.G., Espie, W.E. and Mullan, W.M.A. (1992). Evaluation of media for the isolation of leuconstocs from fermented products. Milchwissenschaft. 47:637-640. This can be downloaded from www.researchgate.net .

Harrigan, W.F. and McCance, M.E. (1998). Laboratory methods in food and dairy microbiology (3rd ed.). Academic Press, London.

Mullan, W.M.A., Daly, C. and Fox, P.F. (1981). Effect of cheesemaking temperatures on the interactions of lactic streptococci and their phages. J. Dairy Res. 48, 465-471.

Mullan, W.M.A. (2014). Starter Cultures: Importance of Selected Genera. In: Batt,C.A., Tortorello, M.L. (Eds.). Encyclopedia of Food Microbiology, vol 3. Elsevier Ltd, Academic Press, pp. 515–521.

Shankar, P. A. and Davies, F. L. (1977). A note on the suppression of Lactobacillus bulgaricus in media containing β -glycerophosphate and application of the media to selective isolation of Streptococcus thermophilus from yoghurt. Journal Society Dairy Technology. 30, 28-30.

Terzaghi, B.E. and Sandine, W.E. (1975). Improved medium for lactic streptococci and their bacteriophages. Appl. Microbiol. 29, 807-813.

 
How to cite this article

Mullan, W.M.A. (2015). [On-line]. Available from: https://www.dairyscience.info/index.php/cheese-starters/250-isolating-lab.html?tmpl=component&print=1&layout=default . Accessed: 11 December, 2019. Updated June, 2016; September, 2019. 

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