The isolation of lactic acid bacteria from raw and pasteurized milk and from fermented dairy products 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 of all genera of LAB present in raw milk.  

While selective media can be used for some LAB including lactobacilli, and 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 not selective for LAB unless modified. Because 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

                                                                                                      Number of strains*

 

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 are 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 before use. The selective pressure can be increased further by e.g. lowering the pH below 5.5, the 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 there 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

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 are particularly useful in the identification 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). 

However, not all researchers have access to these methods and it is often useful to use older, traditional approaches. However, these must be used with caution and it may be prudent to refer to the genus or species designated as presumptive identifications. Alternatively refer to counts on particular media and qualify the limitations.

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 schemes they are still used and provided the limitations are understood they have some utility.

At a practical level, the author suggests that the use of the simple tests given in Table 3 should be the minimum required to state that an isolate from a cheese or a fermented milk culture could be given a presumptive genus designation.

Table 3: Simple scheme for the presumptive identification of isolates of lactic acid bacteria to genus level

Criterion

Lactococcus

Leuconostoc

Lactobacillus

Pediococcus

Gram reaction

+

+

+

+

Cell morphology

Cocci

Cocci/ coccobacilli

Rods/ coccobacilli

Cocci in pairs or tetrads

Catalase reaction

                           -

         -

-

-

Gas from glucose

-

+

±

-

Gas from fructose

-

+

              ±

-

Gas from trehalose

-

±

±

-

NH3 from arginine

±

-

±

+

Growth on Rogosa agar

-

+

+

+

Dextran production from sucrose

                  -

+

±

-

 

Isolating  lactic acid bacteria from fermented products

Lactococci

Note Lactococcus lactis subspecies cremoris was elevated back to the species level as Lc. cremoris by Li et al. (2021).

Lactococci can be differentiated to the species or biovariant level using the scheme originally developed for lactic streptococci-see here. Note that lactococci will not grow on Rogosa agar (Bille et al., 1992). Differential, but not selective, media are available and can be useful for quality control and strain isolation purposes. The medium, Reddy's Differential Agar, developed by Reddy et al. (1969) is still of value. This medium contains the differential ingredients lactose, calcium citrate, L-arginine and the pH indicator bromocresol purple. This indicator gives yellow and blue/purple colours under acid and alkaline conditions respectively.

Yellow colonies of Lactococcus cremoris on Reddy's medium

Lc.  cremoris (shown in plate 2) gives yellow colonies due to acid production from lactose. Lc. lactis while producing acid also produces ammonia from arginine. The ammonia neutralises the acid and eventually produces an alkaline reaction that results in blue/purple-coloured colonies. Lc. lactis subsp. lactis biovar. diacetylactis also gives a blue/purple colony. Unlike Lc. lactis, however, Lc. lactis subsp. lactis biovar. diacetylactis exhibits zones of clearing around colonies because of citrate utilisation. Because some strains of Lc. lactis possess only weak arginase activity the use of a streaking technique on an improved version of this medium may help identify these strains (Mullan and Walker, 1979). This agar is commercially available as Lactic Streak Agar.

Leuconstocs

Leuconostoc species are important flavour producers in fermented dairy products. They also produce carbon dioxide, which is desirable in some varieties of cheese, e.g. Emmental, and Gruyere. ln other cheese varieties, e.g. Cheddar, gas production is undesirable and may cause faults in the products.

Studies of leuconostoc growth in cheese by the author and colleagues (Billie et al. 1992) revealed that the growth of lactococci was easy to suppress but that none of the published selective media employed suppressed the growth of lactobacilli and pediococci. We determined the selectivity of Rogosa agar, WACCA medium containing 0.3 lU/ml penicillin, the tetracycline medium of McDonough et al., the sodium azide-containing medium of Mayeux et al., Rogosa agar with added sodium chloride (1 % w/v), MRS agar containing-added sodium chloride (1 % w/v) and calclum lactate (5 % w/v) and MRS medium with added vancomycin (100 µg/ml), for selected leuconostocs, lactobacilli, pediococci, and starter lactococci. None of the media tested was selective for members of a particular species, thus limiting their usefulness for isolating leuconostocs from environments containing lactobacilli and pediococci. Attempts to devise an improved medium for leuconostocs, based on modifications of Rogosa and MRS agars were not successful. With further development' modification of Rogosa medium with added sodium chloride, and MRS medium with added sodium chloride and calcium lactate, may prove useful for the enumeration and/ or isolation of pediococci from starters and cheeses. MRS medium with added vancomycin would appear to be useful for determining the numbers of non-starter lactic acid bacteria in starter cultures and cheeses.

Although time-consuming, the most suitable method for isolating leuconostocs in mixed cultures of lactic acid bacteria and in cheeses is to plate samples onto Rogosa, or MRS agar containing 100 µg/ml vancomycin. Presumptive identification of isolates to genus level using Gram-reaction, cell morphology, ammonia production from arginine gas production from glucose, and citrate metabolism should follow. Determination of the lactic acid configuration would be the most appropriate test to confirm genus designation. The use of the API-CHL fermentation system is also useful.

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.

Li, T., Tian, W, and Gu, C. (2021). Elevation of Lactococcus lactis subsp. cremoris to the species level as Lactococcus cremoris sp. nov. and transfer of Lactococcus lactis subsp. tructae to Lactococcus cremoris as Lactococcus cremoris subsp. tructae comb. nov. International journal of systematic and evolutionary microbiology. 71, 1-6.

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. and Walker, A.L. (1979). An agar medium and a simple streaking technique for the differentiation of the lactic streptococci. Dairy Industries, 44 (6):13, 17.

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.

Reddy M. S., Vedamuthu E. R., Washam C. J. and Reinbold G. W. (1969). Differential agar medium for separating Streptococcus lactis and Streptococcus cremoris. Appl. Microbiology 18, 755-759.

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/cheese-starters/250-isolating-lab.html . Accessed: 15 April, 2024. Updated June, 2016; September, 2019, January 2024.