Journal cf Microbiological Me~'hods 13 (1991) 75 - 83 75 Elsevier

MIMET 00417

X-o -Gal-based medium for simultaneous enumeration of olx uuua Lc m and lactic acid

bacteria in milk

Pierre Chevalier, Denis Roy and Luc Savoie

Lactic Acid Bacteria Research Group, Agriculture Canada, Food Research and Development Centre, Saint-Hyacinthe, Qudbec, Canada

(Received 9 March i990; revision received 18 Decembel" 1990; accepted i January 1991)

Summary

A new method using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-c~-D-galactoside was devel- oped for the differential enumeration of Bifidobacterium spp. and lactic acid bacteria used in preparation of fermented and nonfermented dairy products. The methodology is based on the high a-galactosidase ac- tivity of bifidobacteria as compared to that of Lactobacillus or Streptococcus strains. The X-u-Gal-based medium is useful to identify bifidobacteria among Lactobacillus since the enzyme action of ~-galactosidase split~ ,':-a-Gai substrate and releases indol which imparts a blue colour to bifidobacterial colonies on agar plates. The blue colonies can also be assigned to the genus Bifidobacterium using a simple permeabilization procedure with Triton X-100 which allows the reliable determination of fructose-6-phosphate phosphoketol- ase activity.

Key words: Bifidobacterium; Differential enumeration; X-u-Gal; c~-Gaiactosidase; Permeabiiization procedure; Presumptive identification

|~troduction

Bifidobacteria constitute a major part of the intestinal microflora of humans [1]. These bacteria have recently gained prominence in the field of human nutrition be- cause of their potential importance leading to metabolic modifications of the colonic flora [2]. Chronic ingestion of fermented and nonfermented dairy products is a means for administering bifidobacteria and maintaining a balanced intestinal flora that should be favourable to the health of the host [3-4].

The number of bifidobacteria added to milk during preparation of fermented or nonfermented dairy product must be determined from mixed cultures. The use of selec-

Correspondence to: D. Roy, Food Research and Development Centre, 3600 Casavant Bird West, Saint- Hyacinthe, Quebec J2S 8E3, Canada. Food Research and Development Centre Contribution 202.

0167-7012/91/$ 3.50 © 1991 Elsevier Science P,'bUshers B.V.

76

tive and differential agar media for bifidobacteria have been reported in the literature [5 -7] . Selective methods are based, mostly, on the inhibition of the growth o f , :.her bacteria by adding lithium chloride or antibiotics to media [8]. These methods involve mat tt~e m~nber of b~fidooacterla is determined toy substractmg me viable counts of bifidobacteria on selective media from the total number of colony counts on nonselec- tive media [5].

The determination of enzyme profile has shown that all strains of bifidobacteria possess ~x-galactosidase, ~-galactosidase and ot-glucosidase achvities [9]. Although the most direct and reliable characteristic for assigning an organism to the genus Bifidobacterium is based on the demonstration of fructose-6-phosphate phospho- ketola~e (F6PPK) activity, Chevalier et al. [10] noted that the detection of a- galactosidase activ"v from lactic acid bacteria used in yoghurt making may be a simple method for rapid preliminary identification of bifidobacteria.

The present study was designed to develop a medium that should facilitate presump- tive identification of bifidobacteria and clearly differentiate bifidobacterial colonies from those of Lactobaciilus spp. as well as from colonies of S. salivarius thermophilus. The simultaneous enumeration of bifidobacteria and lactic acid from milk samples was based on the splitting of 5-bromo-4-chloro-3-indolyl-o~-D-galactopyranoside (X-tx-Gal) by bifidobacteria. X-tx-Gal has been already used as a chromogenic sub- strate for ot-galactosidase (tx-D-galactosyl galactohydrolase, EC 3.2.1.22) detection permitting the differentiation of ale (blue colonies) and lager (white colonies) strains of Saccharomyces cerevisiae [11].

Materials and Methods

Cultures The strains used in this study are listed in Table 1. Each strain was subcultured in

TABLE 1

BACTERIAL STRAINS USED IN THIS WORK

Species Strain Source

Bifidobacterium B. adolescentis B. bifidum B. breve B. infantis B. Iongum

Lactobacillus L. acidophilus L. delbrueckii bulgaricus L. helveticus

Streptococcus S. salivarius thermophilus

ATCC a 15703 (type strain) ATCC 29521 (type strain) ATCC 15700 (type strain) ATCC 27920 (neotype) ATCC 15707 (type strain)

ATCC 4356 (neotype) FYE 40 b ATCC 15009 (neotype)

ATCC 19258 (type strain)

Infant faeces Infant intestine Infant intestine Infant intestine Adult intestine

Human Yoghurt starter Emmental cheese

Pasteurized milk

a American Type Culture Collection, Rockville, Maryland. b Lacto Labo, Dande Saint Romain, France.

I I

lactobacilli MRS B r n t h (r'~ifi-, 0 I ~ h n r a t o r l o % l '~mrr~t r~i,~h~,-,,~.x . . . . . . . . . . . . . . . . . . . . . . . . . . . . . - ....... ~,,,,,, sui;plemented with 0.05°70 cysteine- HCI, incubated at 37 °C for 18 h under an anaerobic atmosphere con- taining 5°'/0 c02, 10070 H2 and 85070 N 2 (anaerobic glove box; Forma Scientific, Ohio). The anaerobios]s was used mroughout for all strains, even facultative ones, because growm was better under this condition (data not shown). The viability of strains was checked each week by streaking cultures on MRS agar (modified MRS broth with 1.5% agar) and incubated under aerobic and anaerobic conditions for 48 h at 37 °C. Bacteri- al suspensions to be tested were obtained after 1R h of,,,',-,w,r, at 37 °C under anaerobic atmosphere as described above in 10 ml of modified MRS broth (using a 10°70 inocu- lum) before being used for enzyme assays.

X-a-Gal-based medium

The modified MRS agar was used as the medium for the incorporation of X-a-Gal (Boehringer Mannheim, Montr6al, Canada). A final concentration of 100, 150 or 200 #M (40.8, 61.2 or 81.6 mg .ml -! from a 0.2°'/0 N,N'-dimethyl-formamide stock so- lution) X-a-Gal was added to the sterilized MRS medium (121 °C, 15 min) to prevent a chemical hydrolysis. Pure and mixed bacterial strains were spread on plates and in- cubated at 37 °C under anaerobic atmosphere (anaerobic glove box) for 48 h. Plates were then moved to an aerobic atmosphere for 6 - 8 h to allow the colorimetric reaction which is oxidative [11]. The substituted indol, released by ot-galactosidase, oxidatively polymerises to give a blue colour (diazo) which does not diffuse in agar.

The bacterial viability on X-a-Gal-based medium of each strain listed in Table I was done by spread-plating 0.1 ml on lactobacilli MRS agar containing different "encen- trations of X-ot-Gal as mentioned above.

Identification of bac:::ia in blue aad nonblue colonies on X-u-Gal MRS agar was made by picking colonies from plates on the counting dilutions and transferring them to i0 mi modified MRS broth. The purity of blue and nonblue colonies was also checked by streaking them on modified MRS agar. These cultures were incubated at 37 °C for 48 h under anaerobic conditions as described above. Subsequently, cells were removed by centrifugation at 7600×g for 15 min and washed once with 10 ml McIl- vaine buffer, 100 mM, pH 6. The cell pellet was resuspended in 1 ml buffer and per- meabilized with 0.25 ml 5070 Triton X-100 (v/v) (Mallinkrodt) solution in McIlvaine buffer supplemented with 0.6070 NaF and 1070 K-Jodoacetate (Sigma Chemical, St Louis, Missouri). The suspension was mixed by vorte.xing. The enzyme measurement was done according to the method of Chevalier et al. [i0] except that NaF-iodoacetate solutior. (Solution 2) was not added and the sodium phosphate buffer was replaced by the McIlvaine buffer, 100 mM, pH 6. Sonification of cells was also performed ac- cording to the method of Chevalier et al. [10].

Milk preparations B. infantis, L. acidophilus and S. salivarius thermophilus were incubated separately

in modified MRS broth, MRS brotb (Difco) and M17 (Difco), respectively. All culture media were supplemented with lactose. Bifidobacteria and lactobacilli cultures were incubated at 37 °C for 18-24 h while the streptococcus culture wa,~ incubated at 43 °C for 18-24 h. All cultures were incubated under anaerobic conditions as described

78

above. Sterir.,: reconstituted skim milk contai,dng 10070 solids ~¢as inecu!ated (v/v) with ve-ic~s ratios (1:I.~, 5:2:1 and 10:5"1' r;" ,)lfidobacteria, lactobacilli and streptococci to obtain a finai volume o: 5 rm. i ,le inoculated milk was sarnpled after 0 h at 22 °C, 4 h at 43 °C (fermented mi!a) and 24 h at 4 °C (nonfermented milk). Titratable acidit~ was determincd with N/9 NaOH according to the procedore described by Ro~ et al. [121.

Enzyme assay c~-D-galactosidase activity was measured by determining the rate of hydrolysis of

p-nitrophenyl-c~-D-galact,-,pyranos,:de ~NPG) (Sigma Chemical). Bacterial suspe!~- sions were centrifuged at "iO(ggOxg for 10 rain. The cell pellet Was adjusted by spec- trophotometry (600 nm) with 50 mM potassium phosphate buffer, pH 6.5, to obtain a turbidity corresponding to an absorbance of 0.9. The bacterial pellets were per- meabilized by adding a final concentration of 2.5°/0 Triton X-100 (Sigma) prepared in the phosphate buffer [i3]. A portion (1 ml) of 4 mM pNPG was added to 0.5 ml permeabilized cell suspension. The mixture was incubated at 37 °C for 30 min and the reaction was stopped by adding 0.5 ml 1 M Na2CO3. The p-nitrophenol released was determ~.ned spectrophotometrically at an absorbance of 405 rim. The enzymatic activi- ty was expressed as nanokatal (nkat) which is the quantity of enzyme hydrolyzing 1 nmoi substrate, s- ~.

Results and Discussion

Viability of bacteria in modified MRS medium containing various concentrations of the chromogen X-c~-Gal is shown in Table 2. It was found that the aumber of Bifidobacterium spp. decreased at high concentration of X-~-Gal whereas other lactic acid bacteria were not affected, suggesting '&at the uptake of X-o~-Gal by bifidobacter- ia may result in a growth inhibition.

TABLE 2

EFFECTS OF THREE CONCENTRATIONS OF X-c~-GAL ON VIABILITY OF BACTERIA I w'3"D IN THIS WORK

Strains Number of bacteria ( C F U . m l - I )

MRS- MRS + 100 t~M MRS + 150 #M MRS + 200 #M X-ct-Gal X-a-Gal X-c~-Gal

B. adolescentis 3,0 × 109 2.4 × 109 6.7 x 107 3.0 x 106 B. bifidum i .9 :< 109 2.3 × 109 1.8 x 108 < |o o B. breve 3.2 × 109 z.4 x !09 5.0 x 108 1.8 x 107 B. infantis 2.0 × 109 1.7 × 109 7.9 x 107 < 106 B. Iono,m I 2v!fb9 Qc;,,~108 8.4X107 1 x l O 6

L. acidophilus 2.1 x 109 1.8 x 109 2.2 x 109 1.1 x 10 ~ L. d. bulgaricus 1.5 × 109 !.7 × l09 2.0x l09 1.2 x i09 L. helveticus 2.3 × 199 2.0× l09 2.2 x 109 1.9× i09 S. s. thermophilus 2.8 x l08 4.0 x l08 3.1 x l08 2.2 x l08

a Commercial (Difce) MRS supplement with 0.05% cysteine- HCI.

~ A

TABLE 3

RESULTS OF ENZYIVIATIC TESTS AND VIABILITY OF SUBCULTURED r31FIDOBACTERIUM- COLOURED COLONIES US!NG t00 #M (40.8 m g . m l - i ) X-c~-GAL

Strain Colorimetric reaction on Enzyme activity Bacterial viability plate (X-a-Gal) (nkat) a

B. adolescentis Blue 0.9 + B. bifidum Blue 0.8 + B. breve Btue 0.7 + B. infantis Blue 1.4 + B. longum Blue 1.5 + L. acidophilus Light blue 0.2 + L. d. bulgaricus Light blue 0. ! + L. nelveticus White 0.03 + S. s. thermophilus White 0.0 +

a Enzyme activity produced using a previously standardized bacterial suspension (see Materials and Methods)

Table 3 indicates that all strains o f Bifidobacterium spp. gave blue colonies on modi- fied MRS medium supplemented with 1 mM X-u-Gal while colonies of other lactic acid bacteria appeared white or light blue. Determination of o~-galactosidase activity using the PNPG procedure shows that L. acidopld!us and L, delbrueckii bulgancus had low o~-galactosidase activity as compared to bifidobacteria (Table 3).

TABLE 4

COMPARISON OF COLONY COUNTS ON X-tx-GAL MRS, MRS and MI7 MEDIA.. OBTAINED FROM MILK PREPARATIONS

Inoculum(v/v) Colony counts (log CFU. m! - ~

X-ot-Gal MRS MRS MI7

Blue Nonblue

Ratio 1:1:! a 0 h/22 °C 7.09 7.04 7.45 7.17

24 h / 40C 7.12 6.99 7.44 7.18 4 h/43 °C 6.68 8.21 8.19 8.06

Ratio 5:2:1 0 h/22 °C 7.76 7.37 8.00 7.70

24 h / 4cC 7.41 7.33 7.88 7.97 4 h/43 °C NO 7.96 7.70 7.54

Ratio 10:5:! 0 h/22 "C 7.97 7.72 8.27 8.38

24 h / 4cC 7.88 7.87 8.29 8.46 4 h/43 °C 6.00 8 . ~ 8.04 7.97

a B. infuntis : L. aclaopmiu~ : S. s_.." ".'..hermol~,.~lus ratios based on percentage of inoculum (v/v). Viable counts were determined from experiments performect ~ trJpi,~a,c in ~ter;.!e reconstituted skim

milk. NO, not observed.

80

It should be noted that the detection of blue colonies was only possible after a post- incubation time (6 -8 h) under aerobic conditions which is required to oxidize the chromogenic moiety liberated by the a-galactosidase. On the other hand, the total number of colonies should not exceed 100 because the colorimetric reaction was weak when Bifidobacterium spp. colonies were too close or too numerous. Lojda et al. [14] have found a very low K M for this substrate (< 1 mM) using different sources of ot- galactosidase. This high affinity of the enzyme for X-~-Gal might result in a rapid conversion of the 5-bromo-4-chloro-3-indolyl-c~-D-galactopyranoside by colonies growing more rapidly leaving 'ess substrate for slow-growing nearby colonies.

Table 4 shows that the total lmmber of both blue and light blue colonies enumerated on the X-~-Gal-based medium was comparable to that of total plate counts obtained on the modified MRS medium during preparation of nonfermented milk using various ratios of/~ infantis, L. acidob~hil~s and S. salivarius thermophilus. On the other hand, no or low number of blue colonies was detected after fermentation of milk. The enumeration of bifidobacterial and non-bifidobacterial colonies using X-~-Gal is based on the assumption tha'.." these microorganisms can grow on MRS agar even though they might have stressed by adversed conditions such as low pH. Moreover, MRS medium contmning c)steine-HC! is considered as an optimal growth medium for transfers of Bifidobacterium spp. strains of various origin [15]. However, the toler- ance of bifidobacteria toward organic acids is very low as compared to Lactobacillus and Streptococcus strains [16]. Modler et al. [4] noted that bifidobacteria would not be expected to survive storage for any length cf time in acidified dairy products. In addition, the simultaneous enumeration of low number of bifidobacteria in acidified milk could be affected by higher numbers of L. acidophilus and S. salivarites ther- mophilus.

Fig. ! shows .L_ _. . uie distinction b ~.tween bifidobacteria (blue colonies) and lactic acid bacteria (light blue colonies) on the same plate, using X-c~-Gal-based medimn. This method seems superior to a se!ective medium supplemen:.ed with inhibitors, For in- stance, the methods using l;:nium chloride (MGA medium) [5] or antibiotics [6] re- quire ~hat two plate couats must be done: viable counts of bifidobacteria in mixed cultures r ~ust be determi_ned by substrac, ing the colony counts on MGA plates (bi- fidobacteria alone) from the total number of colony counts on another medium (bi- fidobacteria plus lactobacilli).

To our knowledge, the differentiation by a single nonselective mediut~i allowing the growth of both bifidobacteria and lactic acid bacteria has not been reported in the literature. Wijsman et aL [7] used a TOS (transgalactosylated oligo-sacch~rides) agar medium supplemented with inhibitors to enumerate selectively the bifidobacteria. The TOS were described as tri-, tetra-, penta- and hexasaccharides but the exact composi- tion was not given; ho~ wer, TOS could be either o~- or fl-glycosides [17-18], suggesting that hydrolysis by o~-galactosidase would be required. However, Wijsman et al. [7] indi- cated that, due to the relatively i~mhed availability of TOS, other carbohydrate sources must be used. The X-ot-Gal-based medium developed in trUS study allows the distinc- tion of bffidobacteria without the presence of inhibitors or specific sugars since their detection is based on the presence of high a-galactosidase activity.

The addition of X-o~-Ga! to this commercial culture medium also gave the possibility of easily differentiating the genus BoTdobacterium spp. After :~- ,..,~ p~tdneubation

8i

Fig. 1. Use of X-t~-Gal-b~sed medium to differentiate bifidobacteria and lactic acid bacteria. Blue colo- nies: B. infantis; nonblue colonies: L. acidophilus

time, blue and light blue colonies were picked up for further growth or charact ~rization of the organism. Subsequently, the determination of F6PPK was performed using per- meabil[zed cells. 1hble 5 indicates that the permeabilization of Bifidobacterium strains with the anionic detergent Triton X-leo was as reliable as sonification which was pro- posed by Scardovi [19]. Using the permeabi!izauon p~o~.~dul,., k .zs possible to detect the presence of F6PPK f, om ci ttde extract obtained ahcr cuit~vation of blue colonies in modified MRS medium (data not show~). The permcabilization with Triton X-le0 is a simple and useful tool for assigning an organism to the genus Bifidobacterium by the F6PPK test.

The development of a methodology for the in situ colorimetric detection of a- galactosida~c excreted by colonies of bifidobacteria u,~ agar plates was useful in

82

TAR|.F 5

COMPARISON OF F6PPK ACTIVITY OF CRUDE EXTRACTS OBTAINED AFTER SONIFICA- TION OR PERMEABILIZAFION PROCEDURE WITH TRITON X-100

Colour intensity a after

Sonificadon Permeabilization with Triton X-100

B. adolescentis 5 5 B. bifidum 5 5 B. brere 4 5 B. mfantis 2 5 B. longum 4 5

a Determination of F6PPK was expressed on a scale of G (no colour developed) to 5 (maximum colour intensity).

differeatial enumeration, on the same plate, despite the delay required for the colour Qeveiopment and the limitation of the maximum number of colop,.'es allowed on a plate. In addition, by streaking mixed cultures of bacteria iso!ated from dairy products, a simple presumptive identification of bifidobacteria at the genus level should be possible.

Acknowledgements

The author wish to thank Pierre Ward, Lison Blanchette, Manon Proulx and Jean Gagn~ for their stimulating discussions on this subject.

References

! Mitsuoka, 1". (1984) Taxonomy and ecology of bifidobaeteria. Bifidobacteria Microflora 3, 11- 28. 2 Marteau, P., Pochart, P., Flouri~, B., Pellier, P., Sanots, L., Desjeux, J. E and Rambaud, J.C. (1990)

Effect of chronic ingestion of a fermented dairy product containing Lactobaciilus acidophilus and Bi. fidobacterium bifidum on metabolic activities of the colonic flora in humans. Am. J. Clin. Nutr. 52, 685 - 688.

3 Driessen, F. M. and De Boer, R. (1989) Fermented mi~ks with selected intestinal bacteria: a healthy trend in new lsroducts Neth. Milk Dairy J. 43, 367-3~.2.

4 Modler, H.W., McKellar, R.C. and Yaguchi, M. (1990) Bifidobacteria and bifidogeaic factors. Can. inst. Food Sci, Technol. J. 23, 29-41.

5 Cheng, C.-C. and Nagasawa, i. 0983) Associative Relationship'o betwect~ bifidobacteria a,~d lactobacil- li in milk. Jp~ J. Zoetech. gci. 54, 740-747.

6 Munoa, F. J. aad Pares, R. (t988) Selective: medium for isolation and enumeration of Bifidobacterium sp. Appl. Environ. Microbiol. 54, 1715-1718.

7 Wijsman, M. P , Hereijgens, J. L. P. M. and De Groote, J. M. E H. (1989) Selective enumeration of bi- fiA,bacteria in fermented dairy products. Neth. Milk Dairy J. 43, 395-405.

8 Rasic, J. L. and Kurmann, J.A. 0983) ~ifidobacteria and Their Role, pp. 166-189, ~rkauser Verlag, t~asei, Switzer!a,~d.

9 De~jardins, M. L,, Roy, D. and Goulet, J. (1990) Growth of bifidobacteria and their enzyme profiles. .I. Dairy S¢; 13, 299-3G7.

o ~ O..~

10 Chevalier, E, Roy, D. and Ward, P. (1990) Dettction of Bifidobacterium species by enzymatic methods. J. Appl. Bacterio!. 68, 619-624.

11 Tubb R.S. and Liljestrom, P. L. (1986) A co" ony-.~olour metho ! which differentiates o~-galactosidase- positive strains of yeasts. J. Inst. Brew. Lol don 92. 588-590.

12 Roy, D., Dussault, E and Ward, P. (1990) Gro~'ta requirements of Bifidobacterium strains in milk. Milchwissenschaft 45, 500-502.

13 Salmon, J.M. (1984) Application of the technique of cellular permeabilization te the ::udy of the en- zymatic activities of Saccharomyce~ cerevisiae in continuous alcoholic fermentation. Biotechnol. Lett. 6, 43-48.

14 "" ~ 7 c~ . ~ ~- . . . . , j. ,-- -'-~,-- Lojd,~, ~., o,~by, and r.ohns~a, j. (i973) Synthetic subs~rates in ~he histochemieal demor, stration of intestinal disaccharidases. Histochemie 34, 361-369.

! 5 Sykes, G. and Skinner, E A. (1973) Techniques for the isolation and characterization of Actinomyces and Bifidobacteriun': species: report of a panel discussion. In: Appl. Bacteriol. Syrup. Ser. No. 2, Aca- demic Press, New York, pp. 327-333.

16 Desjardins, M. L., Roy, D., Toupin, C. and Goulet, J. (1990) Uncoupling of growth and acids production in Bifidobacterium spp. J. Dairy Sci. 73, 1478-1484.

17 Mitsutomi0 M. and Oktakara, A. (1988) Isolation and identification of oligosaccharides produced from raffinose by transgalactosylation reaction of thermostable a-galactosidase from Pycnoporus cinnabarinus. Agric. Biol. Chem. 52, 2305-2311.

18 Deya, E., Amoya, M., Nc~jiri, K. and lgaroshi, S. (1982) Studies on application of galactozyl tactcse for infant formula. I. Preparation of galactosyl lactose by/~-galactosidase. In: Rep. Res. Lab. No. 78, pp. 19-26, Technical Research I~dtute, Snow Brand Milk Products, Japan.

19 Scardovi, V. (1981) The genus Eiftdobacterium. In: The Procaryotes, a Handbook on Habitats, Isola- tion, and Identification of 13,acteria (Etarr, M.P., Stolip, H., Truper, H.G., Balows, A. and Schlegel. H.G., eds.), pp. 1951-196I.