Method for the differential enumeration of lactic acid bacteria in a mixture in a food product

Abstract

The invention relates to a method for distinguishing between and enumerating strains of lactic acid bacteria or Bifidobacteria present in a food product. This method implements various agar culture media and/or selective culture conditions, combined with various chromogenic substrates.

Claims

1. A method for distinguishing from one another, and counting, strains of lactic acid bacteria or Bifidobacteria in a known mixture, which are present in different population amounts in a dairy product, wherein the diary product to be tested contains at least one strain of Lactobacillus paracasei subsp. paracasei, at least one strain of Lactobacillus delbrueckii subsp. bulgaricus, and at least one strain of Streptococcus thermophilus, wherein the method comprises: (a) inoculating aliquots of said food product in a series of culture dishes each containing a chemically defined agar M1 medium with the following composition: agar 15 g/l, tryptone 2.5 g/l, pepsin-digested meat peptone 2.5 g/l, papain-digested soya peptone 5 g/l, sodium glycerophosphate 19 g/l, lactose 5 g/l, yeast extract 2.5 g/l, meat extract 5 g/l, magnesium sulfate 0.25 g/l, ascorbic acid 0.5 g/l; and at least two chromogenic substrates: 6-chloro-3-indoxyl-?-D-galactopyranoside (salmon Gal) 0.2 g/l and 5-bromo-4-chloro-3-indolyl-?-D-glucopyranoside (X-Glu) 0.1 g/l producing different colorations, each of said substrates being taken up by at least one of said bacterial strains, and not being taken up by at least one other of said strains; and (b) optionally, inoculating aliquots of said food product in a series of culture dishes containing a chemically defined agar M2 medium enabling the growth of the lactic acid bacterial strains present in the product to be tested, which strains cannot grow on the M1 medium, and at least one chromogenic substrate taken up by at least one of said bacterial strains; and c) incubating said dishes for the time necessary to form bacterial colonies, and counting the colonies for each of the colorations observed in each culture dish.

2. The method according to claim 1, wherein the agar medium of a portion of the culture dishes also comprises at least one additive selectively promoting the growth of at least one of said bacterial strains, and/or the agar medium of a portion of the culture dishes also comprises at least one additive selectively inhibiting the growth of at least one of said bacterial strains.

3. The method according to claim 2, wherein a portion of said culture dishes is incubated under selective conditions favorable to the growth of at least one of said bacterial strains, and another portion of the culture dishes is incubated under different selective conditions favorable to the growth of at least one other of said bacterial strains.

4. The method according to claim 2, wherein at least two of the bacterial strains present in the product to be tested belong to the same species and subspecies.

5. The method according to claim 1, wherein a portion of said culture dishes is incubated under selective conditions favorable to the growth of at least one of said bacterial strains, and another portion of the culture dishes is incubated under different selective conditions favorable to the growth of at least one other of said bacterial strains.

6. The method according to claim 5, wherein at least two of the bacterial strains present in the product to be tested belong to the same species and subspecies.

7. The method according to claim 1, wherein at least two of the bacterial strains present in the product to be tested belong to the same species and subspecies.

8. The method according to claim 1, wherein the product to be tested also contains at least one strain of Lactobacillus rhamnosus.

9. The method according to claim 8, wherein to count the bacteria of the species Lactobacillus delbrueckii bulgaricus, a M2 medium with the following composition is used: polypeptone: 10 g/l; yeast extract: 5 g/l; meat extract: 10 g/l; dipotassium phosphate: 2 g/l; sodium acetate: 5 g/l; ammonium citrate: 2 g/l; magnesium sulfate: 0.2 g/l; manganese sulfate: 0.05 g/l; agar: 15 g/l; 5-bromo-4-chloro-3-indolyl-?-D-galactopyranoside (X-Gal): 0.15 g/l.

10. The method according to claim 8, wherein to count the bacteria of the species Lactobacillus paracasei subsp. paracasei, Lactobacillus rhamnosus, and Streptococcus thermophilus, a first portion of the dishes containing the M1 medium does not comprise any additive, a second portion of said dishes comprises vancomycin as an additive, and a third portion of said dishes comprises rhamnose as an additive.

11. The method according to claim 8, wherein to count the bacteria of the species Lactobacillus paracasei subsp. paracasei, Lactobacillus rhamnosus, and Streptococcus thermophilus, the inoculated dishes are incubated for approximately 48 hours under a controlled atmosphere containing from 6 to 16% O.sub.2 and from 2 to 10% CO.sub.2, a portion of said dishes being incubated at approximately 37? C., and another portion at approximately 44? C.

12. The method according to claim 8, wherein to count the bacteria of the species Lactobacillus delbrueckii bulgaricus, the inoculated dishes are incubated for approximately 48 hours at approximately 47? C. under a controlled atmosphere containing less than 1% O.sub.2 and at least 13% CO.sub.2.

Description

EXAMPLE DIFFERENTIAL COUNTING OF LACTIC ACID BACTERIA IN FERMENTED DAIRY PRODUCTS

(1) Materials and Methods:

(2) Composition of the Products Tested

(3) Product 1:

(4) Product 1 is a fermented product containing 5 bacterial strains belonging to three species, namely: 1 probiotic strain of L. casei subsp. paracasei (hereinafter referred to as Lactobacillus paracasei strain 1); 3 technological strains of S. thermophilus; 1 technological strain of L. delbrueckii subsp. Bulgaricus.

(5) The theoretical bacterial loads expected at the start of the self life (D4) and at the end of the, self life (D35) of the product are indicated in table I below.

(6) TABLE-US-00001 TABLE I Theoretical bacterial load (in bacteria/ml) D4 D35 Lactobacillus paracasei strain 1 2 ? 10.sup.8 2 ? 10.sup.8 3 Streptococcus thermophilus >10.sup.8 >10.sup.8 Lactobacillus bulgaricus 1 ? 10.sup.7 10.sup.1 to 10.sup.4
Product 2:

(7) Product 2 is a fermented product containing 7 bacterial strains belonging to three species, namely: 2 probiotic strains of L. casei subsp. paracasei (Lactobacillus paracasei strain 1 and Lactobacillus paracasei strain 2); 1 probiotic strain of L. rhamnosus; 3 technological strains of S. thermophilus; 1 technological strain of L. delbrueckii subsp. Bulgaricus.

(8) The theoretical bacterial loads expected at the beginning of the shelf life (D4) and at the end of the shelf life (D35) of the product are indicated in table II below.

(9) TABLE-US-00002 TABLE II Theoretical bacterial load (in bacteria/ml) D4 D35 Lactobacillus paracasei 2 to 3 ? 10.sup.8 2 to 3 ? 10.sup.8 strain 1 and strain 2 Lactobacillus rhamnosus 1 ? 10.sup.8 l ? 10.sup.8 3 Streptococcus thermophilus 1 ? 10.sup.8 l ? 10.sup.8 Lactobacillus bulgaricus 1 ? 10.sup.7 10.sup.1 to 10.sup.4
Enzymatic Activities of the Strains:

(10) The different strains were tested for their ?-glucosidase and ?-galactosidase activities.

(11) The results are indicated in table III below.

(12) TABLE-US-00003 TABLE III Strain ?-Glucosidase ?-Galactosidase Lactobacillus rhamnosus + + Lactobacillus paracasei 1 + ? Lactobacillus paracasei 2 ? ? Streptococcus thermophilus 1 ? + Streptococcus thermophilus 2 ? + Streptococcus thermophilus 3 ? + Lactobacillus bulgaricus ? +
Bacteria Counting in Products 1 and 2:
Culture Conditions and Media:

(13) The culture conditions and media currently used for counting bacterial strains present in products 1 and 2 are summarized in tables IV and V, respectively, below. These reference culture conditions and media were used as controls.

(14) TABLE-US-00004 TABLE IV Strain studied Medium Temperature Duration Atmosphere Lactobacillus MRS + 37? C. 48 hours CO.sub.2 paracasei 1 vancomycin (1 ?g/ml) Streptococcus sp. M17 44? C. 48 hours Aerobic Lactobacillus Acid MRS 50? C. 48 hours Anaerobic bulgaricus

(15) TABLE-US-00005 TABLE V Strain studied Medium Temperature Duration Atmosphere Lactobacillus MRS + 44? C. 48 hours CO.sub.2 rhamnosus vancomycin (1 ?g/ml) Lactobacillus MRS + 37? C. 48 hours CO.sub.2 rhamnosus + vancomycin Lactobacillus (1 ?g/ml) paracasei 1 + Lactobacillus paracasei 2 Streptococcus sp. M17 44? C. 48 hours Aerobic Lactobacillus Acid MRS 50? C. 48 hours Anaerobic bulgaricus

(16) The MRS, acid MRS and M17 media are conventional commercially available media (AES CHEMUNEX).

(17) For the cultures under a controlled atmosphere, GASPAK? gas generating systems were used to obtain the following conditions: anaerobic conditions: % O.sub.2<1%; % CO.sub.2?13% CO.sub.2: % CO.sub.2>2.5% microaerobic conditions: 6%<% O.sub.2<16%; 2%<% CO.sub.2<10% aerobic conditions: ambient air

(18) Two chromogenic media, hereinafter referred to as M1 and M2, were prepared: the composition of these media is indicated in tables VI and VII below, respectively:

(19) TABLE-US-00006 TABLE VI M1 Medium Concentration Constituent (in g/l) Agar 15 Tryptone 2.5 Pepsin-digested meat peptone 2.5 Papain-digested soya peptone 5 Sodium glycerophosphate 19 Lactose 5 Yeast extract 2.5 Meat extract 5 Magnesium sulfate 0.25 Ascorbic acid 0.5 6-Chloro-3-indoxyl-?-D-galactopyranoside 0.2 (salmon Gal) 5-Bromo-4-chloro-3-indolyl-?-D-glucopyranoside 0.1 (X-Glu)

(20) TABLE-US-00007 TABLE VII M2 Medium Concentration Constituent (in g/l) Polypeptone 10 Yeast extract 5 Meat extract 10 Dipotassium phosphate 2 Sodium acetate 5 Ammonium citrate 2 Magnesium sulfate 0.2 Manganese sulfate 0.05 Agar 15 5-Bromo-4-chloro-3-indolyl-?-D-galactopyranoside 0.15 (X-Gal)

(21) The tests were carried out with 3 different batches of each of the M1 and M2 media.

(22) The culture conditions used for products 1 and 2 are respectively indicated in tables VIII and IX below:

(23) TABLE-US-00008 TABLE VIII Strain studied Medium Temperature Duration Atmosphere Lactobacillus M1 37? C. 48 hours Microaerobic paracasei 1 + Streptococcus sp. Lactobacillus M2 47? C. 48 hours Anaerobic bulgaricus

(24) TABLE-US-00009 TABLE IX Strain studied Medium Temperature Duration Atmosphere Lactobacillus M1 + 37? C. 48 hours Microaerobic paracasei 1 rhamnose (10 g/l) Lactobacillus M1 + 37? C. 48 hours Microaerobic paracasei 2 vancomycin (1 ?g/ml) Lactobacillus M1 44? C. 48 hours Microaerobic rhamnosus + Streptococcus sp. Lactobacillus M2 47? C. 48 hours Anaerobic bulgaricus
Operating Protocol:

(25) Samples were taken at two self life phases of the products: start of life and end of life.

(26) For product 1, start of self life samples were taken at 4, 5 and 6 days after the manufacture of the fermented product, and end of self life samples were taken at 31, 32 and 33 days after the manufacture; for product 2, start of self life samples were taken at 7, 8 and 9 days after the manufacture, and end of self life samples were taken at 28, 29 and 30 days after the manufacture.

(27) For each test specimen, a range of dilutions to 1/10 was carried out in tryptone salt tubes. Three dilutions, chosen as a function of the theoretical populations (cf. Tables I and II), were inoculated on each of the media used. For each dilution, 1 ml of diluted product was inoculated in 1 Petri dish, and 15 ml of the medium used were poured into the dish.

(28) The dishes were incubated under the conditions, and for the durations, indicated in tables IV, V, VIII and IX.

(29) Results:

(30) Identification of the Strains:

(31) On M1 medium: at 44? C., the Lactobacillus rhamnosus strain produces blue-colored colonies, and the Streptococcus thermophilus strains produce magenta-colored colonies; the Lactobacillus paracasei strains 1 and 2 and the Lactobacillus bulgaricus strain do not grow; at 37? C. with addition of rhamnose, the Lactobacillus rhamnosus strain and the Lactobacillus paracasei strain 2 produce colorless colonies; the Lactobacillus paracasei strain 1 produces turquoise-colored colonies and the Streptococcus thermophilus strains produce magenta-colored colonies. The Lactobacillus bulgaricus strain does not grow; at 37? C. with addition of vancomycin, the Lactobacillus rhamnosus strain produces blue colonies; the Lactobacillus paracasei strain 1 produces turquoise-colored colonies and the Lactobacillus paracasei strain 2 produces colorless colonies; the Streptococcus thermophilus strains and the Lactobacillus bulgaricus strain do not grow.

(32) On M2 medium: the Lactobacillus rhamnosus strain produces colorless colonies, and the Lactobacillus bulgaricus strain produces blue colonies. The Streptococcus thermophilus strains and the Lactobacillus paracasei strains 1 and 2 do not grow.

(33) These results are summarized in table X below:

(34) TABLE-US-00010 TABLE X M1 Medium Addition of rhamnose and Addition of Incubation incubation vancomycin and Name of strain at 44? C. at 37? C. incubation at 37? C. M2 Medium Lactobacillus Blue Colorless Blue Colorless rhamnosus Lactobacillus Absence of Turquoise Turquoise Absence of paracasei 1 growth growth Lactobacillus Absence of Colorless Colorless Absence of paracasei 2 growth growth Streptococcus Magenta Magenta Absence of Absence of thermophilus (strains growth growth 1, 2, and 3) Lactobacillus Absence of Absence of Absence of Blue bulgaricus growth growth growth Strain(s) identified Lactobacillus Lactobacillus Lactobacillus Lactobacillus rhamnosus and paracasei 1 paracasei 2 bulgaricus Streptococcus thermophilus
Product 1:

(35) M1 Medium therefore makes it possible to count Lactobacillus paracasei and Streptococcus thermophilus, and M2 medium makes it possible to count Lactobacillus bulgaricus.

(36) Product 2:

(37) M1 Medium therefore makes it possible to count: at 44? C.: Lactobacillus rhamnosus and Streptococcus thermophilus with addition of rhamnose at 37? C.: Lactobacillus paracasei strain 1; with addition of vancomycin at 37? C.: Lactobacillus paracasei strain 2.

(38) M2 Medium makes it possible to count Lactobacillus bulgaricus.

(39) Bacteria Counting Bacteria:

(40) Product 1:

(41) Early in Self Life:

(42) The results are illustrated in table XI.

(43) TABLE-US-00011 TABLE XI Reference medium Species (control) Batch 1 Batch 2 Batch 3 L. paracasei 1 Bacterial load 5.4 ? 10.sup.8 6.1 ? 10.sup.8 5.7 ? 10.sup.8 6.2 ? 10.sup.8 Difference vs. control (log) +0.06 +0.03 +0.07 S. thermophilus Bacterial load 6.4 ? 10.sup.8 5.3 ? 10.sup.8 6.8 ? 10.sup.8 5.5 ? 10.sup.8 Difference vs. control (log) ?0.08 +0.03 ?0.07 L. bulgaricus Bacterial load 1.6 ? 10.sup.6* 3 ? 10.sup.6 3 ? 10.sup.6 2.8 ? 10.sup.6 Difference vs. control (log) +0.27 +0.26 0.23

(44) The variability of the counting method is as follows: On M1 medium: between 5.7?10.sup.8 CFU/ml and 6.2?10.sup.8 CFU/ml for L. paracasei 1, between 5.3?10.sup.8 CFU/ml and 6.8?10.sup.8 CFU/ml for S. thermophilus, On M2 medium: between 2.8?10.sup.6 CFU/ml and 3?10.sup.6 CFU/ml for L. bulgaricus.

(45) Difficulties in counting L. bulgaricus on the reference medium, acid MRS, are to be noted: day 5: difference of one log versus medium M2 and the count from D4, day 6: absence of growth.
End of Self Life:

(46) The results are illustrated in table XII.

(47) TABLE-US-00012 TABLE XII Reference medium Species (control) Batch 1 Batch 2 Batch 3 L. paracasei 1 Bacterial load 4.5 ? 10.sup.8 4.1 ? 10.sup.8 4 ? 10.sup.8 4.5 ? 10.sup.8 Difference vs. control (log) ?0.04 ?0.05 ?0.01 S. thermophilus Bacterial load 1.8 ? 10.sup.8 1.9 ? 10.sup.8 1.9 ? 10.sup.8 2.2 ? 10.sup.8 Difference vs. control (log) +0.02 +0.02 +0.08 L. bulgaricus Bacterial load Absence Absence Absence Absence Difference vs. control (log) N.A. N.A. N.A.

(48) M1 Medium makes it possible to effectively discriminate between Lactobacillus paracasei and Streptococcus thermophilus present in the product. The counts for L. paracasei vary between 4?10.sup.8 CFU/ml and 4.5?10.sup.8 CFU/ml over the three batches and over three days of analysis. The load of S. thermophilus varies between 1.9?10.sup.8 CFU/ml and 2.2?10.sup.8 CFU/ml.

(49) These tests also demonstrate the absence of L. bulgaricus in the product at this lifetime stage, whether on the reference medium or on M2 medium.

(50) Product 2:

(51) Early in Self Life:

(52) The results are illustrated in table XIII.

(53) TABLE-US-00013 TABLE XIII Reference medium Species (control) Batch 1 Batch 2 Batch 3 L. paracasei L. paracasei 1 bacterial load 2 ? 10.sup.8 1.6 ? 10.sup.8 1.8 ? 10.sup.8 1.7 ? 10.sup.8 1 and 2 L. paracasei 2 bacterial load 2.3 ? 10.sup.7 3 ? 10.sup.7 2.8 ? 10.sup.7 Difference vs. control (log) ?0.02 +0.03 0.00 S. thermophilus Bacterial load 8.9 ? 10.sup.8 8.7 ? 10.sup.8 9.1 ? 10.sup.8 9.8 ? 10.sup.8 Difference vs. control (log) ?0.01 +0.01 +0.04 L. rhamnosus Bacterial load 2.3 ? 10.sup.8 2.3 ? 10.sup.8 2.3 ? 10.sup.8 2.3 ? 10.sup.8 Difference vs. control (log) 0.00 0.00 0.00 L. bulgaricus Bacterial load 2.5 ? 10.sup.5 4.6 ? 10.sup.5 2.6 ? 10.sup.5 3.1 ? 10.sup.5 Difference vs. control (log) +0.27 +0.03 0.10

(54) The variability of the counting method is as follows: On M1 medium: between 1.6?10.sup.8 CFU/ml and 1.8?10.sup.8 CFU/ml for L. paracasei 1, between 2.3?10.sup.7 CFU/ml and 3?10.sup.7 CFU/ml for L. paracasei 2, between 8.7?10.sup.8 CFU/ml and 9.8?10.sup.8 CFU/ml for S. thermophilus, 2.3?10.sup.8 CFU/ml for the three batches for L. rhamnosus On M2 medium: between 2.6?10.sup.5 CFU/ml and 5?10.sup.5 CFU/ml for L. bulgaricus.
End of Self Life:

(55) The results are illustrated in table XIV.

(56) TABLE-US-00014 TABLE XIV Reference medium Species (control) Batch 1 Batch 2 Batch 3 L. paracasei L. paracasei 1 bacterial load 2.1 ? 10.sup.8 1.6 ? 10.sup.8 1.5 ? 10.sup.8 1.6 ? 10.sup.8 1 and 2 L. paracasei 2 bacterial load 1.4 ? 10.sup.7 2.2 ? 10.sup.7 2.2 ? 10.sup.7 Difference vs. control (log) ?0.07 ?0.08 ?0.05 S. thermophilus Bacterial load 7.3 ? 10.sup.8 6.8 ? 10.sup.8 6.6 ? 10.sup.8 7.5 ? 10.sup.8 Difference vs. control (log) ?0.03 ?0.04 +0.01 L. rhamnosus Bacterial load 1.8 ? 10.sup.8 2.0 ? 10.sup.8 2.0 ? 10.sup.8 2.0 ? 10.sup.8 Difference vs. control (log) +0.03 +0.04 +0.04 L. bulgaricus Bacterial load Absence Absence Absence Absence Difference vs. control (log) N.A. N.A. N.A.

(57) The counts over the three batches of M1 medium vary between 1.5?10.sup.8 CFU/ml and 1.6?10.sup.8 CFU/ml for L. paracasei 1, between 1.4?10.sup.7 CFU/ml and 2.2?10.sup.7 CFU/ml for L. paracasei 2, between 6.6?10.sup.8 CFU/ml and 7.5?10.sup.8 CFU/ml for S. thermophilus, 2.0?10.sup.8 CFU/ml for the three batches for L. rhamnosus.

(58) The tests also demonstrate the absence of L. bulgaricus in the product at this lifetime stage, whether on the reference medium or on M2 medium.

(59) Use of the method described above makes it possible to discriminate between the three bacterial species present in product 1 and the four present in product 2.