Synergistic fermentation of Lactobacillus rhamnosus and Lactobacillus paracasei subsp paracasei

Abstract

The present invention relates to a process for preparing a fermented product by synergistic co-fermentation of Lactobacillus rhamnosus and Lactobacillus paracasei subsp. paracasei.

Claims

1. A process of preparing a fermented dairy product comprising two Lactobacillus strains, wherein said process comprises: (a) inoculating a dairy fermentation medium with Lactobacillus paracasei subsp. paracasei deposited with the Collection Nationale de Cultures de Micro-organismes (CNCM) under accession number 1-1518 and Lactobacillus rhamnosus deposited with the CNCM under the accession number 1-3690; (b) fermenting the inoculated medium by incubating it at a temperature suitable for growth of said strains, until it reaches a desired target pH or until the global Lactobacillus population reaches a desired target value; and (c) stopping the fermentation and recovering the fermented product wherein the fermentation is stopped when the global Lactobacillus population is of at least 10 times the population inoculated.

2. The process of claim 1, wherein the Lactobacillus strains are inoculated in the dairy fermentation medium in a global quantity of at least 10.sup.6 CFU/ml.

3. The process of claim 1, wherein each individual strain is at least 1% of the global population of inoculated Lactobacillus.

4. The process of claim 1, wherein the fermentation is conducted at a temperature of from 30 C. to 42 C.

5. The process of claim 1, wherein the fermentation is stopped when a target pH of from 3.7 to 4.2 is reached.

6. The process of claim 1, wherein the fermentation is stopped when the global Lactobacillus population is of at least 10.sup.8 CFU/ml.

7. The process of claim 1, wherein the global Lactobacillus population in the recovered fermented product is of from 10.sup.6 to 10.sup.9 CFU/ml.

8. The process of claim 1, wherein the fermented dairy product further comprises Lactobacillus paracasei subsp. paracasei deposited with the CNCM under the accession number 1-3689.

9. The process of claim 1, wherein the fermentation is stopped when the global Lactobacillus population is of at least 100 times the population inoculated.

Description

EXAMPLE 1. SYNERGISTIC GROWTH OF TWO OR THREE LACTOBACILLUS STRAINS

(1) Material and Methods:

(2) 1) Strains:

(3) The following strains were studied:

(4) Lactobacillus rhamnosus strains:

(5) Rhamno A

(6) Rhamno B

(7) HN001

(8) CNCM I-3690

(9) Lactobacillus paracasei subsp. paracasei strains

(10) Paracasei A

(11) Paracasei B

(12) CNCM I-1518

(13) CNCM I-3689

(14) HN001, CNCM I-3690, CNCM I-1518 and CNCM I-3689 are known probiotic strains, while Rhamno A, Rhamno B, Paracasei A and Paracasei B are ordinary strains chosen at random.

(15) 2) Cryotubes:

(16) The strains were conserved in cryotubes (2 ml) which were previously made up according to the protocol below.

(17) Strain culture was performed with Man Rogosa Sharpe (MRS) broth and incubated 16 h at 37 C.; sterilized DMSO (5.3%) has been added to improve the strain stability during the storage phase. The cryotube was frozen with nitrogen steam during 30 nm. The strain purity was checked with API gallery 50CH+methylen blue. The cryotubes were stored at 80 C.

(18) 3) Subculture with Growth Medium:

(19) Each strain was cultured individually. The cryotubes were defrosted under sterile conditions and 1 ml (1%) of culture was added to 100 ml of sterile MRS broth used as growth medium, and incubated at 37 C. until the culture reaches the stationary phase (pH around 4.00). The subculture was centrifuged at 5000 rpm during 15 nm at 4 C. Supernatant was replaced by sterile tryptone salt.

(20) 4) Fermentation in Dairy Medium:

(21) The dairy medium contains skimmed milk powder (120 g) and distilled water (930 g). After 1 h rehydratation, the mix was sterilized by autoclaving for 15 nm at 115 C.

(22) The bacteria from the subcultures were inoculated at a global bacterial population of 210.sup.7 cell/ml in 200 ml of dairy medium. When 2 strains were used, each strain represented of the global bacterial population; when 3 strains were used, each strain represented of the bacterial population. Fermentation was conducted at 37 C., and monitored by measuring the decrease of pH in the culture medium. Fermentation was stopped when a target pH of 3.80 was reached.

(23) The cell count analysis is performed by serial dilutions of the fermented medium with tryptone salt solution, and plating on Petri dishes with MRS agar. Petri dishes are incubated at 37 C. during 72 H in anaerobic conditions.

(24) Results:

(25) The results are shown in Tables I and II and FIGS. 1 to 3

(26) Table I shows the total bacterial count when target of pH 3.80 is reached.

(27) Table II shows the decrease in fermentation time needed to reach the target pH of 3.80 (average of 2 independent experiments). The decrease is calculated by the difference between the time necessary to reach pH=3.80 for the strain which shows the fastest fermentation rate when cultivated alone, and the time to reach pH=3.80 for the strain combination. We consider that a synergistic effect is present when the fermentation time reduction with combination is of at least 1%, preferably with at least the same minimum level of population.

(28) TABLE-US-00001 TABLE I N.sup.o of Total count combination Strain combination CFU/g 1 CNCM I-1518 + CNCM I-3689 3 10e9 2 CNCM I-1518 + Paracasei B 3 10e9 3 CNCM I-1518 + Rhamno A 2.8 10e9 4 CNCM I-1518 + HN001 3 10e9 5 CNCM I-3689 + CNCM I-3690 3.1 10e9 6 CNCM I-1518 + Paracasei A 4 10e9 7 CNCM I-1518 + CNCM I-3690 3.1 10e9 8 CNCM I-1518 + CNCM I-3689 + Rhamno 4 10e9 B 9 CNCM I-1518 + CNCM I-3689 + Rhamno 2.5 10e9 A 10 CNCM I-1518 + CNCM I-3689 + HN001 1.4 10e9 11 CNCM I-1518 + CNCM I-3690 + CNCM 3 10e9 I-3689 12 CNCM I-1518 + Paracasei B + CNCM 2.75 10e9 I-3690 13 CNCM I-1518 + Paracasei A + CNCM 3.2 10e9 I-3690

(29) Table I, shows total count results. The target count (same as for the fermentation of each individual strain=at least 110e9 CFU/g) is obtained for each combination tested in Table II.

(30) TABLE-US-00002 TABLE II Decrease in fermentation time (average of 2 Decrease in N.sup.o of independent fermentation combination Strain combination experiments) time (%) 1 CNCM I-1518 + CNCM 1 hr 1% I-3689 2 CNCM I-1518 + Paracasei B 1 hr 1% 3 CNCM I-1518 + Rhamno A 4 hrs 10% 4 CNCM I-1518 + HN001 6 hrs 13% 5 CNCM I-3689 + CNCM 6 hrs 12% I-3690 6 CNCM I-1518 + Paracasei A 7 hrs 15% 7 CNCM I-1518 + CNCM 10 hrs 19% I-3690 8 CNCM I-1518 + CNCM 4 hrs 12% I-3689 + Rhamno B 9 CNCM I-1518 + CNCM 6 hrs 15% I-3689 + Rhamno A 10 CNCM I-1518 + CNCM 7 hrs 16% I-3689 + HN001 11 CNCM I-1518 + CNCM 12 hrs 29% I-3690 + CNCM I-3689 12 CNCM I-1518 + Paracasei 14 hrs 28% B + CNCM I-3690 13 CNCM I-1518 + Paracasei 20 hrs 40% A + CNCM I-3690

(31) Table II clearly demonstrates that all the combinations of L. rhamnosus and/or L. casei give an unexpected synergistic increase in growth, reflected by a decrease in fermentation time of at least 1 hour. This synergistic effect is even stronger when three strains are co-fermented.

(32) FIG. 1 shows the fermentation curves under the same culture conditions, of the 2 individual strains CNCM I-3689 and CNCM I-3690 and of the 2 strains in co-culture (combination 5). The co-fermentation results in a decrease of 6 hours in the fermentation time.

(33) FIG. 2 shows the fermentation curves under the same culture conditions, of the 2 individual strains CNCM I-1518 and CNCM I-3690 and of the 2 strains in co-culture (combination 7). The co-fermentation results in a decrease of 10 hours in the fermentation time.

(34) FIG. 3 shows the fermentation curves under the same culture conditions, of the 3 individual strains CNCM I-1518, CNCM I-3689, and CNCM I-3690 and of the 3 strains in co-culture (combination 11). The co-fermentation results in a decrease of 12 hours in the fermentation time.

(35) These results confirm those presented in Table II, showing a synergistic effect of the co-fermentation of strains on the growth rate, and that the combination of three different strains is the most effective in shortening the fermentation time.