WO2008003810A1

WO2008003810A1 - Method for the differentiation and quantification of lactic bacteria and bifidobacteria in fermented milks, using selective antibiotic-free culture media

Info

Publication number: WO2008003810A1
Application number: PCT/ES2007/070123
Authority: WO
Inventors: Raquel Tabasco Rentero; Torsten Paarup; Carolina Janer Otero; Carmen PELÁEZ MARTÍNEZ; Teresa REQUENA ROLANÍA
Original assignee: Consejo Superior De Investigaciones Científicas
Priority date: 2006-07-03
Filing date: 2007-06-29
Publication date: 2008-01-10

Abstract

The invention relates to a method for the differentiation and quantification of lactic bacteria and bifidobacteria in fermented milks, using selective antibiotic-free culture media. The method can be used for the differential and selective quantification of four species of lactic bacteria (S. thermophilus, L. bulgaricus, L. casei, L. acidophilus) and B. lactis in mixed cultures present in fermented milks. The invention is novel and competitively advantageous in that it can reliably and simultaneously distinguish and quantify three different species of lactobacilli in relation to one another and other lactic bacteria, such as S. thermophilus, and bifidobacteria present in mixtures in commercial fermented milks. The specificity of the media and the different incubation conditions enable the colonies to be differentiated without the addition of antibiotics that can affect growth and, consequently, quantification. The method has been validated on the basis of specificity, reproducibility, recovery of the tested population and interspecies competition.

Description

TITLE:

PROCEDURE FOR DIFFERENTIATING AND QUANTIFY LACTIC BACTERIA AND BIFIDOBACTERIES IN FERMENTED MILKS THAT USES SELECTIVE CULTURE MEDIA FREE OF ANTIBIOTICS

SECTOR OF THE TECHNIQUE:

The technique is part of the Agrifood Area, in the Dairy Sector, and develops a procedure for the analysis of mixed populations of lactic bacteria and bifidobacteria in yogurts and fermented milks.

STATE OF THE TECHNIQUE:

The analysis of the microorganisms used in the manufacture of yogurts and fermented milks refers to both Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus, which are the only species accepted for the manufacture of yogurt [Quality Standard for yogurt or yogurt, RD 179/2003 of February 14; BOE of February 18, 2003, pp. 6448-6450], as well as to different probiotics that have been incorporated in the manufacture of new fermented milks and that mainly include bacteria of the Lactobacillus and Bifidobacterium genera. In both cases, yogurt and fermented milks, there are requirements that make it necessary to analyze the viability of the microorganisms in the product. Within the aforementioned Quality Standard for yogurt, L. bulgaricus and S. thermophilus are required to be viable in the finished product and present in a minimum quantity of 1 x 10 ⁷ colony forming units (ufe) per g mL. In the case of fermented milks that contain probiotic microorganisms, there is a generally accepted criterion that establishes that for these bacteria to exert a beneficial effect on the health of the consumer they must also be viable and in high populations, estimating a population recommended in the product in the moment of its consumption of at least 10 ⁶ cfu per go mL [Roy, D. (2001) Int. J. Food Microbiol. 69: 167-182].

For the quantification of yogurt bacteria, there is an International Standard ISO / IDF [ISO / FDIS 9232 | IDF146: 2002] that establishes conditions for the quantification of these microorganisms and is based on a colony count technique of S. thermophilus on M-17 agar [Terzaghi, BE and Sandine, WE (1975) Appl. Microbiol 29: 807-813], added with lactose, after incubation in aerobiosis at 37 ° C for 48 h. For L. bulgaricus, the colony count is established on MRS agar [de Man, J. C, Rogosa, M. and Sharpe, E. (1960) J. Appl. Bacterial 23: 130-135] with glucose and acidified to pH 5.4, after incubation in anaerobiosis at 37 ° C for 72 h. In relation to the quantification of other types of microorganisms present in fermented milks mixed with the yogurt microorganisms, there are no International Standards or standards, although there are working groups that currently carry out collaborative studies for the development of these official standards. In this sense, the International Dairy Federation (IDF / FIL) is working on the validation of a method of quantification of L. acidophilus [ISO / CD 20128 / IDF 192] but with the addition of clindamycin, and a proposal for Ia formation of a working group for the quantification of Bifidobacterium. In the same sense, there are different studies in the scientific literature that analyze the possible differentiation and quantification of the new species of bacteria added to yogurt, mainly L. acidophilus, Bifidobacterium spp. and L. casei [reviews of these studies: Charteris, WP, Nelly, PM, Morelli, L. and Collins, K (1997) Int. J. Food Microbiol. 35: 1-27; Roy, 2001]. In general, the means proposed for the differentiation of the three bacterial groups mentioned between each other and against the yogurt bacteria base their selectivity on the different growth conditions of the species under study and basically on the addition of different antibiotics (vancomycin, acid nalidixic, dicloxacillin, etc.) [Vinderola, CG and Reinheimer, JA (2000) 271-275; Bonaparte, C, Klein, G., Kneifel, W. and Reuter, G. (2001) Lait 81: 227-235; Tharmaraj, N. and Shah, NP (2003) J. Dairy Sci. 86: 2288-2296]. Recently, a study has been published comparing different selective and differential culture media described in the literature for the quantification in commercial yogurts of the same five species object of the present invention [Talwalkar, A. and Kailasapathy, K. (2004) Int Dairy J. 14: 143-149]. The study concludes that none of the selective means described in the literature It allows reliable quantification of these microorganisms when they are mixed in different combinations in the fermented milks analyzed. Additionally, the use of antibiotics in the culture media usually causes a certain degree of reduction in the counts obtained in the selected population [Roy, 2001], which can lead to the underestimation of said bacterial population and, where appropriate, It could compromise the assessment of the quality of the product tested and / or its potential probiotic benefit.

There are published works that describe the development of selective or differential means for the quantification of the cited species based only on the differentiation of the species under study for their ability to ferment the carbon source present in the culture medium, for example raffinose for Ia selection of Bifidobacterium [Hartemink, R., Kok, BJ, Weenk, GH and Rombouts, FM (1996) J. Microbiol. Meth. 27: 33-43] or use of selective incubation conditions p. ex. selection of L. casei by incubation at 15 ° C [Champagne, C. P., Roy, D. and Lafond, A. (1997) Biotechnol. Techniques 11: 567-569]. Although the means mentioned have proven effective, in both cases the species tested was alone in mixture with the microorganisms of yogurt or in other examples in mixtures only of thermophilic species of Lactobacillus [Camaschella, P., Mignot, O., Pirovano, F , Sozzi, T. (1998) Lait 78: 461-467].

In general, many of the works published in scientific journals that describe the quantification of lactobacilli and / or bifidobacteria in mixed cultures present in fermented milks lack the analysis of the different parameters that allow the validation of the methods developed and / or the necessary studies for the confirmation of the authenticity of the results obtained.

In relation to the existing international patents related to the subject matter of the present invention, the patents JP60114188 and JP92029359, published in 1983, and the patent JP11028098, published in 1997, developed by the company Yakult Honska KK, which are characterized by using galacto-oligosaccharides as a carbon source to select bifidobacteria when they are mixed with lactic bacteria. However, there is no patented method for the reliable discriminative detection of yogurt bacteria in admixture with probiotic lactobacilli and bifidobacteria, a situation usually prevalent in commercial fermented milks.

DESCRIPTION OF THE INVENTION: - Brief description of the invention

A procedure has been developed that allows the selective and differential quantification of four species of lactic bacteria (S. thermophilus, L. bulgaricus, L. casei, L. acidophilus) and B. lactis in mixed cultures present in fermented milks. The novelty of the procedure and what is advantageous with respect to the existing ones is that it can simultaneously distinguish three different species of lactobacilli from each other and against another lactic bacterium such as S. thermophilus and bifidobacteria, all based on the use of different incubation conditions and / or in the differentiation by morphology of the colonies, but without the addition of antibiotics to the culture media that could compromise the viability of the species under study. The validation of the procedure has been carried out based on the study of the selectivity of the media, the accuracy and precision to recover the population tested, the reproducibility of the procedure and the analysis of the possible competition between the species. The matrix effect has also been analyzed by means of the quantification of the populations in acidified milk in comparison with the reference culture media. The confirmation of the identity of the colonies obtained in the proposed means has been carried out according to the procedure described in the patent. Procedure for the detection and specific identification of lactic bacteria and bifidobacteria in fermented milks and in starter cultures for fermented milks. - Detailed description of the invention

The procedure of selective and differential quantification of bacterial species S. thermophilus, L. bulgaricus, L. casei, L. acidophilus and B. lactis in mixed cultures present in fermented milks is based on the use of selective growth characteristics and differences morphological of the colonies obtained under the proposed culture conditions. In no case are antibiotics used as selective growth agents. The basal culture media used were M-17 for S. thermophilus and MRS for fermentation for lactobacilli and B. lactis, described in the Official Standard for Analysis of Yogurt Bacteria [ISO / FDIS 9232 | IDF146: 2002]. Its commercial origin has been from Laboratorios Conda, Madrid.

Differential growth has been based on the selective use of carbon sources such as glucose, lactose, raffinose, maltose and fructose, at different temperatures and incubation times such as 30, 37 and 45 ° C for 24 and 72 hours and in the presence of oxygen: aerobiosis, partial atmosphere of CO ₂ , anaerobiosis.

The species have also been differentiated by the size and morphology of the colonies obtained in the media, only colonies of size equal to or greater than 2 mm being considered positive.

In this way, the quantification of S. thermophilus has been carried out by growth in M-17 lactose agar under incubation conditions in aerobiosis, for 24 h and at 45 ° C.

L. bulgaricus is quantified using MRS agar with fructose and incubation in anaerobiosis for 72 h at 45 ° C. The colonies obtained are lentil-shaped and larger than 2 mm in size, clearly differentiable from the rough colonies corresponding to L. acidophilus and the punctiform colonies of B. lactis.

For the quantification of L. acidophilus, MRS agar with maltose is used and incubation in an atmosphere of 20% CO ₂ for 72 h at 37 ° C. The formation of rough and translucent colonies larger than 2 mm in size differentiate this species from L. casei, characterized by the formation of round and white colonies.

The quantification of L. casei is carried out by growth in MRS agar with glucose and incubation in aerobiosis for 72 h at 30 ° C.

Finally, B. lactis is selected by the fermentation of raffinose present in MRS agar, the tolerance to LiCI and by the conditions of incubation in anaerobiosis for 72 h and at 45 ° C.

For the validation of the quantification procedure of each species, recommendations of the ISO / TR 13843: 2000 Standard have been followed and the following parameters have been determined:

Precision and Accuracy: The proposed culture methods have been evaluated against the optimal growth conditions of each species that have been considered as reference methods. The values of the counts obtained for each species have been compared with the reference methods and those obtained with the methods proposed by the Student t-test for which the tabulated t has been compared for n-1 degrees of freedom and 95% of level of confidence with the t calculated from the results obtained. The results come from at least three repetitions and have indicated that there are no significant differences between the reference values and those obtained with the proposed methods, since for all species the calculated t has been lower than the tabulated t. In addition, recovery rates for each species greater than 90% have been obtained with the proposed methods.

The precision of the proposed methods is high since the relative standard deviation of the differences between the values of the proposed method and the reference values for each species has been less than 0.2 logarithmic units and always better than that obtained from a distribution of Poisson The accuracy of the procedure was also analyzed based on the reproducibility of results by analysis of the same samples by two different operators. In addition, the effect of the matrix was incorporated into the analysis, in this case acidified milk at pH 4.6, and the effect of different levels of inoculum, at a low dose (1 x 10 ⁴ cfu / ml) and at a high dose (1 x 10 ⁶ cfu / ml). The relative standard deviation of reproducibility of the results obtained by the two operators was equal to or less than 0.02 logarithmic units for the species analyzed.

Selectivity.- For the evaluation of the selectivity of the proposed media and the effect of the presence of competitors, all species were combined at the same level of inoculum (1 x 10 ⁷ cfu / ml) and it was observed that all selective or differential media they presented a selectivity of around 100% for each of the species analyzed. When each of the species was added to acidified milk at pH 4.6 at a low dose (1 x 10 ⁴ cfu / ml) together with the rest of the other four species, each at a high dose (1 x 10 ⁶ cfu / ml), a specificity of 100% for each of the species evaluated was obtained for the proposed methods. The results indicate, therefore, that the quantification of each of the species in the respective means proposed is not affected by the presence of the other species.

Identification.- The verification of the identity of the species was carried out by identifying 10% of colonies obtained in the different selective and differential media according to the procedure described in the patent Procedure for the specific detection and identification of lactic bacteria and bifidobacteria in fermented milks

The main industrial application of the procedure developed is the quantitative microbiological analysis of different species of lactic bacteria and bifidobacteria in yogurts and fermented milks that contain mixed cultures of these bacteria, in order to determine the levels of viability in the product they respond, by a on the other hand, to the values required by the legislation for yogurt bacteria and, on the other hand, to those recommended for probiotic bacteria. Detailed description of the content of the figures. - Brief description of the content of the figures.

Fig. 1 shows the morphology differences between the colonies of L. bulgaricus, L. acidophilus and B. lactis. Fig. 2 shows the morphology differences between the colonies of L. acidophilus and L. casei.

Figure 1: morphology differences between the colonies of L. bulgaricus, L. acidophilus and B. lactis. The colonies obtained from a dilution of the mixture of S. thermophilus, L. bulgaricus, L. acidophilus, L. casei and B. lactis seeded in depth and after incubation in MRS agar with fructose for 72 hours are shown. ° C and in anaerobiosis. The colonies correspond to L. bulgaricus with lentil morphology and size greater than 2 mm in diameter, L. acidophilus that forms rough colonies and size greater than 2 mm and to punctate colonies corresponding to B. lactis.

Figure 2: morphology differences between the colonies of L. acidophilus and L. casei.

The colonies obtained from a dilution of the mixture of S. thermophilus, L. bulgaricus, L. acidophilus, L. casei and B. lactis seeded on the surface and after incubation in MRS agar with maltose for 72 hours are shown 37 ° C and in CO ₂ stove. The transparent and rough colonies of at least 2 mm in diameter correspond to L. acidophilus and the round and white colonies larger than 2 mm correspond to L. casei.

EXAMPLE OF EMBODIMENT OF THE INVENTION:

Example of selective and differential quantification of S. thermophilus, L. bulgaricus, L. acidophilus, L. casei and B. lactis in fermented milk analyzed during 28 days of storage at 4 ° C.

The procedure for the selective and differential counting of the mixture of these five species has been applied to a commercial fermented milk indicated on the label containing said species. The fermented milk sample (1 ml) was homogenized with 9 ml of Ringer's solution, containing 0.5 g / L of cysteine, to obtain the first decimal dilution from which serial dilutions were made and three dilutions were inoculated and in duplicate under the following conditions:

• S. thermophilus: 1 ml deep seeding of the appropriate dilutions in Petri dishes to which 15 ml of M-17 agar (Conda) containing 5 g / L of lactose was added. Plates were incubated 24 hours in aerobiosis at 45 ° C. The medium is selective and all colonies are counted, whose appearance is lentil size equal to or greater than 2 mm.

• L. bulgaricus: 1 ml deep seeding of the appropriate dilutions in Petri dishes to which 15 ml of MRS agar was added for fermentation (Conda) containing 10 g / L fructose, 8 g / L casamino acids, 2 g / L of Tween 80 and 0.5 g / L of cysteine. The plates were incubated 72 h in anenerobiosis at 45 ° C. The medium is differential and the lentil-shaped colonies with a size equal to or greater than 2 mm are counted, perfectly differentiable from the rough colonies larger than 2 mm corresponding to L. acidophilus and the punctate colonies corresponding to B. lactis ( see photo of Fig. 1). • L. acidophilus: planting 0.1 ml on the surface of the appropriate dilutions in Petri dishes containing 15 ml of MRS agar for fermentation (Conda) to which 5 g / L of maltose, 8 g / L of casamino acids and 0.5 g / L cysteine. The plates were incubated 72 h in a CO ₂ oven at 37 ° C. The medium is differential and the rough and translucent colonies of size equal to or greater than 2 mm are counted, perfectly differentiable from the round and white colonies equal to or greater than 2 mm corresponding to L. casei (see photo of Fig. 2) .

• L. casei: 1 ml deep seeding of the appropriate dilutions in Petri dishes to which 15 ml of MRS agar for fermentation (Conda) containing 5 g / L glucose was added. Plates were incubated 72 h in aerobiosis at 30 ° C. The medium is selective and all colonies are counted, whose appearance is lentil size equal to or greater than 2 mm. • B. lactis: 1 ml deep seeding of the appropriate dilutions in Petri dishes to which 15 ml of MRS agar for fermentation (Conda) containing 5 g / L of raffinose, 8 g / L of casamino acids, 0 , 5 g / L LiCI and 0.5 g / L cysteine. The plates were incubated 72 h in anenerobiosis at 45 ° C. The medium is selective and all colonies are counted, whose appearance is lentil size equal to or greater than 2 mm.

The counts that were obtained from fermented milks in different stages of conservation (beginning, middle and end of expiration) appear in Table 1.

Table 1. Counts of S. thermophilus, L. bulgaricus, L. acidophilus, L. casei and B. lactis in fermented labeled milk containing these species and analyzed until their expiration date (conservation at 4 ° C for 28 days).

Species Recounts (cfu / ml) in fermented milk preserved at 4 ° C

1 week 2 weeks 3 weeks 4 weeks

S. thermophilus 2.12 x 10 ⁹ 1, 51 x 10 ⁹ 1, 73 χ 10 ⁹ 1, 67 x 10 ⁹

L. bulgaricus 3.01 x 10 ⁷ 1, 77 x 10 ⁷ 4.30 x 10 ⁷ 6.42 x 10 ⁶

L acidophilus 1, 55 x 10 ⁷ 1, 61 x 10 ⁷ 8.03 x 10 ⁶ 3.79 x 10 ⁶

L casei 3.24 χ 10 ⁶ 3.55 x 10 ⁶ 3.19 x 10 ⁶ 3.27 χ 10 ⁶

B. lactis 1, 24 x 10 ⁸ 1, 21 ^χ 10 ⁸ 1, 02 ^χ 10 ⁸ 1, 35 ^χ 10 ⁸

Claims

CLAIMS:

1. Procedure for differentiating and quantifying lactic and bifidobactehas bacteria in fermented milks and fermentation milks starting cultures from a milk or culture sample characterized by using culture media with specific components and free of antibiotics in combination with specific growth conditions for S. thermophilus, L. bulgaricus, L. acidophilus, L. casei and B. lactisi are both in a mixture of the five species and in the different binary, ternary or quaternary combinations obtained from the five species, so that the medium, culture conditions and morphology of the colonies determine the species and the quantification is carried out by decimal dilutions.

2. Procedure for differentiating and quantifying lactic and bifidobactehas bacteria in fermented milks and fermentation milks starting cultures from a milk or culture sample according to claim 1 characterized in that the specific components of the culture medium comprise carbon sources by which The indicated species have different fermentative capacity.

3. Procedure for differentiating and quantifying lactic and bifidobactehas bacteria in fermented milks and fermentation milks starting cultures from a milk or culture sample according to claims 1 and 2 characterized in that the culture conditions comprise the environmental incubation conditions relative to duration, temperature and percentage of oxygen in the atmosphere.

4. Procedure for differentiating and quantifying lactic bacteria and bifidobactehas in fermented milks and fermentation milks starting cultures from a milk or culture sample according to claims 1 to 3 characterized in that three dilutions of the sample are used for duplicate sowing in the specific media.

5. Method for differentiating and quantifying lactic and bifidobactehas bacteria in fermented milks and fermentation milks starting cultures from a milk or culture sample according to claims 1 to 4 characterized by comprising a. The identification and quantification of colonies with a lentil form and size equal to or greater than 2 mm corresponding to S. thermophilus when the sample is incubated in lactose medium and under aerobic culture conditions, for 24 hours and at a temperature of 45 ° C. b. The identification and quantification of colonies with a lentil form and size equal to or greater than 2 mm corresponding to L. bulgaricus when the sample is incubated in medium with fructose and 2 g / L of Tween 80 under anaerobiosis culture conditions, during 72 h and a temperature of 45 ° C. C. The identification and quantification of rough and translucent colonies with a size equal to or greater than 2 mm corresponding to L. acidophilus when the sample is incubated in maltose medium and under culture conditions of 20% CO ₂ , for 72 hours and at a temperature of 37 ° C. At the same time, rough colonies of size equal to or greater than 2 mm corresponding to L. acidophilus in case b.) Could be identified and quantified. D. The identification and quantification of colonies with the shape of a lentil with a size equal to or greater than 2 mm corresponding to L. casei when incubating the sample in a medium with glucose under aerobic culture conditions, for 72 hours and at a temperature of 30 ° C. At the same time, round and white colonies with a size equal to or greater than 2 mm corresponding to L. casei in case c.) E could be identified and quantified. The identification and quantification of colonies with the form of a lentil with a size equal to or greater than 2 mm corresponding to B. lactis when incubating the sample in the middle with raffinose and 0.5 g / L of LiCI under anaerobiosis culture conditions, for 72 h and at a temperature of 45 ° C.

6. Method for differentiating and quantifying lactic and bifidobactehas bacteria in fermented milks and fermentation milks starting cultures from a milk or culture sample according to claims 1 to 4 characterized by comprising a. The identification and quantification of colonies with the form of a lentil size equal to or greater than 2 mm corresponding to S. thermophilus by incubating 1 ml of the dilution of the sample in 15 ml of M-17 agar containing

5 g / L of lactose by deep seeding and under aerobiosis culture conditions, for 24 h and at a temperature of 45 ° C b. The identification and quantification of colonies with a lentil form and size equal to or greater than 2 mm corresponding to L. bulgaricus when incubating 1 ml of the dilution of the sample in 15 ml of MRS agar for fermentation containing 10 g / L of fructose, 8 g / L of casamino acids, 0.5 g / L of cysteine and 2 g / L of Tween 80 by deep seeding and under anaerobic culture conditions, for 72 h and at a temperature of 45 ° C. C. The identification and quantification of rough and translucent colonies with a size equal to or greater than 2 mm corresponding to L. acidophilus when incubating 0.1 ml of the dilution of the sample in 15 ml of MRS agar for fermentation containing 5 g / L maltose , 8 g / L of casamino acids and 0.5 g / L of cistern by surface seeding and under cultivation conditions of 20% CO ₂ , for 72 h at a temperature of 37 ° C in a CO ₂ stove. and quantify rough colonies of size equal to or greater than 2 mm corresponding to L. acidophilus of case b.) d. The identification and quantification of colonies with a lentil form of a size equal to or greater than 2 mm corresponding to L. casei by incubating 1 ml of the dilution of the sample in 15 ml of MRS agar for fermentation containing 5 g / L of glucose by seeding in depth and under aerobic conditions, for 72 hours and at a temperature of 30 ° C. At the same time, round and white colonies with a size equal to or greater than 2 mm corresponding to L. casei in case c.) E could be identified and quantified. The identification and quantification of colonies with a lentil form and size equal to or greater than 2 mm corresponding to B. lactis when incubating 1 ml of the dilution of the sample in 15 ml of MRS agar for fermentation containing 5 g / L of raffinose, 8 g / L of casamino acids, 0.5 g / L of LiCI and 0.5 g / L of cysteine by deep seeding and under anaerobic culture conditions, for 72 h and at a temperature of 45 ° C

7. Procedure for differentiating and quantifying lactic and bifidobactehas bacteria in fermented milks and fermentation milks starting cultures from a sample of milk or culture according to claims 1 to 6 characterized in that it has been validated following recommendations of Standard ISO / TR 13843 : 2000.

8. Procedure for differentiating and quantifying lactic and bifidobactehas bacteria in fermented milks and fermentation milks starting cultures from a milk or culture sample according to claim 7 characterized in that the relative recovery in all species is greater than 90% of the quantification obtained.

9. Procedure for differentiating and quantifying lactic and bifidobactehas bacteria in fermented milks and fermentation milks starting cultures from a milk or culture sample according to claim 7 characterized in that the selective quantification is effective (100%) when all species are combined at the same level of inoculum (1 x 10 ⁷ cfu / ml).

10. Procedure for differentiating and quantifying lactic and bifidobactehas bacteria in fermented milks and fermentation milks starting cultures from a milk or culture sample according to claim 7, characterized in that the selective quantification is effective (100%) when evaluating each one of the species in acidified milk at pH 4.6 (matrix effect) at a low dose (1 x 10 ⁴ cfu / ml) combined with the rest of the other four species, each at a high dose (1 x 10 ⁶ cfu / ml)

1 1. Procedure for differentiating and quantifying lactic and bifidobactehas bacteria in fermented milks and fermentation milks starting cultures from a milk or culture sample according to claim 7 characterized in that the quantification does not differ from the reference methods according to the analysis of T comparison of

Student, n-1 degrees of freedom and 95% confidence level, its relative standard deviation is equal to or less than 0.2 logarithmic units and because it is better than that obtained from a Poisson distribution.

12. Procedure to differentiate and quantify lactic and bifidobactehas bacteria in fermented milks and fermentation milks starting cultures from a milk or culture sample according to claim 7 characterized in that when the matrix effect is studied, the relative standard deviation of reproducibility of The results, obtained by two operators, is equal to or less than 0.02 logarithmic units when each of the species is analyzed in acidified milk at pH 4.6 at a low dose (1 x 10 ⁴ cfu / ml).

13. Procedure for differentiating and quantifying lactic and bifidobactehas bacteria in fermented milks and fermentation milks starting cultures from a milk or culture sample according to claim 7 characterized in that when the matrix effect is studied, the relative standard deviation of reproducibility of the results, obtained by two operators, is equal to or less than 0.02 Logarithmic units when each species is analyzed in acidified milk at pH 4.6 at high dose (1 x 10 ⁶ cfu / ml).