Liquid Concentrate Of Bacteria That Are Adapted And Fit For Alimentary Use

Abstract

The invention relates to a liquid concentrate of bacteria that are adapted and fit for alimentary use. In a preferred but not limited manner, the bacteria produced are lactic bacteria.

Description

LEGEND OF THE FIGURES

FIG. 1: representation of the viability of adapted and non-adapted Lactobacillus casei strains in a yogurt-type food product over a 28-day period;

FIG. 2 a: histogram of size distribution of adapted and non-adapted Lactobacillus casei strains before being subjected to acid stress.

FIG. 2 b: histogram of size distribution of adapted and non-adapted Lactobacillus casei strains after being subjected to acid stress.

FIG. 3: Curves showing the adaptation of Lactobacillus casei with respect to the temperature of the culture medium (37 and 39° C.). The relative permittivity ε_R (dimensionless amount equal to the permittivity ε, expressed in pF/cm, divided by the permittivity of the vacuum ε₀) is expressed according to the age of the bacteria (in hours)

FIG. 4: Curves showing the adaptation of Lactobacillus casei as a function of the osmotic pressure (glucose concentration of 20, 40, and 80 g/L, respectively in light grey, dark grey and black). The permittivity ε, expressed in pF/cm, is expressed as a function of the optical density (OD).

EXAMPLES

Example 1

Consequences of the Adaptation of L. casei Strains on Their Viability

The consequences of a step of adaptation of Lactobacillus casei strains on their viability are to be evaluated.

To do this, a batch of Lactobacillus casei control bacteria is prepared and placed in culture in a MRS medium (special medium allowing for the growth of Lactobacilli, developed by Man, de Rogosa and Sharpe).

Simultaneously, a batch of Lactobacillus casei bacteria is prepared, which, after being placed in culture in a MRS medium, is adapted by a step of natural acidification.

To do this, after 17 hours of culture, a reduction in the pH is achieved by natural acidification over one hour to change it from pH 6.5 to pH 5.

Then, the two batches of bacteria are washed and concentrated in bacteria by tangential microfiltration.

These two bacteria concentrates are separately added to a yogurt mass at pH 5.5 and at a temperature of 10° C.

The amount of living bacteria at D+1 is measured in the two yogurt batches.

Then, every day, a sample is taken from the two batches of yogurt to which the control bacteria concentrate and the adapted bacteria concentrate, respectively, have been added, and the number of surviving strains is quantified with respect to the number of living strains at D+1. A mass count is used for this.

For each of the viability measurements during the period of preservation of the final product, the latter is well homogenized before the sample is collected. A sterile sample of 1 ml of product is collected. A serial dilution by factors of 10 is performed. The various dilutions of the product are placed in a Petri dish and a liquid agar medium (since previously heated at 50° C.) is poured over these fractions of the product. The medium to be poured will be selected according to the type of bacteria to be counted. The agar medium hardens. The Petri dishes are then placed in incubation for a few days (2 to 5 d) at 37° C. The results are shown in FIG. 1.

After 7 days, it is observed that the number of surviving bacteria in the control bacteria batch is 80% and that in the adapted bacteria batch is 105% (there was slight bacterial growth).

After 14 days, the number of surviving bacteria in the control bacteria batch is 58% and that in the adapted bacteria batch is 110% (there was slight bacterial growth).

After 28 days, the number of surviving bacteria in the control bacteria batch is 42% and that in the adapted bacteria batch is 110% (there was slight bacterial growth).

To conclude, the bacteria adaptation step causes a decrease in the mortality of the bacteria on the order of 60% with respect to a batch of non-adapted control bacteria, after 28 days in yogurt.

Example 2

Change in the Size of Adapted Bacteria and Non-Adapted Bacteria Subjected to Acid Stress

The change in size of adapted bacteria and non-adapted bacteria subjected to acid stress is to be monitored.

To do this, a batch of Lactobacillus casei control bacteria is prepared and placed in culture in an MRS medium (special medium allowing for the growth of Lactobacilli, developed by Man, de Rogosa and Sharpe).

Simultaneously, a batch of Lactobacillus casei bacteria is prepared, which, after being placed in culture in an MRS medium, is adapted by a step of natural acidification.

To do this, after 17 hours of culture, a reduction in the pH is achieved by natural acidification over one hour to change it from pH 6.5 to pH 5.

Then, the two batches of bacteria are washed and concentrated in bacteria by tangential microfiltration.

Then, a sample of the bacteria is collected and their size is measured by flow cytometry. A histogram of adapted and non-adapted (control batch) bacteria size distribution is thus established (FIG. 2a). It is observed that the bacteria size distribution of the two lots is very similar.

These two batches of bacteria are then subjected to acid stress by adding bacteria to a medium having a Ph of 3.

Then a sample of the bacteria is collected and their size is measured by flow cytometry. A histogram of adapted and non-adapted (control batch) bacteria size distribution after acid stress is thus established (FIG. 2b). It is observed that the bacteria size distribution of the two lots is very different. In the batch of adapted bacteria, the largest size frequency is 3.2 μm (frequency of 0.016). In the batch of non-adapted bacteria, the largest size frequency is 5.45 μm (frequency of 0.012).

To conclude, the bacteria adaptation step causes a decrease in the bacteria size on the order of 60% when they are subjected to acid stress, with respect to a batch of non-adapted control bacteria. It is therefore possible to show the adaptation of bacteria by measuring their size.

Example 3

Determination of the Adaptation of Bacteria by Measuring the Influence of the Parameter of “Temperature” of the Culture Medium

The adaptation of bacteria is to be determined by measuring the influence of the temperature parameter of the culture medium.

To do this, two batches of Lactobacillus casei bacteria are prepared from the same inoculum. These two batches are then placed in culture in two MRS media (special medium allowing for the growth of Lactobacilli, developed by Man, de Rogosa and Sharpe).

A biomass sensor enabling the relative permittivity ε_R to be measured is used. The probes that can be used for this purpose are known to a person skilled in the art (see in particular FR 2835921). The relative permittivity ε_R is a dimensionless amount equal to the permittivity ε (expressed in pF/cm) divided by the permittivity of the vacuum ε₀ (ε₀=8.854187×10⁻² pF/cm). The change in relative permittivity is measured over time. The relative permittivity will be dependent on the number of living cells and the size of these cells.

The two strictly identical culture media comprising the same number of bacteria are cultivated, one at 37° C. and the other at 39° C.

The number of strains increases over time. This is normal.

The inventors were able to verify that the total number of cells was no different between the two culture media. A conventional technique for measuring the optical density (absorption spectrometry) can be used to this end, or a Wedgewood optical probe can be used (system measuring the optical density of microbial suspensions in the near infrared). The absorbency of the medium measured by a spectrometer will be dependent on the total number of cells in the medium. Techniques for counting in Petri dishes can also be used.

With the biomass sensor used, it was possible to demonstrate that the bacteria changed shape and size, and therefore adapted according to the temperature of the culture medium. FIG. 3 shows this observation.

Example 4

Determination of the Adaptation of Bacteria by Measuring the Influence of the Parameters of “Temperature” and “pH” of the Culture Medium

The adaptation of bacteria is to be determined by measuring the influence of two parameters of the culture medium, which are temperature and pH.

To do this, three batches of Lactobacillus casei bacteria are prepared from the same inoculum.

These three culture media are cultivated at three different temperatures (35° C., 37° C. and 39° C.) while subjecting the bacteria each time to acid stress by lowering the pH of the medium to a pH of 3.

The change in size of the cells will result in their adaptation to the conditions of the medium. This size will be measured by microscopy.

In Table 1, the results obtained clearly show that the bacteria adapt according to the temperature and pH parameters of the medium since these bacteria change size.

TABLE 1
Influence of the temperature and pH on the size
of the bacteria (expressed in μm)
		Average size	Average size	Average size
		of bacteria	of bacteria	of bacteria
	Temperature	during the	before change	after change
	(° C.)	preculture	in pH	in pH
	35	3	3	2
	37	4	5	4
	39	3	7	5

Example 5

Determination of the Adaptation of Bacteria by Measuring the Influence of the Parameter of “Osmotic Pressure” of the Culture Medium

The adaptation of bacteria is to be determined by measuring the influence of the parameter of osmotic pressure of the culture medium.

To do this, three batches of Lactobacillus casei bacteria are prepared from the same inoculum. These batches are placed in culture in an MRS media (special medium allowing for the growth of Lactobacilli, developed by Man, de Rogosa and Sharpe).

The three culture media comprising the same number of bacteria respectively contain amounts of 20, 40, and 80 g of glucose per litre of culture medium. The higher the glucose concentration of the medium is, the higher the osmotic pressure of said medium is.

A sensor enabling the relative permittivity to be measured is used. The probes that can be used for this purpose are known to a person skilled in the art (FR 2835921). The change in this relative permittivity over time is measured. The permittivity ε (expressed in pF/cm) is calculated by multiplying the measured relative permittivity ε_R by the permittivity of the vacuum ε₀ (ε₀=8.854187×10⁻² pF/cm).

A conventional technique for measuring the optical density (absorption spectrometry), or a Wedgewood optical probe (system measuring the optical density of microbial suspensions in the near infrared), is used to measure the change in optical density of the medium over time. The optical density (OD) value obtained will be dependent on the total number of cells in the medium.

By expressing the permittivity as a function of the OD, the resulting curve (FIG. 4) makes it possible to determine the change in viability (expressed by the permittivity measurement) and the size of the cells as a function of the osmotic pressure of the medium. The results show that the bacteria change size. Indeed, if there is no change in size of the cells, the results observed in FIG. 4 would be straight lines. In this case, curves can be seen (nonlinear correlation).

The bacteria therefore adapt according to the osmotic pressure of the culture medium.

Claims

1. A bacteria concentrate in liquid form comprising adapted and viable bacteria at a concentration between 5.1010 and 5.1011 ufc/ml, said adapted and viable bacteria being more resistant to various physiochemical stresses.

2. The concentrate according to claim 1, wherein the bacteria are lactic bacteria, Lactobacillus spp., Bifid bacterium spp., Streptococcus spp. or Lactococcus spp. genera.

3. The concentrate according to claim 1, wherein the adapted bacteria have at least one of the following characteristics when they are added to a food product: i) a survival rate above 80% after 14 days in a food product at a temperature between 4 C and 45 C, with said food product having a pH between 3 and 7, orii) a survival rate above 60% and advantageously above 80% after 28 days in a food product at a temperature between 4 C and 45 C, with said food product having a pH between 3 and 7.

4. The concentrate according to claim 1, wherein the bacteria have both characteristics i) and ii).

5. The concentrate according to claim 1, wherein the food product is a dairy product and/or a drink.

6. The concentrate according to claim 1, wherein the bacteria are viable for a period of between 4 and 6 weeks.

7. The concentrate according to claim 1, wherein it is capable of being obtained by the method including the successive steps of propagation of the bacteria in a culture medium, adaptation of the bacteria, washing of the culture medium containing the adapted bacteria by tangential microfiltration, and concentration of bacteria in the washed medium by tangential microfiltration.

8. The concentrate according to claim 1, wherein the adaptation of the bacteria is determined by measuring parameters of the bacteria culture medium and/or parameters of the bacteria.

9. The concentrate according to claim 8, wherein the parameters of the culture medium are the pH, the osmotic pressure and/or the temperature.

10. The concentrate according to claim 9, wherein the parameter of the culture medium is the pH and the adaptation step is performed by reducing the pH by natural acidification.

11. The concentrate according to claim 1, wherein the bacteria are adapted by a tangential microfiltration method.

12. The concentrate according to claim 8, wherein the parameter of the bacteria is the size thereof.

13. The concentrate according to claim 12, wherein the distribution of lengths of each bacterium of said concentrate are primarily between 0.1 and 10 micrometers.

14. The concentrate according to claim 1, wherein its pH is between 3 and 6.

15. The concentrate according to claim 1, wherein it is preserved at a temperature between −50° C. and 4 C after packaging.

16. The concentrate according to claim 15, wherein it is reheated to a temperature between 25 C and 45 C, before being used.

17. A food additive comprising the concentrate according to claim 1.

18. (canceled)

19. A flexible, hermetically sealed and sterile bag containing the concentrate according to claim 1.

20. A food product comprising the liquid concentrate of adapted and viable bacteria according to claim 1.

21. The food product to claim 20, wherein it is a dairy product or a drink.

22. A method for producing a the food product according to claim 20, comprising adding the liquid concentrate of adapted and viable bacteria to the food product at the end of the production line and before packaging of the food product.

23. The method according to claim 22, wherein the liquid concentrate of adapted and viable bacteria is added to the food product in the line by pumping.