WO2001088150A1

WO2001088150A1 - Probiotic lactic acid bacteria, unable to utilize lactose

Info

Publication number: WO2001088150A1
Application number: PCT/EP2001/004941
Authority: WO
Inventors: Jacques Edouard Germond; Beat Mollet
Original assignee: Societe des Produits Nestle SA; Nestle SA
Current assignee: Societe des Produits Nestle SA; Nestle SA
Priority date: 2000-05-15
Filing date: 2001-05-02
Publication date: 2001-11-22
Anticipated expiration: 2002-11-15
Also published as: AU2001262250A1

Abstract

The present invention pertains to a Lactic acid bacterium having probiotic properties and being deficient in using lactose as a carbon source for the preparation while still retaining the capability of translating β-galactosidase. In particular, the present invention relates to the use of such micro-organisms for the preparation of a food product containing lactose. Moreover, the present invention also relates to food compositions containing such micro-organisms.

Description

PROBIOTIC LACTIC ACID BACTERIA, UNABLE TO UTILISE LACTOSE

The present invention pertains to a Lactic acid bacterium having probiotic properties and being deficient in using lactose as a carbon source for the preparation while still retaining the capability of translating β-galactosidase. In particular, the present invention relates to the use of such jmicro-organisms for the preparation of a food product containing lactose. Moreover, the present invention also relates to food compositions containing such micro-organisms.

Micro-organisms producing lactic acid upon growth in a lactose containing medium have been known for long and are generically summarized under the group "Lactic acid bacteria". This group comprises micro-organisms exhibiting quite different phentoypic traits, such as Lactococci, Bifidobacteria, Lactobacilli, Pediococci etc..

In the food industry Lactic acid bacteria have been utilized for different purposes such as for the preservation of food taking benefit of a low pH and the action of fermentation products generated during the fermentative activity thereof. Also for the direct preparation of a variety of different foodstuff such as cheese, yogurt and other fermented dairy products, use is made of such bacteria.

In the recent past some strains of Lactic acid bacteria have been isolated which are capable to contribute to the well-being of man and animals. Most of these strains have been found to be able to colonize the intestinal mucosa thus preventing the infestation of the individual's gut by deleterious micro-organisms and/or to be able to produce compounds that stimulate the individual's immune system. In the food industry these strains are presently used for the preparation of so called "probiotic" products. Probiotics are considered to be viable microbial preparations which promote the individual's health by preserving the natural micro-flora in the intestine. Yet, a crucial prerequisite for their action resides in that they have to reach the guts' mucosa in an essentially proper and viable form and do riot get destroyed in the upper part of the gastrointestinal tract, especially by the influence of the low pH prevailing in the stomach.

In WO 97/00078 such a strain, Lactobacillus GG (ATCC 53103), is described. This micro-organism shall be employed in a method of preventing or treating food induced hypersensitivity reactions. To this end, it is administered to a recipient together with a food material that has been subjected to a hydrolysis treatment. The Lactobacillus strain selected is described to exhibit a protease enzyme system, thus being capable to further hydrolyze the food stuff by means of proteases secreted by the specific Lactobacillus strain.

In EP 0 577 903 a strain of Lactic acid bacteria is described that has the capability of displacing Heliobacter pylori, a micro-organism known to cause ulcer, on intestinal or gastric cells. This micro-organism is proposed to be included in an ingestable carrier to be used for the therapeutic or prophylactic treatment of an ulcer associated with the action of Heliobacter pylori.

Further, in EP 99 104 922.2 specific strains of Lactic acid bacteria are described that have the capability to prevent colonization of an individual's gut by pathogenic bacteria, that cause diarrhea, such as particular strains of E. coli or salmonella.

In order to provide such beneficial strains and the properties thereof, respectively, to consumers they are most often included in dairy products, such as in a yoghurt. Yet, most of the probiotic Lactic acid bacteria strains known so far do not grow sufficiently well in milk to be capable to produce some lactic acid and, hence, at least partly to acidify the milk products. However, in most cases, other lactic acid bacteria are added to the product with the probiotic strains to complete the acidification process which leads to the final fermented end-product. Rapid fermentation brings about an increased concentration of live probiotic bacteria in the final product and ensures a prolonged shelf life of the fermented product in respect to its raw material, milk.

Though, with a view to the shelf life and maintenance of the product, the lactose fermentative activity of the micro-organisms, once the fermentation process has been completed, is not desirable. On the other hand it is a desired trait that the microorganisms should assist in the degradation of lactose in the gut, when lactose containing food material is incorporated.

Thus, there is a need in the art for avoiding the shortcomings of the state of the art and to enable the provision of probiotic milk based products, that do not acidify the product during processing and storage, but still provide active beta-galactosidase to the benefit of the consumer upon consumption of the product.

This problem has been solved by providing Lactic acid bacteria as such probiotic strains, that are deficient in using lactose as a carbon source and that are still capable to translate the β-galactosidase gene.

The present inventors have now found that when using probiotic Lactic acid bacteria that are deficient in using lactose as a carbon source, such that no lactic acid is produced, the acidification and maintaining quality of products to which such strains are added are not essentially changed.

The probiotic Lactic acid bacteria are preferably selected from the group comprising Lactobacillus johnsonii, L. gasseri, L. acidophilus, L. rhamnosus, L. casei, and L. reuteri. In order to attain the desired traits, being unable to utilize lactose as a carbon source but still retaining the capability of translating the β-galactosidase gene, the Lactic acid bacterium preferably has a mutation in the lac-operon such that lactose may not be utilized as the carbon source. This mutation may relate to a regulatory region, or gene or to one or more of the structural genes selected from, e.g. the permease gene.

In a particular preferred embodiment the Lactic acid bacteria are CNCM - I 2369 and CNCM - 1 2474, which micro-organisms have been deposited according to the Budapest Treaty with the Pasteur Institute, Paris, France on December 17^th 1999 and May 9^th, 2000, respectively.

It has surprisingly been recognized that by using such strains in products containing lactose, such as milk, the probiotic properties of said strains can be included in an increased number of products. It is now feasible to provide so called "value added" food products, i.e. products to which probiotic strains have been added, which probiotic strain do, however, not acidify or ferment the product. As an example for such a "value added" product common milk, supplemented with a health beneficial probiotic strain, may be mentioned. That is, the probiotic strain being deficient in using lactose as a carbon source is added to normal skimmed or unskimmed milk in a desired concentration, so that the consumer will incorporate the probiotic strain with drinking the milk, which strain will then exert its beneficial actions within the intestinal tract or the consumer.

In addition, since the strains are still able to translate the β-galactosidase gene and produce the corresponding polypeptide they may assist in the digestion of lactose in the gut, since upon lysis of the micro-organisms in the gut the cellular material inclusive the β-galactosidase will be set free and will exert its enzymatic activity.

The probiotic strain will, however, not grow in the lactose containing food product, such as milk, yoghurt etc, such that the product may be stored in the refrigerator for the same time period as the food product without being complemented with the probiotic strain.

In order to achieve the desired goal a probiotic Lactic acid bacteria strain may be constructed, that has a mutation in the lac-operon such that lactose may not be utilized as the carbon source but still translates the β-galactosidase gene. Preferably, the mutation lies within a structural gene, that is within the lactose operon, e.g. the permease, but it may well lie within a regulatory region of the Lac operon or a control gene outside of the Lac operon.

The construction of probiotic strains deficient in utilizing lactose as the carbon source may be effected by means of recombinant technology. To this end e.g. a structural gene selected from the permease gene that has been mutated such that the gene product thereof is not or is essentially not functional any more, is inserted into the location of the natural structural gene, by means of e.g. homologous recombination, with the effect that lactose may not be utilized by the micro-organism any more. Methods to genetically engineer Lactic acid bacteria are within the general knowledge of the skilled artisan.

However, such strains are preferably produced by common methods, such as mutation and selection or by simply selecting for a spontaneous mutation exerting a selective pressure on a culture of Lactic acid bacteria.

According to a particularly preferred embodiment the probiotic strains are the micro- organisms deposited under the accession no. CNCM 1-2369 and CNCM 1-2474. These strains have been derived from the lactic acid bacterium CNCM 1-1225, available from the Pasteur Institute, that has the capability to displace Heliobacter pylori from intestinal cells.

The products to which the strains described herein are to be added are preferably milk derived products, since milk contains mainly lactose as the carbon source for lactic acid bacteria. To this end "value added" products, such as milk, yoghurt, curd, cheese, fermented milks, ice-cream etc. may be produced by simply adding the probiotic Lactic acid bacteria, deficient in using lactose as the carbon source to the product.

The following examples are for illustrative purposes only without limiting the invention thereto.

Example 1: Isolation of mutants of Lai (CNCM - 1225)

Natural mutants of Lai were prepared in a two step process. In a first step the bacterial strain was grown in a medium containing 1 % yeast extract, 1 % tryptone, 1 % lactose and different concentrations of penicillin (2.5 g/ml).

The ratio behind this procedure was that bacteria capable to use lactose will eventually grow and will be killed by the antibiotic. In contrast thereto, bacteria unable to use lactose will not grow and consequently not incorporate the antibiotic and stay alive.

In a second step surviving bacteria were plated on MRS (Difco Laboratories, MI, USA) plates containing X-gal, a substrate for β-galactosidase. This substrate is known to produce a blue coloured product upon cleavage by β-galactosidase. Thus, bacteria still capable to use lactose, should transcribe the structural polypeptides of the lac-operon, inclusive β-galactosidase, and will develop a blue colour, while mutants unable to use lactose should remain white. From 200 colonies, several mutants (light blue colonies) could be isolated. Example 2:

Stability of the mutants

Five colonies that remained light blue during the experiment according to example 1 (and showed a β-galactosidase activity) were further investigated for the genetic stability of the mutation. To this end, the corresponding strains were cultivated in a volume of 10 ml of MRS milk supplemented with yeast extract (0.2%). After 12 hours of incubation at 37 °C an aliquot of 0.05 ml of the culture was used to inoculate another volume of 10 ml of MRS supplemented milk as above. Each transfer of 0.1 ml to another volume was determined to be 6 generations. The stability was assessed on the basis of the natural mutants bSing unable to acidify the supplemented milk supplemented with yeast extract (0.2%)as above upon incubation, which can be visualized by the coagulation of casein micelles occurring at lower pH values.

The stability was assessed on the basis of the natural mutants being able to acidify the supplemented milk as above upon incubation, which can be visualized by the coagulation of casein micelles occurring at lower pH values. It could be shown that the five natural mutants did not acidify the supplemented milk even after 100 generations

The bacteria were further investigated whether they were derived from the original starter culture Lai. In this respect total DNA was isolated from both the mutant and the starter strain and were digested with the restriction enzyme Muni under the conditions recommended by the supplier (Boehringer, Mannheim).

The DNA was transferred to an agarose gel (0.8%) and ran for six hours. According to the pattern of DNA fragments generated it could be shown that the DNA of Lai and the mutants were essentially identical indicating that the isolated mutants were not contaminating bacteria. Example 3:

Properties of Lai mutants

Two mutants were selected for further characterization and deposited at the Institute Pasteur with the accession nos. I - 2369 and 1-2474.

Bacteria were grown in milk supplemented with 0.2% yeast extract in the presence or absence of 1 % glucose and incubated overnight. Growth of bacteria was determined by measurement of the pH of the fermented products (table 1).

NCC CNCM - 1 PH pH glu OD₄₂₀

533 (Lai) 1225 4.1 4.2 .24

2655 2369 5.8 4.3 .67

2680 2474 6.0 4.4 .07

Table legend:

Internal reference (NCC) Accession no at the pasteur institute (CNCM) pH of fermented milk (pH), pH of fermented milk supplemented with 1 % glucose (pH glu); and β-galactosidase activity (OD₄₂₀) of Lai and its mutants.

The two mutants are unable to grow in milk. Hence, they can not ferment lactose, and have a lactose-negative phenotype. In the presence of glucose, bacteria can grow and growth is comparable to that of the wild type Lai .

The β-galactosidase activity was determined by incubating equivalent amounts of the bacteria (by optical density at 600nm) in a phosphate buffer (100mm sodium phosphate pH 7.0, 1 mM MgCl₂) in the presence of ONPG (0.5 mg/ml ortho-nitrophenyl-β-D- galactopyranoside) after permeabilisation with 0.1% Triton X-100 (30 min at room temperature). Although that both mutants have a Lac-minus phenotype, they are genetically different. NCC 2680 is unable to metabolise ONPG after permeabilisation. This result indicates that the mutation had affected the β-galactosidase gene or a region/gene directly regulating its expression. The expressed β-galactosidase activity of strain NCC 2680 is strongly reduced. NCC2655 is still able to metabolise ONPG. In fact, the results (table 1) showed that its β-galactosidase is even more expressed than that of its mother strain, Lai. In this case, β-galactosidase is still active and another gene of the lac operon or its regulation as beenaffected to explain the inability ofstrain NCC2655 to metabolise lactose.

Example 4:

Product prepared with Lai mutants

UHT processed milk at 3.7% fat was inoculated with the mutants of Lai (CNCM 1-2369 or CNCM 1-2474) (2% in a volume of frozen concentrated culture). The frozen concentrate contained 5x10⁹ cells/ml. The inoculated milk was mixed, distributed in 150ml plastic jars or tetra-pack type packaging and directly stored at temperature of 8°C.

A milk product was obtained which did not acidify during a shelf life of 14 days.

In summary from the above it may clearly be seen that the mutants described in the present specification are particularly suitable as an addition to milk products, since they impart the probiotic properties to said products, while not negatively affecting the shelf life of the resulting product.

TRAITE DE BUDAPEST SUR LA RECONNAISSANCE INTERNATIONALE DU DEPOT DES MICRO-ORGAN1SMES AUX BINS DE LA PROCEDURE EN MATIERE DE BREVETS

ΪORMULE INTERNATIONALE

D DEESTINATAIRE ^~ι RECEPISSE EN CAS DE DEPOT INITIAL, delivre en vertu de la regie 7.1 par

Sociέtέ des Produits Nestlέ S.A. 1'AUTORITE DE DEPOT INTERNATIONALE Patents Department identif ee au ba^'s de cette page Avenue Nestlέ 55 CH-1800 Vevey

L NOM ET ADRESSE DU DEPOSANT J

Formule BP/ (page unique) CNCM Paris, May 09, 2000

National Collection of cultures of micro-organisms INSTITUT PASTEUR 25, Rue du Docteur Roux M. Roman Vuille F-75724 PARIS CEDEX 15 vice-president

SOCIETE DES PRODUITS NESTLE S.A.

Tel: (33-1) 45 68 82 50 Patents Department Fax: (33-1) 45 68 82 36 Avenue Nestle 55

CH-1800 VEVEY (Switzerland)

Via fax to numbers (0041) 21 924 28 80 / (0041) 21 785 89 25

Our Ref. (N/R) : CNCM 7460.005 Obj. Registration of a bacterium with regard to a deposit for the purposes of the Treaty of Budapest

C/C Mr. J.-E. GERMOND and Mrs. Aline MAMIN

Nestle Research Center, Vers-chez-les-Blanc, Lausanne

Sir,

with the present letter, we confirm to you that we have received today with regard to an initial deposit according to the rule 6.1 of the treaty of Budapest twelve lyophilized samples relating to the here-below identified bacterium.

Your deposit project has been registered at the CNCM with date of May 9, 2000 under the following number:

Identification reference Registration number CNCM

NCC 2680 1-2474

If a deposit is accepted, the order number given by the CNCM is identical with the registration number and the date of the deposit is the date of the registration.

Remaining at your disposition,

I ask you to trust, Sir, in the assurance of my distinguished appreciation.

Georges WAGENER

Claims

1. Probiotic lactic acid bacterium, which is deficient in using lactose as a carbon source and which still essentially translates the β-galactosidase gene.

2. The Lactic acid bacterium according to claim 1, which is selected from Lactobacillus johnsonii, Lactobacillus gasseri, Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus casei and Lactobacillus reuteri.

3. The Lactic acid bacterium according to claim 1 or claim 2, wherein the lactic acid bacterium has a mutatiori such that lactose may not be utilized as the carbon source.

4. The Lactic acid bacterium according to claim 3, wherein the mutation lies in a structural gene of the Lac operon, in a gene regulating the expression of the Lac operon or in a control region of the Lac operon.

5. The Lactic acid bacterium according to claim 4, wherein the mutation lies in any of the permease gene.

6. The Lactic acid bacterium according to claim 1, which is CNCM 1-2369 and CNCM 1-2474.

7. Use of a Lactic acid bacterium according to any of the preceding claims for the preparation of a food product.

8. The use according to claim 7, wherein the food product is a milk derived product.

9. The use according to claim 8, wherein the product is selected from milk, yoghurt, curd, cheese, fermented milks, ice-cream.

10. Food product, containing a probiotic lactic acid bacterium according to any of the claims 1 to 6.

11. The food product according to claim 10, which is selected from milk, yoghurt, curd, cheese, fermented milks or ice-cream.