US-20260125635-A1 - NEW LACTIC ACID BACTERIA

US20260125635A1US 20260125635 A1US20260125635 A1US 20260125635A1US-20260125635-A1

Abstract

The invention relates to a polynucleotide comprising a lacZ gene (lacZ FS ) encoding a β-galactosidase characterized by a particular profile regarding its efficiency of hydrolysis of lactose. The invention is also directed to a Streptococcus thermophilus strain comprising a lacZ FS allele and bacterial composition thereof, and their use to obtain fermented milk not undergoing post-acidification.

Inventors

Anais JEDRZEJOWSKI
Christophe Fremaux
Sabine VAN DILLEN
Thomas DESFOUGERES
Max Charles JODEAU
Damien LUGAND

Assignees

INTERNATIONAL N&H DENMARK APS

Dates

Publication Date: 20260507
Application Date: 20250915
Priority Date: 20190516

Claims (9)

1 . A polynucleotide encoding a β-galactosidase FS , which consist of an amino acid sequence identified by SEQ ID NO:4; and which is defined as a lacZ allele which increases the ratio of the activity of lactose importation of the LacS permease at pH 4.5 over the activity of lactose hydrolysis of the beta-galactosidase at pH 4.5 above 8 in a DGCC715 derivative, said DGCC715 derivative being a strain DGCC715 (deposited at the DSMZ on Feb. 12, 2019 under the accession number DSM33036), into which its lacZ gene was replaced by said polynucleotide encoding a β-galactosidase FS .
2 . A Streptococcus thermophilus strain comprising an allele of the lacZ gene as defined in SEQ ID NO:3, which is a lacZ FS allele encoding a β-galactosidase FS according to claim 1 .
3 . A bacterial composition comprising the Streptococcus thermophilus strain of claim 2 , and one or more further lactic acid bacteria selected from the group consisting of Streptococcus, Lactococcus, Lactobacillus, Leuconostoc, Pediococcus, Enterococcus , Oenococcus and Bifidobacterium.
4 . A food or feed product comprising the Streptococcus thermophilus strain of claim 2 or a bacterial composition comprising the Streptococcus thermophilus strain of claim 2 , wherein the food or feed product is a dairy, meat or cereal food or feed product.
5 . A method for manufacturing a fermented product, comprising: a) inoculating a substrate with the Streptococcus thermophilus strain of claim 2 or a bacterial composition comprising the Streptococcus thermophilus strain of claim 2 ; and b) fermenting the inoculated substrate obtained from step a) to obtain a fermented product.
6 . A method according to claim 5 , for manufacturing stirred yoghurt, comprising: a) fermenting a milk substrate inoculated with the Streptococcus thermophilus strain of claim 2 or a bacterial composition comprising the Streptococcus thermophilus strain of claim 2 , to obtain a stirred yoghurt with a pH from 4.2 to 4.7; b) cooling the stirred yoghurt; c) packing the stirred yoghurt; and d) transferring the packages of step c) into a storage cold room; wherein the temperature of cooling and packing is at least 24° C.
7 . A method according to claim 5 , for manufacturing stirred yoghurt, comprising: a) fermenting a milk substrate inoculated with the Streptococcus thermophilus strain of claim 2 or a bacterial composition comprising the Streptococcus thermophilus strain of claim 2 , to obtain a stirred yoghurt with a pH from 4.2 to 4.7; b) packing the stirred yoghurt; and c) transferring the packages of step b) into a storage cold room; wherein the process does not comprise any cooling step between end of fermentation and packing.
8 . A method according to claim 5 , for manufacturing set yoghurt, comprising: a) packing a milk substrate inoculated with the Streptococcus thermophilus strain of claim 2 or a bacterial composition comprising the Streptococcus thermophilus strain of claim 2 ; b) fermenting the inoculated milk substrate to obtain a set yoghurt with a pH from 4.2 to 4.7; and c) directly transferring the packages of step b) into a storage cold room, wherein said process does not comprise a cooling step in a cooling room after the fermentation step b).
9 . A method to prepare a Streptococcus thermophilus strain with a full STOP phenotype, comprising: a) providing a Streptococcus thermophilus strain, having a ratio of the activity of lactose importation of the LacS permease at pH 4.5 over the activity of lactose hydrolysis of the beta-galactosidase at pH 4.5 which is less than 5; b) replacing the allele of the lacZ gene of said Streptococcus thermophilus strain of step a) with a polynucleotide according to claim 1 , or replacing a part of the allele of the lacZ gene of said Streptococcus thermophilus strain of step a) with a polynucleotide comprising a part of at least 100 nucleotides of the sequence of said β-galactosidase FS comprising or consisting of an amino acid sequence identified by SEQ ID NO:4, wherein said nucleotide part encompasses the codon corresponding to the residue 354 of said β-galactosidase FS , or modifying the sequence of the lacZ gene of said Streptococcus thermophilus strain of step a) to have a lacZ FS allele with the same sequence as a polynucleotide according to claim 1 ; and c) recovering the Streptococcus thermophilus strain(s) with a full STOP phenotype when used to ferment milk.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS The present application is a Continuation of U.S. application Ser. No. 17/437,255, filed Sep. 8, 2021, which is a U.S. national stage application under 35 U.S.C. § 371 of International Application No. PCT/EP2020/056766, filed on Mar. 13, 2020, entitled “NEW LACTIC ACID BACTERIA,” and claims priority from EP Application No. 19162856.9, filed Mar. 14, 2019, CN Application No. 201910406044.7, filed May 16, 2019, and EP Application No. 19214119.0, filed Dec. 6, 2019, the contents of which are incorporated by reference in their entirety. FIELD OF THE INVENTION The invention relates to a polynucleotide comprising a lacZ gene (lacZFS) encoding a β-galactosidase characterized by a particular profile regarding its efficiency of hydrolysis of lactose. The invention is also directed to a Streptococcus thermophilus strain comprising a lacZFS allele and bacterial composition thereof, and their use to obtain fermented milk not undergoing post-acidification. INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 20250912_NB41527USPCN_SequenceListing.xml, created on Sep. 12, 2025, which is 76,618 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety. BACKGROUND TO THE INVENTION The food industry uses bacteria in order to improve the taste and the texture of food or feed products. In the case of the dairy industry, lactic acid bacteria are commonly used in order to, for example, bring about the acidification of milk (by fermentation of lactose) and to texturize the product into which they are incorporated. For example, the lactic acid bacteria of the species Streptococcus thermophilus (S. thermophilus) are used extensively, alone or in combination with other bacteria, in the manufacture of fresh fermented dairy products, such as cheese or yoghurt. One of the limitations of the use of lactic acid bacteria in dairy technology is post-acidification, i.e. the production of lactic acid by the lactic acid bacteria after the target pH (the one required by the technology) has been obtained. Thus, to avoid this post-acidification phenomenon, dairy product manufacturers are required to rapidly cool the fermented product right after the target pH is obtained. Thus, dairy product manufacturers lack flexibility in the manufacturing process, while having the possibility of keeping the fermented product at the fermentation temperature for some time would be an advantage. Moreover, the cooling step is energy-consuming, such that bypassing the cooling step, would be both an economical and environmental advantage. WO90/05459 describes Lactobacillus bulgaricus mutant strains, selected based on their color phenotype on X-gal-containing medium. The application reports the identification of temperature conditional L. bulgaricus mutants (blue at 37° C., but white at 4° C.) and pH sensitive L. bulgaricus mutants (blue at pH 7 but white at pH 4.5 or 5). However, WO90/05459 is silent about any mutation in the lacZ gene. Moreover, WO90/05459 describes mutants characterized by enzyme which has an activity of at least 90% the activity of a wild type enzyme in production conditions (processing temperature or processing pH), while having an activity reduced of at least 20% as compared to the activity of a wild type enzyme in storage conditions. However, the teaching of WO90/05459 is insufficient regarding any enzyme activity and in particular regarding the beta-galactosidase activity; indeed, as shown in examples 4 and 5 of the present application, there is neither admitted reference beta-galactosidase activity in strains, at pH 4.5 or pH 6. Therefore, the characterization of the mutants described in WO90/015459 is not possible without any reference value or reference strain. WO2010/139765 describes a method to manufacture a fermented dairy product using a weakly post-acidifying culture based on specific Lactobacillus bulgaricus strains. Because the culture is characterized by a weak production of lactic acid at fermentation temperature, the pH is substantially steady and the cooling step can be avoided. However, WO2010/139765 does not characterize the exemplified Lactobacillus bulgaricus strains. WO2015/193459 proposes other solutions to overcome the post-acidification issue: controlling the concentration of lactose in the milk before fermentation for example by adding lactase, providing lactic acid bacteria which are not able to hydrolyze lactose (lactose-deficient lactic acid bacteria). These solutions are however not satisfactory for dairy product manufacturers, since they require either the addition of exogenous enzyme (such as lactase) in the milk before fermentation rendering the manufacturing process more complex and more expensive, or the addition of a carbohydrate into the milk (such as sucrose) what is n