Lactic acid bacteria

Patent metadata

Field	Value
Publication number	US-12441977-B2
Application number	US-202017437255-A
Country	US
Kind code	B2
Filing date	Mar 13, 2020
Priority date	Mar 14, 2019
Publication date	Oct 14, 2025
Grant date	Oct 14, 2025

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.

First claim

The invention claimed is:

1. A polynucleotide encoding a β-galactosidase FS , which comprises an amino acid sequence which has at least 95% identity with SEQ ID NO:2; 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. The polynucleotide according to claim 1 , encoding a β-galactosidase FS comprising an amino acid suppression, an amino acid addition, an amino acid substitution or an amino acid suppression and addition, relative to a β-galactosidase selected from the group consisting of:
a) a β-galactosidase having an amino acid sequence as defined in SEQ ID NO:2; and
b) a β-galactosidase variant comprising an amino acid sequence having at least 95% identity with SEQ ID NO:2, encoded by a lacZ variant allele which does not increase the ratio LacS pH4.5 over LacZ pH4.5 in a DGCC715 derivative to 5 or more than 5, said DGCC715 derivative being a strain DGCC715 into which its lacZ gene was replaced by said lacZ variant allele.

3. The polynucleotide according to claim 1 , wherein the sequence of said β-galactosidase FS does not comprise an arginine at position 354.

4. The polynucleotide according to claim 1 , wherein the sequence of said β-galactosidase FS comprises:
a) an amino acid sequence which is otherwise as defined in SEQ ID NO:2, but which does not comprise an arginine at position 354;
b) an amino acid sequence which has at least 95% identity with SEQ ID NO:2 and does not comprise an arginine at position 354;
c) an amino acid sequence which is otherwise as defined as the one of a β-galactosidase variant protein having at least 95% identity with SEQ ID NO:2, but which does not comprise an arginine at position 354.

5. The polynucleotide according to claim 1 , wherein the sequence of said β-galactosidase FS comprises:
a) an amino acid sequence which is otherwise as defined in SEQ ID NO:2, but which comprises a cysteine or an equivalent amino acid thereof at position 354;
b) an amino acid sequence which has at least 95% identity with SEQ ID NO:2 and comprises a cysteine or an equivalent amino acid thereof at position 354;
c) an amino acid sequence which is otherwise as defined as the one of a β-galactosidase variant protein having at least 95% identity with SEQ ID NO:2, but which comprises a cysteine or an equivalent amino acid thereof at position 354.

6. A polynucleotide comprising a part of at least 100 nucleotides of the polynucleotide according to claim 1 , wherein said nucleotide part encompasses a codon corresponding to the residue 354 of said β-galactosidase FS .

7. A Streptococcus thermophilus strain comprising an allele of the lacZ gene which is a lacZ FS allele encoding a β-galactosidase FS according to claim 1 .

8. A Streptococcus thermophilus strain according to claim 7 , characterized by a difference of efficiency of hydrolysis (ΔEH) of the imported lactose which is less than −0.5 calculated by the following formula (I):

Δ
⁢

⁢
EH

=

ln
[

LacS

p
⁢
H
⁢
6

LacZ

p
⁢
H
⁢
6

]

-

ln
[

LacS

pH
⁢

⁢
4.5

LacZ

pH
⁢

⁢
4.5

]

(
I
)

in which formula (I), LacS pH6 and LacS pH4.5 represent the activity of lactose importation of the LacS permease at pH 6 and at pH 4.5 respectively, and LacZ pH6 and LacZ pH4.5 represent the activity of lactose hydrolysis of the beta-galactosidase at pH 6 and at pH 4.5 respectively.

9. A bacterial composition comprising the Streptococcus thermophilus strain of claim 7 , and one or more further lactic acid bacteria selected from the group consisting of Streptococcus, Lactococcus, Lactobacillus, Leuconostoc, Pediococcus, Enterococcus, Oenococcus and Bifidobacterium.

10. A food or feed product comprising the Streptococcus thermophilus strain of claim 7 or a bacterial composition comprising the Streptococcus thermophilus strain of claim 7 , wherein the food or feed product is a dairy, meat or cereal food or feed product.

11. A method for manufacturing a fermented product, comprising:
a) inoculating a substrate with the Streptococcus thermophilus strain of claim 7 or a bacterial composition comprising the Streptococcus thermophilus strain of claim 7 ; and
b) fermenting the inoculated substrate obtained from step a) to obtain a fermented product.

12. A method according to claim 11 , for manufacturing stirred yoghurt, comprising:
a) fermenting a milk substrate inoculated with the Streptococcus thermophilus strain of claim 7 or a bacterial composition comprising the Streptococcus thermophilus strain of claim 7 , 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.

13. A method according to claim 11 , for manufacturing stirred yoghurt, comprising:
a) fermenting a milk substrate inoculated with the Streptococcus thermophilus strain of claim 7 or a bacterial compo

Assignees

• Int N&H Denmark Aps

Inventors

• Jedrzejowski Anais
• Fremaux Christophe
• Van Dillen Sabine
• Desfougeres Thomas
• Jodeau Max Charles
• Lugand Damien

Classifications

• A23L13/74

  using microorganisms or enzymes · CPC title

• A23V2400/249

  Thermophilus · CPC title

• G01N33/5038

  involving detection of metabolites per se · CPC title

• C12P1/04

  by using bacteria · CPC title

• C12N15/746

  for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus) · CPC title