EP4642896A1 - Bioprotectrice lacticaseibacillus rhamnosus à faible postacidification - Google Patents

Bioprotectrice lacticaseibacillus rhamnosus à faible postacidification

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Publication number
EP4642896A1
EP4642896A1 EP23838018.2A EP23838018A EP4642896A1 EP 4642896 A1 EP4642896 A1 EP 4642896A1 EP 23838018 A EP23838018 A EP 23838018A EP 4642896 A1 EP4642896 A1 EP 4642896A1
Authority
EP
European Patent Office
Prior art keywords
dsm
rhamnosus
milk
cfu
lacticaseibacillus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23838018.2A
Other languages
German (de)
English (en)
Inventor
Solvej PRECHT
Mads Lund
Helle Skov Guldager
Vera Kuzina POULSEN
Jeorgos TRIHAAS
Lise Soendergaard KRISTENSEN
Anisha GOEL
Sophia HARRAND
Louise Essendrup STEFFENSEN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chr Hansen AS
Original Assignee
Chr Hansen AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chr Hansen AS filed Critical Chr Hansen AS
Publication of EP4642896A1 publication Critical patent/EP4642896A1/fr
Pending legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C19/00—Cheese; Cheese preparations; Making thereof
    • A23C19/02—Making cheese curd
    • A23C19/032—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin
    • A23C19/0323—Making cheese curd characterised by the use of specific microorganisms, or enzymes of microbial origin using only lactic acid bacteria, e.g. Pediococcus and Leuconostoc species; Bifidobacteria; Microbial starters in general
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; PREPARATION THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1238—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt using specific L. bulgaricus or S. thermophilus microorganisms; using entrapped or encapsulated yoghurt bacteria; Physical or chemical treatment of L. bulgaricus or S. thermophilus cultures; Fermentation only with L. bulgaricus or only with S. thermophilus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/175—Rhamnosus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/46—Streptococcus ; Enterococcus; Lactococcus

Definitions

  • the present application relates to Lacticaseibacillus rhamnosus bacteria with low postacidification in fermented milk products and having antifungal effects.
  • Lactic acid bacteria have been used over decades for increasing the shelf life of food products. During fermentation, lactic acid and other organic compounds are produced by the lactic acid bacteria, thereby reducing the pH of the food product and consequently making it unfavorable to the growth of undesired microorganisms, such as yeast and mold.
  • Bioprotection is defined as the extension of shelf life and enhanced safety of foods using natural or controlled antimicrobial compounds.
  • spoilage by mold and yeast cells is one of the major problems negatively affecting shelf life.
  • considerable efforts have been invested to explore the bioprotective potential of LAB, to identify new strains with bioprotective properties from various food sources, as well as to elucidate the mechanisms behind the observed antifungal activity. Numerous metabolites produced by LAB have been identified as having antifungal activities.
  • the fast milk acidifying phenotype of Streptococcus thermophilus can be acquired by natural transformation of the genomic island encoding the cell-envelope proteinase PrtS.” Microbial cell factories. Vol. 10. No. SI. BioMed Central, 2011), it is expected that strains exhibiting reduced post-acidification will also have reduced bioprotective effects.
  • WO2021239574 discloses Lactobacillus rhamnosus DSM 33515, a bioprotective strain that was described to exhibit a combination of reduced post-acidification and high bioprotective effects.
  • the present application therefore provides a bacterium of the species Lacticaseibacillus rhamnosus deposited as DSM 34195 or a mutant Lacticaseibacillus rhamnosus of the deposited bacteria.
  • the mutant may exhibit substantially the same or improved bioprotective effect against yeast and mold as well as reduced post-acidification compared to DSM 34195.
  • the mutant is obtained by using as starting material DSM 34195.
  • the mutant is different from DSM 33515 and DSM 23035.
  • the mutant may have retained or improved properties compared to DSM 34195, and may have improved properties compared to DSM 33515.
  • the properties discussed in this paragraph and in the present disclosure are antifungal effect or activity (in other words: bioprotective effect against yeast and mold) and reduced post-acidification.
  • the invention additionally provides a composition comprising bacteria of the species Lacticaseibacillus rhamnosus as described above.
  • the composition further comprises cryoprotectants, lyoprotectants, antioxidants, nutrients, fillers, flavorants or mixtures thereof.
  • the bacteria are in a concentration of at least 10 9 colony forming units (CFU)/g, or in a concentration of at least 10 10 CFU/g, or in a concentration of at least 10 11 CFU/g in the composition.
  • the composition is frozen or freeze-dried.
  • the composition further comprises a starter culture.
  • the invention further provides a method of producing a fermented milk product comprising adding bacteria of the species Lacticaseibacillus rhamnosus strain as described above or a composition comprising the same to milk or to a milk product and fermenting the mixture at a temperature between about 22°C and about 43°C, such as between 22°C and 43°C, until a pH of 4.6 or less than 4.6 is reached.
  • the invention provides a fermented milk product comprising bacteria of the species Lacticaseibacillus rhamnosus as described above.
  • the fermented milk product is obtained by the method as mentioned above.
  • the fermented milk product maintains a pH above 3.8 when stored for at least 28 days at 25°C.
  • the bacteria of the species Lacticaseibacillus rhamnosus are present in a concentration of at least 10 7 CFU/g.
  • the invention provides food, feed or pharmaceutical product comprising the bacteria of the species Lacticaseibacillus rhamnosus as described above or a composition comprising the same.
  • the food, feed or pharmaceutical product is obtained by the method as mentioned above.
  • Figure 1 shows post acidification of L. rhamnosus DSM 34195 and DSM 33515 at four different final inoculation dosages in 96 low-well plate assay.
  • Figure 2 shows mold inhibition of L. rhamnosus DSM 34195 and DSM 33515 at four different final inoculation dosages in 96 low-well plate assay.
  • Figure 3 shows yeast inhibition of L. rhamnosus DSM 34195 and DSM 33515 at four different final inoculation dosages in 96 low-well plate assay.
  • Figure 4 shows the growth of yeasts prepared from milk fermented with a starter culture only (Reference), or with a starter culture in combination with L. rhamnosus bacteria DSM 33515 or with a starter culture in combination with L. rhamnosus DSM 34195 (3 different dosages).
  • Figure 5 shows the growth of six different molds added as contaminant to agar plates made from milk fermented with starter culture only (Reference), or starter culture in combination with L. rhamnosus bacteria DSM 33515 or a starter culture in combination with L. rhamnosus DSM 34195 (3 different dosages).
  • Figure 6 shows the growth of three different molds added as contaminant to agar plates made from milk (with or without 10% sucrose) fermented with starter culture only (YF- 812 or Advance 2.0) (Reference), or starter culture in combination with L. rhamnosus bacteria DSM 33515 or starter culture in combination with L. rhamnosus DSM 34195 (3 different dosages).
  • Figure 7 shows the growth of yeasts prepared from milk (with 10% sucrose) fermented with the starter culture only (Reference), or with a starter culture in combination with L. rhamnosus bacteria DSM 33515 or with a starter culture in combination with L. rhamnosus DSM 34195 (3 different dosages).
  • Figure 7 A and 7B show the results using the starter culture YF-L812 and Advance 2.0, respectively.
  • Figure 8 shows the growth of yeasts prepared from milk (without sucrose) fermented with the starter culture only (Reference), or with a starter culture in combination with L. rhamnosus bacteria DSM 33515 or with a starter culture in combination with L. rhamnosus DSM 34195 (3 different dosages).
  • Figure 8A and 8B show the results using the starter culture YF-L812 and Advance 2.0, respectively.
  • Figure 9 shows the growth of three different molds added as contaminant to agar plates made from milk fermented with starter culture only (YoFlex Mild 1.0 or YoFlex Premium 1.0) (Reference), or starter culture in combination with L. rhamnosus bacteria DSM 33515 or a starter culture in combination with L. rhamnosus DSM 34195 (3 different dosages).
  • Figure 10 shows the growth of yeast prepared from milk fermented with the starter culture only F-DVS YoFlex Mild 1.0 or YoFlex Premium 1.0) (Reference), or with a starter culture in combination with L. rhamnosus bacteria DSM 33515 or with a starter culture in combination with L. rhamnosus DSM 34195 (3 different dosages).
  • Figure 1OA and 1OB show the growth of Debaryomyces (D.) hansenii and Torulaspora delbrueckii, respectively.
  • Figure 11 shows the pH development in fermented milk products over time when stored at 25 ⁇ 1°C for 28 days.
  • the products were fermented with starter culture only (Reference), or with starter culture in combination with DSM 33515 or with starter culture in combination with DSM 34195.
  • Figure 11A and 11B show the results using the starter culture YF-L812 and Advance 2.0, respectively.
  • Figure 12-14 shows the growth of six different molds added as contaminant to agar plates made from crema acida without L. rhamnosus (Reference) or with L. rhamnosus bacteria DSM 33515 or L. rhamnosus DSM 34195 and incubated at different temperatures.
  • Figure 15 shows the growth of D. hansenii yeast in crema acida prepared without L. rhamnosus (Reference) or with L. rhamnosus bacteria DSM 33515 or L. rhamnosus DSM 34195.
  • Figure 16-18 shows the pH development in crema acida over time when stored at at 7°C, 12°C and 25°C for 28 days. The products were without L. rhamnosus (Reference) or with L. rhamnosus bacteria DSM 33515 or L. rhamnosus DSM 34195.
  • Bioprotective cultures offering a safe add on solution for traditionally fermented products are available. These bioprotective cultures are used in combination with normal starter cultures to co-ferment milk to a fermented product. During the fermentation, it will exert bioprotective effects and thus provide an extended shelf-life of the fermented products against molds and yeasts. Fermentation of many dairy products, such as yoghurt, are stopped and the product is cooled down at a specific pH, after fermentation the bacteria are frequently still active during storage. Further lactate is produced and the process is known as post-acidification. The resulting lower pH of the final product has a negative sensory impact on the product and is therefore undesirable.
  • mutant should be understood as a strain derived from a strain of the invention, for example by means of e.g. genetic engineering, radiation and/or chemical treatment. It is preferred that the mutant is a functionally equivalent mutant, e.g. a mutant that has substantially the same, or improved, properties in particular in relation to the effects on reducing post-acidification and/or bioprotection, as the deposited strain. Respective mutants represent embodiments of the present application.
  • mutant in particular refers to a strain obtained by subjecting a strain of the invention to any conventionally used mutagenization treatment including treatment with a chemical mutagen such as ethane methane sulphonate (EMS) or N- methyl-N'-nitro-N-nitroguanidine (NTG), UV light or to a spontaneously occurring mutant.
  • a mutant may have been subjected to several mutagenization treatments (a single treatment should be understood one mutagenization step followed by a screening/selection step), but it is presently preferred that no more than 20, or no more than 10, or no more than 5, treatments (or screening/selection steps) are carried out.
  • a presently preferred mutant less than 5%, or less than 1% or even less than 0.1% of the nucleotides in the bacterial genome have been shifted with another nucleotide, or deleted, compared to the mother strain.
  • strain DSM 34195 Extensive screening of 10,000 mutants of DSM 23035 led to the identification of strain DSM 34195.
  • This strain can be used at lower inoculation dosage while achieving low post-acidification and high antifungal activity. Furthermore, the strain has been shown to be combinable with different starter cultures and milk bases, showing better compatibility than DSM 33515. Furthermore, DSM 34195 exhibits good sensorial properties, with its clean and mild taste and lack of off-flavor notes.
  • the present application thus provides the Lacticaseibacillus rhamnosus strain DSM 34195 and mutants maintaining the advantageous properties of DSM34195, when compared under the same conditions.
  • the comparison can be carried out for example as described below: preparing a homogenized milk base consisting of 2.8% protein, 1.2% fat and 10% sucrose, heat-treating the milk base at 95 ⁇ 1°C for 5 min and cooling immediately, inoculating the milk base with the starter culture YF-L812 at 500U per 2500L, inoculated the milk base with L. rhamnosus DSM 34195 and, in parallel, a mutant to be compared, in total concentration of 1 x 10 7 CFU/g,
  • the comparison can be carried out for example as described below: preparing a homogenized milk base consisting of 2.8% protein, 1.2% fat and 10% sucrose, heat-treating the milk base at 95 ⁇ 1°C for 5 min and cooling immediately, inoculating the milk base with the starter culture YF-L812 at 500U per 2500L, inoculated the milk base with L. rhamnosus DSM 34195 and, in parallel, a mutant to be compared, in total concentration of 1 x 10 7 CFU/g,
  • Lacticaseibacillus rhamnosus strains of present application have particular advantages as they reduce the risk of post-acidification while exhibiting antifungal activity, thus improves the storage stability of food products made with these bacteria, in particular the storage stability under conditions above refrigeration temperatures.
  • a "mold” is a fungus that grows in the form of multi-cellular filaments called hyphae.
  • the term “inhibit” in relation to molds refers to a decrease in the growth or sporulation or a reduction in the number or in the concentration of molds, for example in food products and/or on the surface of food products comprising the bacteria of the present application in relation to food products which do not comprise such bacteria.
  • the extent of inhibition provided by the Lacticaseibacillus rhamnosus strain of present application is preferably determined by growth on agar solidified fermented milk in the presence and absence of Lacticaseibacillus rhamnosus bacteria.
  • molds are members of the genus Penicillium, such as Penicillium solitum, Penicillium brevicompactum, Penicillium crustosum, Penicillium roqueforti, Penicillium paneum and Penicillium carneum.
  • Yeasts are fungi growing as single cells.
  • the Lacticaseibacillus rhamnosus strain of the present application i.e., the strain deposited as DSM 34195 and mutants maintaining the advantageous properties inhibit growth of molds and can further inhibit growth of yeasts.
  • the term "inhibit" also refers to a decrease in the growth or a reduction in the number or in the concentration of yeasts, for example in food products and/or on the surface of food products comprising the bacteria of the present application in relation to food products which do not comprise such bacteria.
  • the extent of inhibition provided by the Lacticaseibacillus rhamnosus strain of present application is preferably determined by growth on agar solidified fermented milk in the presence and absence of Lacticaseibacillus rhamnosus bacteria.
  • yeasts are members of the genus Debaryomyces and Torulaspora, such as Debaryomyces hansenii and Torulaspora delbrueckii.
  • Respective compositions may comprise numerous further bacteria including LABs.
  • lactic acid bacteria or "LAB” is used to refer to food-grade bacteria producing lactic acid as the major metabolic end-product of carbohydrate fermentation. These bacteria are related by their common metabolic and physiological characteristics and are usually Gram-positive, low-GC, acid tolerant, non-sporulating, non-respiring, rodshaped bacilli or cocci. During the fermentation stage, the consumption of lactose by these bacteria causes the formation of lactic acid, reducing the pH and leading to the formation of a protein coagulum. These bacteria are thus responsible for the acidification of milk and for the texture of dairy product.
  • lactic acid bacteria encompasses, but is not limited to, bacteria belonging to the genus of Lactobacillus spp., Bifidobacterium spp., Streptococcus spp., Lactococcus spp., such as Lactobacillus delbrueckii subsp.
  • compositions of the present application are therefore characterized in that the composition further comprises at least one further bacterium selected from one or more of the following genera and species Lactobacillus spp., Bifidobacterium spp., Streptococcus spp., Lactococcus spp., such as Lactobacillus delbrueckii subsp.
  • further bacterium selected from one or more of the following genera and species Lactobacillus spp., Bifidobacterium spp., Streptococcus spp., Lactococcus spp., such as Lactobacillus delbrueckii subsp.
  • compositions of the present application comprise bacteria of the species Lacticaseibacillus rhamnosus deposited as DSM 34195 or a mutant Lacticaseibacillus rhamnosus obtainable from the deposited bacteria and one or more further bacteria. In one embodiment, several different strains of the Lacticaseibacillus rhamnosus bacteria are combined.
  • the composition of the present application may additionally comprise cryoprotectants, lyoprotectants, antioxidants, nutrients, fillers, flavorants or mixtures thereof.
  • the composition may be in frozen or freeze-dried form.
  • the composition preferably comprises one or more of cryoprotectants, lyoprotectants, antioxidants and/or nutrients, more preferably cryoprotectants, lyoprotectants and/or antioxidants and most preferably cryoprotectants or lyoprotectants, or both.
  • protectants such as cryoprotectants and lyoprotectants are known to a skilled person in the art.
  • Suitable cryoprotectants or lyoprotectants include mono-, di-, tri-and polysaccharides (such as glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch and gum arabic (acacia) and the like), polyols (such as erythritol, glycerol, inositol, mannitol, sorbitol, threitol, xylitol and the like), amino acids (such as proline, glutamic acid), complex substances (such as skim milk, peptones, gelatin, yeast extract) and inorganic compounds (such as sodium tripolyphosphate).
  • mono-, di-, tri-and polysaccharides such as glucose, mannose, xylose, lactose, sucrose, trehalose, raffinose, maltodextrin, starch and gum arabic (acacia) and
  • Suitable antioxidants include ascorbic acid, citric acid and salts thereof, gallates, cysteine, sorbitol, mannitol, maltose.
  • Suitable nutrients include sugars, amino acids, fatty acids, minerals, trace elements, vitamins (such as vitamin B-family, vitamin C).
  • the composition may optionally comprise further substances including fillers (such as lactose, maltodextrin) and/or flavorants.
  • starter cultures are most commonly added to milk in the form of a starter culture.
  • starter or “starter culture” as used in the present context refers to a culture of one or more food-grade microorganisms, in particular to lactic acid bacteria, which are responsible for the acidification of the milk base. Starter cultures may be fresh but are most frequently frozen or freeze-dried. These products are also known as "Direct Vat Set” (DVS) cultures and are produced for direct inoculation of a fermentation vessel or vat for the production of a dairy product, such as a fermented milk product or a cheese.
  • Respective starter cultures are commercially available from numerous sources, including Premium 1.0, YF-L812, Mild 1.0, Advance 2.0, which are commercially available from Chr.
  • the starter culture comprises Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus.
  • compositions in the form of a solid frozen or freeze-dried starter culture comprising lactic acid bacteria in a concentration of at least 10 9 colony forming units (CFU)/g, or in a concentration of at least IO 10 CFU/g, or in a concentration of at least 10 11 CFU/g.
  • These starter cultures further comprise bacteria of the species Lacticaseibacillus rhamnosus deposited as DSM 34195 or a mutant Lacticaseibacillus rhamnosus obtainable from the deposited bacteria.
  • milk is broadly used in its common meaning to refer to liquids produced by the mammary glands of animals or by plants.
  • the milk may have been processed and the term “milk” includes whole milk, skim milk, fat-free milk, low fat milk, full fat milk, lactose-reduced milk, or concentrated milk.
  • Fat-free milk is non-fat or skim milk product.
  • Low-fat milk is typically defined as milk that contains from about 1% to about 2% fat.
  • Full fat milk often contains 2% fat or more.
  • milk is intended to encompass milks from different mammal and plant sources. Mammal sources of milk include, but are not limited to cow, sheep, goat, buffalo, camel, lama, mare and deer.
  • Plant sources of milk include, but are not limited to, milk extracted from soybean, pea, peanut, barley, rice, oat, quinoa, almond, cashew, coconut, hazelnut, hemp, sesame seed and sunflower seed.
  • milk derived from cows is most preferably used as a starting material for the fermentation.
  • milk also includes fat-reduced and/or lactose-reduced milk products. Respective products can be prepared using methods well known in the art and are commercially available. Lactose-reduced milk can be produced according to any method known in the art, including hydrolyzing the lactose by lactase enzyme to glucose and galactose, or by nanofiltration, electrodialysis, ion exchange chromatograph and centrifugation.
  • milk product or “milk base” is broadly used in the present application to refer to a composition based on milk or milk components which can be used as a medium for growth and fermentation of LAB.
  • the milk product or base comprises components derived from milk and any other component that can be used for the purpose of growing or fermenting LAB.
  • the milk substrate Prior to fermentation, the milk substrate may be homogenized and pasteurized according to methods known in the art.
  • Homogenizing as used herein means intensive mixing to obtain a soluble suspension or emulsion. If homogenization is performed prior to fermentation, it may be performed so as to break up the milk fat into smaller sizes so that it no longer separates from the milk. This may be accomplished by forcing the milk at high pressure through small orifices.
  • “Pasteurizing” as used herein means treatment of the milk substrate to reduce or eliminate the presence of live organisms, such as microorganisms.
  • pasteurization is attained by maintaining a specified temperature for a specified period of time. The specified temperature is usually attained by heating. The temperature and duration may be selected in order to kill or inactivate certain bacteria, such as harmful bacteria. A rapid cooling step may follow.
  • the present application further provides methods of, wherein the fermented product is stored at a temperature above 7°C, preferably at a temperature between 7°C and 25°C.
  • the product may be stored at any time but is preferably stored for a period of at least 14 days and wherein the pH of the fermented milk product is maintained above pH 4.0 during storage.
  • the invention further provides methods of producing a food, feed or pharmaceutical product comprising a method of producing a fermented milk product as described above and the food, feed or pharmaceutical product obtainable by this method.
  • Fermentation is carried out to produce food products, feed products or pharmaceuticals.
  • the terms "fermented milk product”, “food” or “feed” product refer to products obtainable by the fermentation methods of the present application and include cheese, yoghurt, fruit yoghurt, yoghurt beverage, strained yoghurt (Greek yoghurt, Labneh), quark, fromage frais and cream cheese.
  • the term food further encompasses other fermented food products, including fermented meat, such as fermented sausages, and fermented fish products. Examples of fermented milk products include crema acida and sour cream.
  • the term “cheese” is understood to encompass any cheese, including hard, semi-hard and soft cheeses.
  • Examples of cheeses include cottage cheese, tvorog, quarg, feta, Cheddar, parmesan, mozzarella, emmentaler, danbo, gouda, edam, feta-type like UF- feta, soft cheese, pasta filata, continental cheese, blue cheeses, brine cheeses like white brined cheese, queso fresco Camembert and Brie.
  • the person skilled in the art knows how to convert the coagulum into cheese, methods can be found in the literature, see e.g., Kosikowski, F. V., and V. V. Mistry, "Cheese and Fermented Milk Foods", 1997, 3rd Ed. F. V. Kosikowski, L. L. C. Westport, CT.
  • a cheese which has a NaCI concentration below 1.7% (w/w) is referred to as a "low-salt cheese”.
  • yoghurt refers to products comprising Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus and optionally other microorganisms such as Lactobacillus delbrueckii subsp. lactis, Bifidobacterium animalis subsp. lactis, Lactococcus lactis, Lactobacillus acidophilus and Lactobacillus paracasei, or any microorganism derived therefrom.
  • lactis lactis, Bifidobacterium animalis subsp. lactis, Lactococcus lactis, Lactobacillus acidophilus and Lactobacillus paracasei, or any microorganism derived therefrom.
  • lactis lactis
  • lactis Bifidobacterium animalis subsp. lactis
  • Lactococcus lactis Lactobacillus acidophilus
  • Lactobacillus paracasei or any microorganism derived therefrom
  • yoghurt encompasses set yoghurt, stirred yoghurt, drinking yoghurt, Petittreu, heat treated yoghurt, strained or Greek style yoghurt characterized by a high protein level and yoghurt-like products.
  • dairy raw materials e.g. Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus
  • Yoghurts may optionally contain added dairy raw materials (e.g.
  • the yoghurt meets the specifications for fermented milks and yoghurts of the AFNOR NF 04- 600 standard and/or the codex StanA-IIa-1975 standard.
  • the product In order to satisfy the AFNOR NF 04-600 standard, the product must not have been heated after fermentation and the dairy raw materials must represent a minimum of 70% (m/m) of the finished product.
  • Lacticaseibacillus rhamnosus is now known as Lacticaseibacillus rhamnosus as described in
  • Table 1 The applicant has made the following deposits at a Depositary institution having acquired the status of international depositary authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the
  • Mutant strains were selected from the mutant pool by high-throughput screening, which included mutant hit picking from agar, growth of mutants in broth, milk acidification and post-acidification, selection of mutants with low post-acidification antifungal assessment.
  • MRS-Difco agar pH 6.5 was poured into low profile square bioassay dish (Corning 431301). The mutant pool was spread on agar using sterile glass beads and incubated for 2 days at 37°C anaerobically. Approximately 10,000 colonies were hit- picked using QPix2 colony picker (Genetix) into 200-pl 96-well micro-titer plates containing MRS-Difco broth. After an overnight incubation of the micro-titer plates under anaerobic conditions at 37°C, 1 % of the overnight inoculum was used to inoculate milk in 2-ml 96-well micro-titer plates.
  • QPix2 colony picker Genetix
  • the selected mutant strains were collected in 200-pl 96-well microtiter plates and tested for their antifungal activity.
  • the mutant strains have been grown in BD5-1 media overnight at 37°C.
  • 10 pl of the preculture was used to inoculate into 2 ml B-milk (ISO 26323:2009) supplemented with 2 % sucrose and the starter culture YF-812 in 96 micro-titer plates.
  • the milk was incubated at 40°C for approx. 6 hours until a pH of 4.55 was reached in control wells containing wild-type strains.
  • the micro-titer plates were stored at 4°C overnight.
  • a homogenized milk base consisting of 2.8% protein, 1.2% fat and 10% sucrose was heat-treated at 95 ⁇ 1°C for 5 min and cooled immediately. To follow the acidification, a pH indicator was added to the milk and the color change was measured as previously described in section 1.2 but the color values were not converted to pH.
  • a commercial starter culture containing Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus (F-DVS YF-L812) was used to inoculate at 0.02% (v/w) and 270 pL was pipetted into each well. Individual wells were then inoculated with 30pL of L. rhamnosus DSM 34195 or DSM 33515 to four different final inoculation dosages: 5 x 10 6 CFU/g, 1 x 10 7 CFU/g, 2 x 10 7 CFU/g and 3 x 10 7 CFU/g. This was done in 12 replicates across multiple 96 low-well plates. Reference wells were also prepared only containing starter culture ("Blank").
  • the 96 low-well plates were incubated at 43 ⁇ 1°C until an eguivalent of pH 4.55 ⁇ 0.5 was reached. pH was monitored using the added pH indicator as a pH eguivalent. The plates were scanned from the bottom and color values were monitored until a color value eguivalent of 4.55 was reached. Separate plates were made for monitoring post-acidification and antifungal effect.
  • Post-acidification was monitored at 25 ⁇ 1°C by incubating plates on top of scanners and scanning continuously every 2 hours for 21 days. The change in color value (ApH eguivalent) during the incubation was then used to indicate post-acidification and shown in Figure 1.
  • P. roqueforti DSM 33518 was spotted on the surface of the fermented yogurt in the 96 low-well plates in concentration of 50 spores per well. The plates were then incubated at 7 ⁇ 1°C, with a scanner scanning the surface of the plates, for 52 days. The resulting color changes on the surface was used to indicate P. roqueforti inhibition. Time to outgrowth is estimated from the color values and shows the inhibition of P. roqueforti.
  • L. rhamnosus DSM 34195 inhibits P. roqueforti at all examined dosages
  • L. rhamnosus DSM 33515 shows low inhibition at 5 x 10 6 CFU/g and 1 x 10 7 CFU/g, almost full inhibition at 2 x 10 7 CFU/g and full inhibition at 3 x 10 7 CFU/g.
  • a homogenized milk base consisting of 2.8% protein, 1.2% fat and 10% sucrose was heat-treated at 95 ⁇ 1°C for 5 min and cooled immediately.
  • a commercial starter culture F-DVS YF-L812 was used to inoculate at 500U per 2500 L, and the inoculated milk was distributed into 200 ml bottles.
  • L. rhamnosus DSM 34195 was used to inoculate in total concentrations of 5 x 10 6 CFU/g, 1 x 10 7 CFU/g, 2 x 10 7 CFU/g and 3 x 10 7 CFU/g, respectively.
  • L. rhamnosus DSM 33515 was used to inoculate in total concentration of 3 x 10 7 CFU/g.
  • Bottles only inoculated with the starter culture were used as reference. All bottles were incubated in a water bath at 43 ⁇ 1°C and fermented at these conditions until pH of 4.55 ⁇ 0.1 was reached. After fermentation, the bottles were vigorously stirred to break the coagulum and immediately cooled in a cooling chamber.
  • fermented milk samples were inoculated with 50 cfu/g the target contaminant D. hansenii CHCC16374.
  • the inoculated samples were stored at 7°C for 24 days and five times during storage the yeast growth was evaluated as total yeast count.
  • the 200 ml fermented milk was warmed to a temperature of 40°C and 40 ml of a 5% sterile agar solution that had been melted and cooled down to 60°C.
  • This solution of fermented milk and agar was then poured into sterile Petri dishes and the plates were dried in a LAF bench for 30 min.
  • Spore suspension of the following six different molds were spotted in concentration of 500 spores/spot onto the agar plates (3 molds per plate as indicated in Figure 5): P. brevicompactum DSM 32094, P. crustosum DSM 33517, P. solitum DSM 32093, P. carneum DSM 33520, P.
  • paneum DSM 33519 and P. roqueforti DSM 33518 Three molds were spotted on each plate and the target contaminants were added in concentrations of 500 spores/spot. Plates were incubated at 22 ⁇ 1°C for 8 days and regularly examined for mold growth.
  • rhamnosus DSM 33515 was used to inoculate in total concentration of 3 x 10 7 CFU/g. Bottles only inoculated with the starter culture were used as reference. All bottles were incubated in a water bath at 43 ⁇ 1°C and fermented at these conditions until pH of 4.55 ⁇ 0.1 was reached. After fermentation, the bottles were vigorously stirred to break the coagulum and immediately cooled in a cooling chamber.
  • the 200 ml fermented milk was warmed to a temperature of 40°C and added with 40 ml of a 5% sterile agar solution that had been melted and cooled down to 60°C.
  • This solution of fermented milk and agar was then poured into sterile Petri dishes and the plates were dried in a LAF bench for 30 min.
  • Spore suspension of the following three different molds were spotted in concentration of 500 spores/spot onto the agar plates: P. brevicompactum DSM 32094, P. crustosum DSM 33517, P. solitum DSM 32093.
  • the three molds were spotted on one plate and the target contaminants were added in concentrations of 500 spores/spot. Plates were incubated at 22 ⁇ 1°C for 11 days and regularly examined for mold growth.
  • fermented milk samples were inoculated with 50 cfu/g the target contaminant D. hansenii CHCC16374.
  • the inoculated samples were stored at 7°C for 27 days and six times during storage the yeast growth was evaluated as total yeast count.
  • L. rhamnosus DSM 33515 dosed at 3 x 10 7 CFU/g or L. rhamnosus DSM 34195 dosed at 1 x 10 7 CFU/g and 3 x 10 7 CFU/g.
  • L. rhamnosus DSM 34195 shows consistent and high inhibition of D. hansenii at both inoculation levels in combination with the two starter cultures and in the two milk bases. Less consistent inhibition is observed with L. rhamnosus DSM 33515 especially in the fermented product 10% sucrose and with YF- L812 as starter culture (7A).
  • rhamnosus DSM 34195 or L. rhamnosus DSM 33515 was used to inoculate in total concentrations of 1 x 10 7 CFU/g and 3 x 10 7 CFU/g, respectively.
  • Two buckets were used as a reference and was only inoculated with one of the starter cultures. All buckets were incubated in a water bath at 43 ⁇ 1°C and fermented at these conditions until pH of 4.60 ⁇ 0.1 was reached. After fermentation, the buckets were vigorously stirred to break the coagulum and cooled to 25 ⁇ 1°C, dispensed into 250 ml cups and immediately placed at cold storage at 5-7°C.
  • the 200 ml fermented milk was warmed to a temperature of 40°C and added with 40 ml of a 5% sterile agar solution that had been melted and cooled down to 60°C.
  • This solution of fermented milk and agar was then poured into sterile Petri dishes and the plates were dried in a LAF bench for 30 min.
  • Spore suspension of the following three different molds were spotted in concentration of 500 spores/spot onto the agar plates: P. carneum DSM 33520, P. paneum DSM 33519 and P. roqueforti DSM 33518.
  • the three molds were spotted on one plate and the target contaminants were added in concentrations of 500 spores/spot. Plates were incubated at 7 ⁇ 1°C for 34 days and regularly examined for mold growth.
  • fermented milk samples were inoculated with 50 cfu/g the target contaminants Debaryomyces hansenii CHCC16374 or Torulaspora delbrueckii CHCC17090 (Chr. Hansen Culture Collection).
  • the inoculated samples were stored at 7°C for 28 days and five times during storage the yeast growth was evaluated as total yeast count.
  • Figure 10 shows the growth curves for D. hansenii (10A) and T. delbreuckii (10B) during 28 days of storage at 7°C.
  • the growth of the two target contaminants was inhibited to the highest degree by of L. rhamnosus DSM 34195 compared to L. rhamnosus DSM 33515, even at an inoculation level at 1 x 10 7 CFU/g compared to 3 x 10 7 CFU/g which was used for L. rhamnosus DSM 33515. Higher inhibition can be observed in both cases using the starter culture F-DVS YoFlex Mild 1.0 or YoFlex Premium 1.0.
  • L. rhamnosus DSM 34195 shows higher and more consistent anti-yeast and anti-mold effect than L. rhamnosus DSM 33515 in combination with different starter cultures and in different milk bases.
  • This example demonstrates the impact of post-acidification of L. rhamnosus DSM 34195 compared to L. rhamnosus DSM 33515.
  • a homogenized milk base consisting of 2.8% protein, 1.2% fat and 10% sucrose was heat-treated at 95 ⁇ 1°C for 5 min and cooled immediately.
  • Commercial starter culture F-DVS YF-L812 or F-DVS YoFlex Advance 2.0 was used to inoculate at 500U per 2500L, and the inoculated milk was distributed into 3-liter buckets.
  • One bucket was inoculated with L. rhamnosus DSM 33515 in total concentration of 3 x 10 7 CFU/g, one bucket was inoculated with the L.
  • rhamnosus DSM 34195 in total concentration of 1 x 10 7 CFU/g, and one bucket was used as a reference and was only inoculated with the starter culture. All buckets were incubated in a water bath at 43 ⁇ 1°C and fermented at these conditions until pH of 4.60 ⁇ 0.1 was reached. After fermentation, the buckets were vigorously stirred to break the coagulum, cooled in cold water to 25 ⁇ 1°C, dispensed into 200 ml bottles and immediately placed at cold storage at 5-7°C.
  • the 2x three fermented milk samples (starter- only, starter + DSM 34915 and starter-i- DSM 33515) were stored at 25 ⁇ 1°C for 28 days and pH was measured on day 1, 7, 14, 21 and 28.
  • Figure 11 shows the effect on post-acidification when combined with F-DVS YF-L812 (11A) or F-DVS YoFlex Advance 2.0 (11B).
  • L. rhamnosus DSM 33515 induced more post-acidification than L. rhamnosus DSM 34195.
  • a homogenized milk base consisting of 2.8% protein, 14-31% fat and 4% carbohydrates was heat-treated at 85 ⁇ 1°C for 15 sec, followed by homogenization and immediate cooling.
  • the crema acida was then chemically acidified with a citric acid solution to adjust the pH to 4.6 and distributed into 200 ml bottles.
  • L. rhamnosus DSM 34195 was used to inoculate a total concentration of 1 x 10 7 CFU/g.
  • L. rhamnosus DSM 33515 was used to inoculate a total concentration of 3 x 10 7 CFU/g.
  • Bottles without addition of L. rhamnosus were used as a reference. All bottles were stored after inoculation at 7°C.
  • fermented milk samples were inoculated with 50 cfu/g the target contaminant D. hansenii CHCC16374.
  • the inoculated samples were stored at 7°C for 24 days and five times during storage the yeast growth was evaluated as total yeast count.
  • the 200 ml fermented milk was warmed to a temperature of 40°C and 40 ml of a 5% sterile agar solution that had been melted and cooled down to 60°C.
  • This solution of fermented milk and agar was then poured into sterile 6-well plates and the plates were dried in a LAF bench for 30 min.
  • Spore suspension of the following six different molds were spotted in concentration of 500 spores/spot onto the plates (One mold per well as indicated in Figure 12): P. brevicompactum DSM 32094, P. crustosum DSM 33517, P. solitum DSM 32093, P. carneum DSM 33520, P. paneum DSM 33519 and P.
  • roqueforti DSM 33518 and plates were incubated 12 ⁇ 1°C for 15 days and 7 ⁇ 1°C for 22 days regularly examined for mold growth. Plates incubated at 22 ⁇ 1°C for 7 days, were inoculated with two molds, including P. crustosum DSM 33517 and P. roqueforti DSM 33518.
  • DSM 34195 has higher anti-yeast and anti-mold effect and lower post-acidification than DSM 33515 in crema acida, even when DSM 34195 is applied at lower dosage.

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Abstract

La présente application concerne le domaine de la technologie laitière. La présente invention concerne la bactérie Lacticaseibacillus rhamnosus DSM 34195 ou ses mutants, ainsi que son utilisation pour la production de denrées alimentaires, d'aliments pour animaux ou de produits pharmaceutiques, plus particulièrement des produits laitiers fermentés. La bactérie Lacticaseibacillus rhamnosus DSM 34195 présente une faible post-acidification dans les produits et présente des effets antifongiques. L'invention concerne également des compositions comprenant Lacticaseibacillus rhamnosus DSM 34195 ou ses mutants.
EP23838018.2A 2022-12-27 2023-12-21 Bioprotectrice lacticaseibacillus rhamnosus à faible postacidification Pending EP4642896A1 (fr)

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