WO2025032004A1

WO2025032004A1 - Symbiotic milk products containing a probiotic and galacto-oligosaccharides

Info

Publication number: WO2025032004A1
Application number: PCT/EP2024/072052
Authority: WO
Inventors: Jan Knol; Patricia LEVY; Beatriz ROSON; Natasha MOTA MACHADO
Original assignee: Gervais Danone SA
Current assignee: Gervais Danone SA
Priority date: 2023-08-04
Filing date: 2024-08-02
Publication date: 2025-02-13
Anticipated expiration: 2026-02-04
Also published as: MX2026001383A; WO2025032353A1

Abstract

The present invention relates to fermented milk products containing galacto-oligosaccharides (GOS) and a lactic acid bacterium such as a probiotic L casei or L. paracasei, compositions for producing the same and methods for producing the same.

Description

SYMBIOTIC MILK PRODUCTS CONTAINING A PROBIOTIC AND GALACTO-OLIGOSACCHARIDES Field of the invention The present invention relates to fermented milk products containing galacto-oligosaccharides (GOS) and a lactic acid bacterium such as a probiotic Lactobacillus casei or Lactobacillus paracasei, compositions for producing the same and methods for producing the same. Technical Lactobacillus casei and Lactobacillus paracasei (also known as Lacticaseibacillus (para)casei) are lactic acid bacteria which are commonly used as probiotics. Probiotics are defined as "living microorganisms, which when consumed in adequate amounts, have a beneficial effect on the health of the host" (Report of the joint expert consultation CAM/OMS on the evaluation of the health and nutritional properties of probiotics in foods, 2001). This definition implies that, in order to exert its beneficial effect, the microorganism concerned must be present in a sufficient quantity in the ready-to-eat product and should stay alive after consumption. The maintenance of the viability and of the capacity to growth of probiotic bacteria after consumption thus constitutes an essential element of the quality of these products. Prebiotics, such as galacto-oligosaccharides (GOS), are ingredients providing health benefits when fermented by the native microflora in the gastrointestinal tract of a subject. Combination of probiotics and prebiotics can give rise to a synergistic effect. In particular, the effect of combined GOS and probiotics can be beneficial for modulation of gut microbiota composition and for modulation of immune response. It has been shown that this synergistic effect depends on the ability of probiotic bacteria to grow on and consume GOS in the gastrointestinal tract of the subject. The effect of such symbiotic products have been evidenced in particular in infants, so that several infant formula compositions comprising GOS and probiotic bacteria, for example, have been used commercially. There is a constant need to provide further symbiotic compositions. In particular, there is a need for products that can improve the efficiency of a probiotic by facilitating its survival or its growth after its ingestion by an individual. Summary of the invention The present invention allows the production in a simple process of a symbiotic milk product, i.e. a milk product comprising a probiotic and GOS fibers which will be used as a growth substrate in the gastrointestinal tract by the probiotic. The present invention follows from the unexpected finding made by the Inventors that the fermentation of a milk substrate by a probiotic, which is Lactobacillus casei or Lactobacillus paracasei, in presence of a beta-galactosidase having transgalactosylating activity allows the production of stable GOS, which are not digested by the probiotic during the production or shelf life of the product but can be used as a growth substrate by the probiotic in the human gastrointestinal tract. The GOS produced by the enzymatic reaction simultaneously with the fermentation will not be digested by the probiotic in the acidic conditions of the fermentation, but they will be consumed when the pH is less acid in the gastrointestinal tract of the consumer. In addition, the invention allows a reduction of lactose content, which is particularly advantageous for individuals with lactose intolerance. An aspect of the invention thus relates to a composition for producing a fermented milk product containing galacto-oligosaccharides (GOS) fibers comprising: - a milk substrate containing lactose, - a lactic acid bacterium, such as a probiotic selected from the group comprising Lactobacillus casei and Lactobacillus paracasei, and - a β-galactosidase having a transgalactosylating activity, said β-galactosidase being able to convert lactose into GOS fibers that can be metabolized by the probiotic. Another aspect of the invention relates to a method for producing a fermented milk product containing GOS fibers comprising: - a step of fermentation of the composition as defined above in conditions allowing the β-galactosidase having transgalactosylating activity to convert lactose into GOS fibers. Another aspect of the invention relates to a fermented milk product containing GOS fibers obtained by the method as defined above. Another aspect of the invention relates to a fermented milk product containing GOS fibers comprising: - a fermented milk substrate, - a lactic acid bacterium, such as a probiotic selected from the group comprising Lactobacillus casei and Lactobacillus paracasei, - a β-galactosidase having a transgalactosylating activity, said β-galactosidase being able to convert lactose into GOS fibers that can be metabolized by the probiotic, and - GOS fibers resulting from the conversion of lactose originally present in the milk. Detailed description of the invention As used herein, the words “comprise”, “comprising” and the like are to be construed in an inclusive sense, that is to say, in the sense of “including, but not limited to”, opposed to an exclusive or exhaustive sense. As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the term “x% (w/w)” or “x% w/w” is equivalent to “x g per 100 g”. Unless indicated otherwise, all % value shall be taken to indicate x% w/w. Unless defined otherwise, all technical and scientific terms have and should be given the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the expression “fermented milk product” shall be taken to mean a product or a composition derived from dairy milk by the acidifying action of at least one lactic acid bacterium, such as a yogurt (e.g., a set, stirred or drink yogurt), or a fresh cheese such as a white cheese or a “petit- Suisse”. It can also be a strained fermented milk such as a strained yoghurt (e.g., a concentrated or Greek-style yoghurt). It can also be a fermented milk product wherein the total protein content is between 0 and 10 g/100g of product and the fat content between 0 and 5 g/100g of product. As used herein, the terms "galacto-oligosaccharides" or "GOS" refer to oligosaccharides composed of different galactosyl residues (usually from 2 to 9 units) and a terminal glucose linked by (3-glycosidic bonds, such as (β-(1-2), (β-(1-3), (β-(1-4), and (β-(1-6). As used herein, the term "beta-galactosidase having transgalactosylating activity" refers to one or more enzymes with enzymatic activity of enzyme class EC 3.2.1.23, also called beta-D-galactoside galactohydrolase, exo-(1->4)-beta-D-galactanase or lactase, which catalyses the hydrolysis of terminal non-reducing beta-D-galactose residues in beta-D-galactosides, as well as transgalactosylation by transferring a galactose moiety of a beta-D-galactoside to another sugar molecule. In presence of a milk substrate, the enzyme works with two main mechanisms: firstly, it hydrolyses the lactose into 2 monosaccharaides, one molecule of glucose and one molecule of galactose; secondly, it polymerizes several galactoses on lactose to obtain GOS and glucose remains as not polymerized. As used herein, EC (Enzyme Committee) numbers refer to the definition of enzymatic activity and nomenclature given by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology as in force on 3rd July 2019. As used herein, the word "fiber" refers to carbohydrate polymers with three or more monomeric units, which are neither digested nor absorbed in the human small intestine. Examples of fibers are inulin, oligosaccharides such as galactooligosaccharides (GOS), fructo-oligosaccharides (FOS), xylo- oligosaccharides (XOS), manno-oligo-saccharides, gluco-oligosaccharides, polydextrose, natural gums such as guar gum and acacia gum, mucilages, pectins, arabinogalactan, beta-glucans, tagatose, and resistant starch. Composition for producing a fermented milk product In an aspect, the invention relates to a composition for producing a fermented milk product containing galacto-oligosaccharides (GOS) fibers comprising: - a milk substrate containing lactose, - a lactic acid bacterium, such as a probiotic selected from the group comprising Lactobacillus casei and Lactobacillus paracasei and - a β-galactosidase having a transgalactosylating activity, said β-galactosidase being able to convert lactose into GOS fibers that can be metabolized by the probiotic. The lactic acid bacterium according to the invention preferably belongs to an Aerococcaceae, Carnobacteriaceae, Enterococcaceae, Lactobacillaceae, Leuconostocaceae, Streptococcaceae or Bifidobacteriaceae family and more preferably to an Aerococcus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus, Weissella or Bifidobacterium genus. More preferably, the lactic acid bacterium according to the invention belongs to a Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus delbruckei, in particular L. delbruckei supsb. bulgaricus or lactis, Lactobacillus diolivorans, Lactobacillus fermentum, Lactobacillus fructivorans, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus jensenii, Lactobacillus kunkeei, Lactobacillus mali, Lactobacillus nagelii, Lactobacillus paracasei, en particulier L. paracasei subsp. paracasei, Lactobacillus plantarum, Lactobacillus vini, Lactobacillus rhamnosus, Streptococcus thermophilus, Streptococcus lactis, Streptococcus raffinolactis, Streptococcus cremoris, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, or Bifidobacterium longum species. In a preferred embodiment, the lactic acid bacterium is a probiotic, more preferably a probiotic selected from the group comprising Lactobacillus casei and Lactobacillus paracasei. As used herein, the term “probiotic” designates any lactic acid bacterium that has a beneficial effect on the health of the host. In an embodiment, the milk substrate may be a milk, i.e. non-human milk, preferably cow milk. For example, the milk substrate may be skimmed milk, semi-skimmed milk and/or whole milk. In an embodiment, the milk substrate may be obtained from milk which has been modified in composition by adjustment of fat content, protein content, lactose content and/or water content. In an embodiment, the milk substrate may correspond to an aqueous composition comprising one or more dairy ingredients. The term "dairy ingredients" relates to ingredients derived from a non-human mammal milk, such as cow milk, goat milk, ewe milk, camel milk, donkey milk. For avoidance of doubt, the term "dairy ingredients" includes milk, i.e. non-human mammal milk, preferably cow milk. Preferably, the dairy ingredients are ingredients derived from cow milk. Examples of dairy ingredients include milk, milk fat, milk powder, skim milk, milk proteins, dairy curd, cream, buttermilk, condensed milk and combinations thereof. The milk may be a whole milk, semi-skimmed milk or a skimmed milk. In an embodiment, the milk substrate corresponds to an aqueous composition obtained by reconstituting milk powder, lactose, whey permeate and/or sweet whey into an aqueous liquid, such as water and/or cow milk. By "milk powder", it is understood non-human mammal milk powder, preferably cow milk powder. For example, the milk substrate may be skimmed milk powder, semi- skimmed milk powder and/or whole milk powder reconstituted into water. The cow milk may be skimmed milk, whole milk, semi-skimmed milk or combination thereof. In an embodiment, the milk substrate comprises at least 4 % (w/w) lactose by dry weight of the milk substrate. Preferably, the milk substrate comprises at least 8 % (w/w), at least 10 % (w/w) or at least 15 % (w/w) lactose by dry weight of the milk substrate. More preferably, the milk substrate comprises at least 20 % (w/w), preferably at least 25 % (w/w) or at least 30 % (w/w) lactose by dry weight of the milk substrate. The concentration of lactose in the composition impacts positively the GOS production and prevents for longer time their degradation, as enough lactose still remain in the medium. In an embodiment, the β-galactosidase having a transgalactosylating activity is a microbe derived β- galactosidase, in particular derived from bacteria or fungi. In an embodiment, the β-galactosidase having a transgalactosylating activity is a Bifidobacterium derived β-galactosidase. As an illustrative example, a β-galactosidase having a transgalactosylating activity appropriate to be used in the present invention is disclosed in WO 01/90317. In an embodiment, the β-galactosidase is a protein having at least 90% sequence identity with the amino acid sequence SEQ ID NO: 1. SEQ ID NO: 1 MAVRRLGGRI VAFAATVALS IPLGLLTNSA WAVEDATRSD STTQMSSTPE VVYSSAVDSK QNRTSDFDAN WKFMLSDSVQ AQDPAFDDSA WQQVDLPHDY SITQKYSQSN EAESAYLPGG TGWYRKSFTI DRDLAGKRIA INFDGVYMNA TVWFNGVKLG THPYGYSPFS FDLTGNAKFG GENTIVVKVE NRLPSSRWYS GSGIYRDVTL TVTDGVHVGN NGVAIKTPSL ATQNGGDVTM NLTTKVANDT EAAANITLKQ TVFPKGGKTD AAIGTVTTAS KSIAAGASAD VTSTITAASP KLWSIKNPNL YTVRTEVLNG GKVLDTYDTE YGFRWTGFDA TSGFSLNGEK VKLKGVSMHH DQGSLGAVAN RRAIERQVEI LQKMGVNSIR TTHNPAAKAL IDVCNEKGVL VVEEVFDMWN RSKNGNTEDY GKWFGQAIAG DNAVLGGDKD ETWAKFDLTS TINRDRNAPS VIMWSLGNEM MEGISGSVSG FPATSAKLVA WTKAADSTRP MTYGDNKIKA NWNESNTMGD NLTANGGVVG TNYSDGANYD KIRTTHPSWA IYGSETASAI NSRGIYNRTT GGAQSSDKQL TSYDNSAVGW GAVASSAWYD VVQRDFVAGT YVWTGFDYLG EPTPWNGTGS GAVGSLAVAE ELVLRHRRHR RLPEDTYYFY QSQWNDDVHT LHILPAWNEN VVAKGSGNNV PVVVYTDAAK VKLYFTPKGS TEKRLIGEKS FTKKTTAAGY TYQVYEGSDK DSTAHKNMYL TWNVPWAEGT ISAEAYDENN RLIPEGSTEG NASVTTTGKA AKLKADADRK TITADGKDLS YIEVDVTDAN GHIVPDAANR VTFDVKGAGK LVGVDNGSSP DHDSYQADNR KAFSGKVLAI VQSTKEAGEI TVTAKADGLQ SSTVKIATTA VPGTSTEKTV RSFYYSRNYY VKTGNKPILP SDVEVRYSDG TSDRQNVTWD AVSDDQIAKA GSFSVAGTVA GQKISVRVTM IDEIGALLNY SASTPVGTPA VLPGSRPAVL PDGTVTSANF AVHWTKPADT VYNTAGTVKV PGTATVFGKE FKVTATIRVQ RSQVTIGSSV SGNALRLTQN IPADKQSDTL DAIKDGSTTV DANTGGGANP SAWTNWAYSK AGHNTAEITF EYATEQQLGQ IVMYFFRDSN AVRFPDAGKT KIQISADGKN WTDLAATETI AAQESSDRVK PYTYDFAPVG ATFVKVTVTN ADTTTPSGVV CAGLTEIELK TATSKFVTNT SAALSSLTVN GTKVSDSVLA AGSYNTPAII ADVKAEGEGN ASVTVLPAHD NVIRVITESE DHVTRKTFTI NLGTEQEFPA DSDERDYPAA DMTVTVGSEQ TSGTATEGPK KFAVDGNTST YWHSNWTPTT VNDLWIAFEL QKPTKLDALR YLPRPAGSKN GSVTEYKVQV SDDGTNWTDA GSGTWTTDYG WKLAEFNQPV TTKHVRLKAV HTYADSGNDK FMSASEIRLR KAVDTTDISG ATVTVPAKLT VDRVDADHPA TFATKDVTVT LGDATLRYGV DYLLDYAGNT AVGKATVTVR GIDKYSGTVA KTFTIELKNA PAPEPTLTSV SVKTKPSKLT YVVGDAFDPA GLVLQHDRQA DRPPQPLVGE QADERGLTCG TRCDRVEQLR KHENREAHRT GLDHLEFVGA ADGAVGEQAT FKVHVHADQG DGRHDDADER DIDPHVPVDH AVGELARAAC HHVIGLRVDT HRLKASGFQI PADDMAEIDR ITGFHRFERH VG Table 1. Example of appropriate β-galactosidase with a transgalactosylating activity In an embodiment, the β-galactosidase is a protein having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the amino acid sequence SEQ ID NO: 1. In an embodiment, the β-galactosidase is a protein consisting of the amino acid sequence SEQ ID NO: 1. In an embodiment, the β-galactosidase is the Nurica^TM enzyme (commercialized by Dupont). In an embodiment, the β-galactosidase is the Zymstar^TM enzyme (commercialized by Dupont). In an embodiment, the β-galactosidase is the NS43104 enzyme (commercialized by Novozyme). The GOS produced by the action of the β-galactosidase and the digestion of said GOS by the probiotic can be measured by methods that are well-known by a skilled person in the art. In the present invention, “Lactobacillus casei” refers to strains belonging to the Lactobacillus casei species, which has been reclassified as the Lacticaseibacillus casei species (Zheng et al., Int J Syst Evol Microbiol 70:2782-2858, 2020). In the present invention, the “Lactobacillus paracasei” species refers to strains previously classified as belonging to the Lactobacillus casei species, which has been reclassified as Lacticaseibacillus paracasei species (Zheng et al., 2020). In a preferred embodiment, the probiotic is a L. paracasei strain. Typically, said given L. paracasei is a probiotic strain with immunomodulatory properties. In a preferred embodiment, the L. paracasei strain is selected from the group comprising L. paracasei CNCM I-1518 and L. paracasei CNCM I-3689. As used herein, the term “CNCM I-“ followed by a 4 digit number shall be taken to refer to a strain deposited at the Collection Nationale de Cultures de Microorganismes (CNCM) 25 rue du Docteur Roux, Paris, France under the Budapest Treaty with an accession number corresponding to said 4 digit number, e.g. CNCM I-1518. L. paracasei CNCM I-1518 is disclosed in EP0794707 and was deposited, according to the Budapest Treaty, at the Collection Nationale de Cultures de Microorganismes (CNCM) 25-28 rue du Docteur Roux, 75724 Paris, France, on September 28, 1994 with the reference CNCM 1-1518. L. paracasei CNCM I-3689 is disclosed in WO2009122042 and was deposited, according to the Budapest Treaty, at deposited at the Collection Nationale de Cultures de Microorganismes 25-28 rue du Docteur Roux, 75724 Paris, France, on November 9, 2006 with the reference CNCM I-3689. CNCM I-1518 and CNCM I-3689 strains both have immuno-modulatory properties (see for example WO2009/130423). L. paracasei strains obtained by mutagenesis or by genetic transformation of CNCM I-1518 or CNCM I- 1518 strains, which keep its probiotic properties, are also encompassed in the present invention. In an embodiment, the composition as defined above further comprises a compound to improve flavor, texture and/or color. In an embodiment, the composition as defined above further comprises dextrose. Dextrose is used to bring caramel notes and color. In a specific embodiment, the invention relates to a composition as defined above comprising: - a skimmed milk powder, - a L. paracasei strain, preferably the L. paracasei strain deposited with the CNCM under the number I-1518, - a β-galactosidase having a transgalactosylating activity, preferably a protein consisting of the amino acid sequence SEQ ID NO 1,and - water. In an embodiment, the invention relates to a composition as defined above, said composition further comprising dextrose. In a specific embodiment, the composition as defined above can be prepared by mixing water, skimmed milk powder and dextrose, pre-heating the mixture, homogenizing the mixture, sterilizing/caramelizing the mixture, adding the probiotic and the β-galactosidase to the mixture. In an embodiment, the composition as defined above comprises only one lactic acid bacterium which is a probiotic, preferably selected from the group comprising L. casei and L. paracasei. In an embodiment, the composition as defined above comprises only one lactic acid bacterium which is a probiotic selected from the group comprising L. paracasei CNCM I-1518 and L. paracasei CNCM I- 3689.

In another aspect, the invention relates to a method for producing a fermented milk product containing GOS fibers comprising: - a step of fermentation of the composition as defined above in conditions allowing the β-galactosidase having transgalactosylating activity to convert lactose into GOS fibers. In a preferred embodiment, the composition is fermented at 37°C +/-1°C, preferably at 37°C +/- 0.5°C, for at least about 3 days, preferably for about 5 days (120h). This temperature allows the fermentation conducted by the probiotic simultaneously with the enzymatic reaction made by the β-galactosidase having transgalactosylating activity. The fermentation step is monitored so that it can be stopped when the desired population of probiotic and/or the desired quantity of GOS fibers and/or the desired pH are reached. In an embodiment, the invention relates to a method as defined above, said method further comprising: - a step of inactivating the β-galactosidase having a transgalactosylating activity when a desired quantity of GOS fibers or reduction of lactose content is reached. In an embodiment, the β-galactosidase having a transgalactosylating activity is inactivated when at least a quantity of 1.4 gram of GOS fibers per 100 grams of composition is reached. In an embodiment, the β-galactosidase having a transgalactosylating activity is inactivated when the lactose content is less than 1% (w/w), preferably less than 0.1% (w/w). In an embodiment, the invention relates to a method as defined above, wherein the β-galactosidase having a transgalactosylating activity is inactivated by the acidification of the composition, preferably when pH reaches 3.8 or less. In an embodiment, the invention relates to a method as defined above, wherein the β-galactosidase having a transgalactosylating activity can be inactivated by heat treatment. In an embodiment, the invention relates to a method as defined above, wherein the β-galactosidase having a transgalactosylating activity is not inactivated by heat treatment. In an embodiment, the fermentation step is stopped when the composition reaches pH 3.6 +/- 0.05. In an embodiment, the invention relates to a method as defined above, wherein a lactase enzyme is added. Such a lactase enzyme can be used to digest residual lactose. In a preferred embodiment, the invention relates to a method as defined above, wherein after the fermentation step, the composition comprises protein 5.7% (w/w), GOS 3.2 % (w/w), lactose 1.2 % (w/w). In an embodiment, the invention relates to a method as defined above, said method further comprising: - a step of adding at least an extra fiber, preferably an extra fiber that is not metabolized by the probiotic but is metabolized by the gut microbiome of a consumer. In an embodiment, the extra fiber is selected from the group comprising inulin, fructo-oligosaccharides (FOS), xylo-oligosaccharides (XOS), mannooligo-saccharides, gluco-oligosaccharides, polydextrose, natural gums such as guar gum and acacia gum, mucilages, pectins, arabinogalactan, beta-glucans, tagatose, and resistant starch, preferably polydextrose. In a preferred embodiment, the extra fiber is added so that the final product comprises at least 1.5 grams, preferably 3.0 grams, more preferably 3.3 grams of total fibers per 100 grams of product. In an embodiment, the invention relates to a method as defined above, said method further comprising: - a step of adding sugar. In an embodiment, the invention relates to a method as defined above, said method further comprising: - a step of adding a syrup. In an embodiment, the syrup comprises water, liquid sugar, polydextrose and citric acid. Fermented milk products containing GOS fibers In another aspect, the invention relates to a fermented milk product containing GOS fibers obtained by the method as defined above. In another aspect, the invention relates to a fermented milk product containing GOS fibers comprising: - a fermented milk substrate, - a lactic acid bacterium, such as a probiotic selected from the group comprising Lactobacillus casei and L. paracasei, - a β-galactosidase having a transgalactosylating activity, said β-galactosidase being able to convert lactose into GOS fibers that can be metabolized by the probiotic, and - GOS fibers resulting from the conversion of lactose originally present in the milk substrate. In an embodiment, the probiotic is selected from the group comprising L. paracasei CNCM I-1518 and L. paracasei CNCM I-3689. In an embodiment, the invention relates to a fermented milk product containing GOS fibers comprising: - a fermented milk substrate, - a probiotic selected from the group comprising L. paracasei CNCM I-1518 and L. paracasei CNCM I- 3689 - a β-galactosidase having a transgalactosylating activity, said β-galactosidase being able to convert lactose into GOS fibers that can be metabolized by the probiotic, and - GOS fibers resulting from the conversion of lactose originally present in the milk substrate. In an embodiment, the invention relates to a fermented milk product as defined above, said product further comprising at least an extra fiber, preferably an extra fiber that is not metabolized by the probiotic but is metabolized by the gut microbiome of a consumer. In an embodiment, the invention relates to a fermented milk product as defined above, wherein said extra fiber is selected from the group comprising inulin, fructo-oligosaccharides (FOS), xylo- oligosaccharides (XOS), mannooligo-saccharides, gluco-oligosaccharides, polydextrose, natural gums such as guar gum and acacia gum, mucilages, pectins, arabinogalactan, beta-glucans, tagatose, and resistant starch, preferably polydextrose. In an embodiment, the invention relates to a fermented milk product as defined above, said product comprising at least 1.5 grams, preferably 3.0 grams, more preferably 3.3 grams, of total fibers per 100 grams of product. In an embodiment, the invention relates to a fermented milk product as defined above, said product comprising at least 1x10⁸ CFU of the probiotic per 100 grams of product. As used herein, the term “cfu” or “CFU” shall be taken to be an abbreviation of the term “colony forming unit”. In an embodiment, the invention relates to a fermented milk product as defined above, said product further comprising added sugar. In an embodiment, the invention relates to a fermented milk product as defined above, said product further comprising a syrup, preferably a syrup comprising water, liquid sugar, polydextrose and citric acid. In an embodiment, the invention relates to a fermented milk product as defined above, wherein the quantity of total carbohydrates does not exceed 15 grams, preferably 11 grams, per 100 grams of product. In an embodiment, the invention relates to a fermented milk product as defined above, which has a low lactose content. In an embodiment, the invention relates to a fermented milk product as defined above, wherein the quantity of lactose does not exceed 1.0 gram, preferably 0.1 gram, per 100 grams of product. In an embodiment, the invention relates to a fermented milk product as defined above, further comprising a lactase enzyme. In a particular embodiment, the invention relates to a fermented milk product as defined above, wherein said product does not comprise added sugar. In a particular embodiment, the fermented milk product comprises 43 % (w/w) of a composition as defined above and 57 % (w/w) of a syrup. In an embodiment, the fermented milk product has a protein content from 1% to 15% (w/w). In an embodiment, the fermented milk product has a fat content from 0% to 5% (w/w). The various features of the present invention referred to in individual aspects above apply, as appropriate, to other aspects mutatis mutandis. Consequently, features specified in a given aspect of the invention may be combined with features specified in other aspects as appropriate. The invention will be further illustrated by the following non-limiting Figures and Examples.

Description of the figures Figure 1. Fermentation of composition A. Figure 2. Evolution of lactose content during fermentation of composition A. Figure 3. Fermentation of composition B. Figure 4. Evolution of lactose content during fermentation of composition B.

Examples Example I - Composition A Production process of the composition A Cooled fluid milk is automatic transferred to the mixing tank. Then, dextrose and skimmed milk powder are manually charged to the mixing tank. Ingredients are dispersed and blended for 15 minutes, and physicochemical analysis performed to check the right mixture composition. The mix is hydrated for 1 hour at 6 °C, under smoothly agitation to guarantee a homogeneous mixture. The standardized mix passes through filters (0.5 mm) to remove any possible physical contamination, then sterilized for food safety security. The Maillard reaction, which gives the specific caramel taste and color to the product, happens right after the sterilization of the standardized mix and takes place in the previously sterilized mass tank. It consists in cooking the mix at 96ºC to reach the color target. At this temperature, the glucose and amino acids from milk proteins react to form « caramel » products and change the mix color (browning). Once the designed color is achieved, the mix is cooled down to the fermentation temperature (37ºC) before inoculation and enzyme addition. The fermentation step takes more than 3 days (>72 hours) for GOS production and 5 days (120 hours) to reach the breaking pH. Fermented mass is cooled in-tank, by circulating chilled water in the double wall jacket, while providing a continuous agitation. This way of cooling the mass, gives the opportunity to use the fermentation tank as a storage tank before mixing with the Sugar syrup. Composition A was prepared with the following ingredients (Table 2). Ingredients Quantity Milk 0.05% fat 5419.93 L 86.887 % Skimmed milk powder 550.00 kg 8.335 % Dextrose 96.75 kg 1.500 % CNCM I-1518 6 packets 0.040 % Enzyme Nurica^TM 2.00 kg 0.0300 % Water 206.92 L 3.208 % Total 6450.00 kg 100% Table 2. Ingredients of composition A. Composition A was fermented at 37°C for 120h (Figure 1). As shown in figure 1, the quantity of GOS remains stable, i.e. they are not digested by the L. paracasei strain at this pH conditions. This is particularly advantageous in that GOS are a substrate for growth for L. paracasei in less acidic conditions (see for example, Watson et al., J Appl Microbiol, 114:1132- 1146, 2012). Consequently, GOS are stable in the fermented product but they will be metabolized by the L. paracasei strain in the gastrointestinal tract when ingested by the consumer. Lactose in the end of fermentation was 1,19% (Figure 2). The β-galactosidase was inactivated when pH reaching 3.8. After fermentation, a syrup has been added to the composition A. The syrup comprises the following ingredients (Table 3). Ingredients Quantity Decorated potable water 5645.00 L 66.026 % Liquid sugar (66.5 % total sucrose) 1698.88 L 26.316 % Polydextrose Litesse Powder 369.36 kg 4.320 % Citric acid granulated 0.855 kg 0.010 % Citric Vanilla flavor 9.542 kg 0.112 % Canne sugar flavor 0.752 kg 0.009 % Water 274.28 L 3.208 % Total 8550.00 kg 100% Table 3. Ingredients of the syrup. The addition of polydextrose is advantageous in that this will be beneficial for the intestinal flora but it will not be consumed by L. paracasei, i.e. the probiotic will not compete with the intestinal microflora as regards with this substrate. A fermented milk product containing galacto-oligosaccharides (GOS) fibers has been obtained by mixing the fermented composition A (43%) with the syrup (57%). Example 2 - Composition B Production process of the composition B Cooled water is automatic transferred to the mixing tank. Then, dextrose and skimmed milk powder are manually charged to the mixing tank. Ingredients are dispersed and blended for 15 minutes, and physicochemical analysis performed to check the right mixture composition. The mix is hydrated for 1 hour at 6 °C, under smoothly agitation to guarantee a homogeneous mixture. The standardized mix passes through filters (0.5 mm) to remove any possible physical contamination, then sterilized for food safety security. The Maillard reaction, which gives the specific caramel taste and color to the product, happens right after the sterilization of the standardized mix and takes place in the previously sterilized mass tank. It consists in cooking the mix at 96ºC to reach the color target. At this temperature, the glucose and amino acids from milk proteins react to form « caramel » products and change the mix color (browning). Once the designed color is achieved, the mix is cooled down to the fermentation temperature (37ºC) before inoculation and enzyme addition. The fermentation step takes more than 3 days (>72 hours) for GOS production and 5 days (120 hours) to reach the breaking pH. Fermented mass is cooled in-tank, by circulating chilled water in the double wall jacket, while providing a continuous agitation. This way of cooling the mass, gives the opportunity to use the fermentation tank as a storage tank before mixing with the Sugar syrup. Composition B was prepared with the following ingredients (Table 4). Ingredients Quantity Decorated potable water 5067.97 L 78.573 % Skimmed milk powder 1075.00 kg 16.649 % Dextrose 96.75 kg 1.500 % CNCM I-1518 6 packets 0.040 % Enzyme Nurica^TM 2.00 kg 0.0300 % Water 206.92 L 3.208 % Total 6450.00 kg 100% Table 4. Ingredients of composition B. Composition B is fermented at 37°C for 111h (Figure 3). As shown in figure 2, the quantity of GOS remains stable, i.e. they are not digested by the L. paracasei strain at this pH conditions. This is particularly advantageous in that GOS are a substrate for growth for L. paracasei in less acidic conditions (see for example, Watson et al., J Appl Microbiol, 114:1132- 1146, 2012). Consequently, GOS are stable in the fermented product but they will be metabolized by the L. paracasei strain in the gastrointestinal tract when ingested by the consumer. Lactose in the end of fermentation was 1,52% (Figure 4). After fermentation, a syrup has been added to the composition B (same syrup as used in Example 1, see Table 3). A fermented milk product containing galacto-oligosaccharides (GOS) fibers has been obtained by mixing the fermented composition B (43%) with the syrup (57%). GOS concentration was monitored in the final product (Table 5). Shelf life time 12 days 51 days Analyses date 03/04/2023 12/05/2023 GOS (%) 2 2 Table 5. Stability of GOS in the final product. GOS concentration was stable in the final product when kept at 10 °C for 51 days. There was no relevant GOS amount variation in the product shelf life. Interestingly, the GOS produced remain stable in the product, with no need of adding a stabilizer.

CLAIMS 1. A composition for a fermented milk product containing galacto-oligosaccharides (GOS) fibers comprising:

- a milk substrate containing lactose, - a lactic acid bacterium, such as a probiotic selected from the group comprising Lactobacillus casei and Lactobacillus paracasei, and - a β-galactosidase having a transgalactosylating activity, said β-galactosidase being able to convert lactose into GOS fibers that can be metabolized by the probiotic. 2. The composition according to claim 1, comprising: - a milk substrate containing lactose, - a probiotic selected from the group comprising Lactobacillus casei and Lactobacillus paracasei, and - a β-galactosidase having a transgalactosylating activity, said β-galactosidase being able to convert lactose into GOS fibers that can be metabolized by the probiotic. 3. The composition according to claim 1 or 2, wherein the β-galactosidase is a microbe derived β- galactosidase, preferably a bacterium derived β-galactosidase, more preferably a Bifidobacterium derived β-galactosidase. 4. The composition according to any one of claims 1 to 3, wherein the β-galactosidase is a protein having at least 90% sequence identity with the amino acid sequence SEQ ID NO 1. 5. The composition according to any one of claims 1 to 4, wherein the probiotic is selected from the group comprising L. paracasei CNCM I-1518 and L. paracasei CNCM I-3689, preferably L. paracasei CNCM I-1518. 6. The composition according to any one of claims 1 to 5, said composition further comprising dextrose. 7. The composition according to any one of claims 1 to 6, said composition comprising: - a skimmed milk powder, - a L. paracasei strain, preferably the L. paracasei strain deposited with the CNCM under the number - a β-galactosidase having a transgalactosylating activity, preferably a protein consisting of the amino acid sequence SEQ ID NO: 1, and - water. 8. A method for producing a fermented milk product containing GOS fibers comprising: - a step of fermentation of the composition as defined in any one of claims 1 to 7, in conditions allowing the β-galactosidase having transgalactosylating activity to convert lactose into GOS fibers. 9. A method according to claim 8, said method further comprising: - a step of inactivating the β-galactosidase having a transgalactosylating activity when a desired quantity of GOS fibers is reached. 10. A method according to claims 8 or 9, said method further comprising: - a step of adding at least an extra fiber, preferably an extra fiber that is not metabolized by the probiotic but is metabolized by the gut microbiome of a consumer. 11. A method according to claim 10, wherein said extra fiber is selected from the group comprising galactooligosaccharides (GOS), fructo-oligosaccharides (FOS), xylo-oligosaccharides (XOS), mannooligo-saccharides, gluco-oligosaccharides, polydextrose, natural gums such as guar gum and acacia gum, mucilages, pectins, arabinogalactan, beta-glucans, tagatose, and resistant starch, preferably polydextrose. 12. A method according to any one of claims 8 to 11, said method further comprising: - a step of adding sugar. 13. A method according to any one of claims 8 to 12, said method further comprising: - a step of adding a syrup, preferably a syrup comprising water, liquid sugar, polydextrose and citric acid. 14. A fermented milk product containing GOS fibers obtained by the method as defined in any one of claims 8 to 14. 15. A fermented milk product containing GOS fibers comprising: - a milk substrate,

Claims

- a lactic acid bacterium, such as a probiotic selected from the group comprising Lactobacillus casei and L. paracasei, - a β-galactosidase having a transgalactosylating activity, said β-galactosidase being able to convert lactose into GOS fibers that can be metabolized by the probiotic, and - GOS fibers resulting from the conversion of lactose originally present in the milk substrate. 16. The fermented milk product according to claim 15, comprising: - a milk substrate, - a lactic acid bacterium, such as a probiotic selected from the group comprising Lactobacillus casei and L. paracasei, - a β-galactosidase having a transgalactosylating activity, said β-galactosidase being able to convert lactose into GOS fibers that can be metabolized by the probiotic, and - GOS fibers resulting from the conversion of lactose originally present in the milk substrate. 17. The fermented milk product according to any one of claims 14 to 16, said product further comprising at least an extra fiber, preferably an extra fiber that is not metabolized by the probiotic but is metabolized by the gut microbiome of a consumer. 18. The fermented milk product according to claim 17, wherein said extra fiber is selected from the group comprising galactooligosaccharides (GOS), fructo-oligosaccharides (FOS), xylo-oligosaccharides (XOS), mannooligo-saccharides, gluco-oligosaccharides, polydextrose, natural gums such as guar gum and acacia gum, mucilages, pectins, arabinogalactan, beta-glucans, tagatose, and resistant starch, preferably polydextrose. 19. The fermented milk product according to any one of claims 14 to 18, said product comprising at least 1.5 grams, preferably 3.0 grams, more preferably 3.3 grams, of total fibers per 100 grams of product. 20. The fermented milk product according to any one of claims 14 to 19, said product comprising at least 1x10⁸ cfu of the probiotic per 100 grams of product. 21. The fermented milk product according to any one of claims 14 to 20, said product further comprising added sugar.

22. The fermented milk product according to any one of claims 14 to 21, said product further comprising a syrup, preferably a syrup comprising water, liquid sugar, polydextrose and citric acid. 23. The fermented milk product according to any one of claims 14 to 20, wherein said product does not comprise added sugar. 24. The fermented milk product according to any one of claims 14 to 23, wherein the quantity of total carbohydrates does not exceed 15 grams, preferably 11 grams, per 100 grams of product. 25. The fermented milk product according to any one of claims 14 to 24, wherein the quantity of lactose does not exceed 1.0 gram, preferably 0.1 gram, per 100 grams of product.

SYNBIOTIC MILK PRODUCTS CONTAINING A PROBIOTIC AND GALACTO-OLIGOSACCHARIDES ABSTRACT The present invention relates to fermented milk products containing galacto-oligosaccharides (GOS) and a lactic acid bacterium such as a probiotic L casei or L. paracasei, compositions for producing the same and methods for producing the same.

22. The fermented milk product according to any one of claims 14 to 21, said product further comprising a syrup, preferably a syrup comprising water, liquid sugar, polydextrose and citric acid.

23. The fermented milk product according to any one of claims 14 to 20, wherein said product does not comprise added sugar.

24. The fermented milk product according to any one of claims 14 to 23, wherein the quantity of total carbohydrates does not exceed 15 grams, preferably 11 grams, per 100 grams of product. 25. The fermented milk product according to any one of claims 14 to 24, wherein the quantity of lactose does not exceed 1.0 gram, preferably 0.1 gram, per 100 grams of product.