Use of cellobiofructose and production method thereof

Abstract

The present invention relates to the use of cellobiofructose (CBF) as a dietary fiber and / or prebiotic component in food products, food product additives, nutraceuticals and / or beverages or for increasing the growth of probiotic microorganisms. The invention further relates to CBF and compositions containing it for use in the prevention and / or treatment of obesity, constipation, high cholesterol, diabetes, hypertension, and / or heart disease. The invention further encompasses a method for the (recombinant) production of cellobiofructose (CBF), as described herein, using a polypeptide having levansucrase activity which comprises or consists of a polypeptide having at least 80 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

Description

[0001] USE OF CELLOBIOFRUCTOSE AND PRODUCTION METHOD THEREOF

[0002] TECHNICAL FIELD

[0003] The present invention relates to the use of cellobiofructose (CBF) as a dietary fiber and / or prebiotic component in food products, food product additives, nutraceuticals and / or beverages or for increasing the growth of probiotic microorganisms. The invention further relates to CBF and compositions containing it for use in the prevention and / or treatment of obesity, constipation, high cholesterol, diabetes, hypertension, and / or heart disease. The invention further encompasses a method for the (recombinant) production of cellobiofructose (CBF), as described herein, using a polypeptide having levansucrase activity which comprises or consists of a polypeptide having at least 80 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0004] BACKGROUND OF THE INVENTION

[0005] A large amount of epidemiological data shows that excessive sugar consumption affects human health beyond the mere intake of calories. Experts have come to believe that high-calorie sugars induce numerous diseases associated with metabolic syndrome. These diseases include obesity, high blood pressure, an increase in triglyceride content due to increased fat synthesis in the liver, insulin resistance, diabetes and ageing processes caused by the non-enzymatic binding of fructose to lipids, proteins and DNA.

[0006] A widespread measure to reduce global sugar consumption is the substitution of high-calorie sugar with low-calorie or calorie-free sugar substitutes. The use of this class of substances, which is divided into sweeteners and sugar substitutes, in finished products and especially soft drinks is steadily increasing. Recently, however, there has been increasing evidence that artificial sweeteners such as saccharin, sucralose and aspartame have a negative effect on the human microbiota and increase the pathogenic properties of Escherichia coli and Enterococcus faecalis. In addition, the synthetic structure of many sweeteners prevents their degradation by natural decomposers or water treatment plants, whereby synthetic sweeteners increasingly accumulate in aquatic systems and disrupt the circular economy principle on which the bioeconomy is based.

[0007] In the context of the rapid rise in obesity and sugar-associated diseases, there is a need for sustainable sugar substitutes that provide consumers with a satisfying taste experience without negatively impacting metabolic and economic processes and the intestinal microbiota, preferably even promoting overall health. In addition, there should be a stronger focus on the degradability of such compounds so that they flow into the natural material cycle and do not accumulate in the environment, in line with bioeconomic objectives.

[0008] In addition to the general trend for sugar substitution in food products and beverages another ongoing trend of consumers or a least a substantial share of consumers is the preference for foods and beverages that are considered to be healthier in that they have prebiotic or probiotic properties. There is thus also need in the art for novel food additives that have prebiotic or probiotic properties.

[0009] It has been found that the levansucrase-mediated fructosylation of cellobiose results in a product, cellobiofructose (CBF), with increased sweetening power. The trisaccharide cellobiofructose (CBF) can be synthesized enzymatically from sustainable raw materials with the help of microbial levansucrases. In the course of the enzymatic reaction, household sugar (sucrose) is split into its monomeric subunits, glucose and fructose, and the fructose component is then transferred to a suitable acceptor molecule. This process is known as transfructosylation. In the presence of the disaccharide cellobiose, this transfructosylation reaction leads to the synthesis of the trisaccharide CBF.

[0010] However, to date methods for enzymatically synthesizing cellobiofructose suffer from the following limitations: a) the yield of biotechnologically produced levansucrase is to small and does not allow for the economic production of fructosylated compounds; b) microbial production of levansucrase requires laborious purifying and isolating of the enzyme, resulting in overall increased production costs; and c) in addition to the primary fructosylation product cellobiofructose various other sucrose-based byproducts, such as kestoses and nystoses, are generated, complicating and hampering purification of cellobiofructose.

[0011] SUMMARY OF THE INVENTION

[0012] The inventors of the present invention have not only developed an improved method of producing CBF but have also found that CBF possesses useful prebiotic properties and thus can be used to foster the growth and / or activity of beneficial microorganisms, such as bifidobacteria, lactobacilli, and commensal microorganisms associated with a healthy gut microbiota. Furthermore, CBF has been found to be completely degraded by enzymes of the human intestinal microbiota or natural decomposers such as fungi or soil microbes. An accumulation of CBF in natural habitats is thus prevented and its degradation products can readily flow back into biological material cycles.

[0013] In a first aspect, the present invention thus provides for the use of cellobiofructose (CBF) as a dietary fiber and / or prebiotic component in a food product, food product additive, nutraceutical and / or beverage.

[0014] In some embodiments of this aspect, cellobiofructose is comprised in the food product in an amount of about 1 to 95 wt.-%, preferably 5 to 90 wt.-%, more preferably 7 to 80 wt.-%, even more preferably 10 to 70 wt.-%, based on the dry weight of the food product; and / or in the food product additive or nutraceutical in an amount of about 1 to 99 wt.-%, preferably 5 to 95 wt.- %, more preferably 7 to 85 wt.-%, even more preferably 10 to 75 wt.-%, based on the dry weight of the food product additive or the nutraceutical, respectively; and / or in the beverage in an amount of about 1 to 50 wt.-%, preferably 2 to 40 wt.-%, more preferably 3 to 30 wt.-%, based on the total weight of the beverage.

[0015] In various embodiments, cellobiofructose modifies the microbiome and / or microbiota of the gastrointestinal tract of the consumer. Specifically, cellobiofructose may modify growth and / or activity of microorganisms, such as bifidobacteria, lactobacilli, and commensal microorganisms associated with a healthy gut microbiota within the gastrointestinal tract of the consumer. Said modification of the growth and / or activity may mean that cellobiofructose increases growth and / or activity of the microorganisms, in particular beneficial microorganisms, such as bifidobacteria, lactobacilli and commensal microorganisms, within the gastrointestinal tract of the consumer. Alternatively or additionally, in various embodiments, cellobiofructose does not influence blood glucose levels of the consumer.

[0016] In a further aspect, the present invention thus also relates to the use of cellobiofructose for modifying, preferably increasing the growth and / or activity of probiotic microorganisms, such as bifidobacteria, lactobacilli, and commensal microorganisms associated with a healthy gut microbiota.

[0017] In some embodiments, the probiotic microorganisms (affected by said properties of CBF) are comprised in or intended to be comprised in a food products composition, food products additive composition, nutraceutical composition and / or beverage composition. In other embodiments, the probiotic microorganisms are part of the microbiome of a host organism.

[0018] In various embodiments, the bifidobacteria are selected from the group consisting of B. actinocoloniiforme, B. adolescentis, B. aemilianum, B. aerophilum, B. aesculapii, B. amazonense, B. angulatum, B. animalis, B. anseris, B. apousia, B. apri, B. aquikefiri, B. asteroids, B. avesanii, B. biavatii, B. bifidum, B. bohemicum, B. bombi, B. boum, B. breve, B. callimiconis, B. callitrichidarum, B. callitrichos, B. canis, B. castoris, B. catenulatum, B. catulorum, B. cebidarum, B. choerinum, B.choladohabitans, B. choloepi, B. colobi, B. commune, B. cricetid, B.cuniculi, B. dentium, B. dolichotidis, B. erythrocebi, B. eulemuris, B. faecale, B. felsineum, B. gallicum, B. gallinarum, B. globosum, B. goeldii, B. hapali, B. indicum, B. infantis, B. italicum, B. jacchi, B. lemurum, B. leontopitheci, B. longum, B. magnum, B.margollesii, B. merycicum, B. miconis, B. miconisargentati, B. minimum, B. mongoliense, B. moraviense, B. moukalabense, B. myosotis, B. oedipodis, B. olomucense, B. panos, B. parmae, B. pluvialisilvae, B. polysaccharolyticum, B. pongonis, B. porcinum, B. primatium, B. pseudocatenulatum, B. pseudoIongum, B. psychraerophilum, B. pullorum, B. ramosum, B. reuteri, B. rousetti, B. ruminantium, B. saguini, B. saguinibicoloris, B. samirii Duranti, B. santillanense, B. scaligerum, B. scardovii, B. simiarum, B. simiiventris, B. stellenboschense, B. subtile, B. thermacidophilum, B. thermophilum B. tibiigranuli, B. tissieri, B. tsurumiense, B. vansinderenii, B. vespertilionis, and S. xylocopae, preferably S. adolescentis, B. breve, B. infantis, and S. longum.

[0019] Alternatively or additionally, In various embodiments, the lactobacilli are selected from the group consisting of Lactobacillus acidophilus, Lactobacillus brevis Lactobacillus easel, Lactobacillus easel immunitas, Lactobacillus casei defensis, Lactobacillus easel shirota, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus lactis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus iners, Lactobacillus paracasei, Lactobacillus plantarum, and Lactobacillus salivarius.

[0020] Alternatively or additionally, in various embodiments, the commensal microorganisms are selected from the group consisting of Lactococcus casei, Lactococcus lactis, Leuconostoc mesenteroides, Oenococcus oeni, Hominimerdicola aceti, Faecalibacterium prausnitzii, Levilactobacillus brevis, Agathobacter rectalis, Phocaeicola vulgatus, Streptococcus salivarius subsp. thermophilus, and Enterococcus faecalis. In still another aspect, the present invention provides for cellobiofructose or a composition comprising CBF for use in the prevention and / or treatment of obesity, constipation, high cholesterol, diabetes, hypertension, and / or heart disease.

[0021] In some embodiments of this aspect, the composition is a food product, food product additive, nutraceutical, beverage, or a pharmaceutical composition.

[0022] In various embodiments of this aspect, the CBF may function as described above for the various uses and the products used may be any one of the compositions described above in relation to the other aspects of the invention.

[0023] In some embodiments, the composition is a pharmaceutical composition comprising CBF in an amount of about 1 to 99 wt.-%, preferably 5 to 95 wt.-%, more preferably 7 to 85 wt.-%, even more preferably 10 to 75 wt.-%, based on the dry weight of the pharmaceutical composition.

[0024] In yet another aspect, the present invention further provides for a method for modifying, preferably increasing, the growth and / or activity of probiotic microorganisms, such as bifidobacteria and lactobacilli, comprising the step of providing cellobiofructose (CBF) as a substrate for said microorganisms.

[0025] In still another aspect, the present invention provides for a method for the production of cellobiofructose (CBF), comprising

[0026] (a) expressing a polypeptide having levansucrase activity in a suitable host cell; and

[0027] (b) contacting said polypeptide having levansucrase activity with a suitable substrate under conditions that allow the enzymatic production of CBF; wherein said polypeptide having levansucrase activity comprises or consists of a polypeptide having at least 80 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 .

[0028] In various embodiments, said polypeptide having levansucrase activity comprises or consists of a polypeptide having at least 80, 81 , 82, 83, 84, 85, 86, 87, 88, 98, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0029] In some embodiments, the polypeptide having levansucrase activity is a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 ; and / or is a polypeptide encoded by a nucleic acid sequence having the nucleic acid sequence set forth in SEQ ID NO: 2.

[0030] In various embodiments of said methods, the host cell is selected from microorganisms of the order Vibrionales, preferably of the family Vibrionaceae, more preferably of the genus Vibrio and / or gammaproteobacteria and / or gram-negative marine bacteria, most preferably from Vibrio natriegens or a Vibrio natriegens strain selected from Vibrio natriegens Vmax or Vibrio natriegens DSM 759.

[0031] In some embodiments, the polypeptide having levansucrase activity is heterologously expressed in said host cell. In some embodiments, step (a) includes a cultivation step that is preferably carried out in the absence of a selection antibiotic.

[0032] In some embodiments, step (b) is carried out using a crude cell extract of the host cells of step (a).

[0033] In some embodiments, the substrate is a combination of cellobiose and sucrose.

[0034] In some particular embodiments, the method is characterized in that:

[0035] (1) the host cell is selected from microorganisms of the order Vibrionales, preferably of the family Vibrionaceae, more preferably of the genus Vibrio and / or gammaproteobacteria and / or gramnegative marine bacteria, most preferably from Vibrio natriegens or a Vibrio natriegens strain selected from Vibrio natriegens Vmax or Vibrio natriegens DSM 759; or the host cell is selected from gram-negative bacteria, preferably of the genus Cupriavidus, such as Cupriavidus necator; the genus Gluconobacter, such as Gluconobacter oxydans; the genus Komagataeibacter, such as Komagataeibacter sucrofermentans; the genus Agrobacterium; or the genus Xanthomonas; or the host cell is selected from gram-positive non-sporulating bacteria, preferably of the genus Acidiproplonibacterium, such as Acidipropionibacterium acidipropionici; of the genus Bifidobacterium, such as Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, or Bifidobacterium longum; of the genus Carnobacterium, such as Carnobacterium divergens; of the genus Companilactobacillus, such as Companilactobacillus alimentarius or Companilactobacillus farciminls; of the genus Corynebacterium, such as Corynebacterium ammoniagenes or Corynebacterium glutamicum; of the genus Fructilactobacillus, such as Fructilactobacillus sanfranciscensis; of the genus Lacticaseibacillus, such as Lacticaseibacillus casei, Lacticaseibacillus huelsenbergensis, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lactiplantibacillus argentoratensis, Lactiplantibacillus paraplantarum, Lactiplantibacillus pentosus or Lactiplantibacillus plantarum; of the genus Lactobacillus, such as Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii or Lactobacillus kefiranofaciens; of the genus Lactococcus, such as Lactococcus lactis; of the genus Lapidilactobacillus, such as Lapidilactobacillus dextrinicus, Latilactobacillus curvatus or Latilactobacillus sakei; of the genus Lentilactobacillus, such as Lentilactobacillus buchneri, Lentilactobacillus diolivorans, Lentilactobacillus hilgardii, Lentilactobacillus kefiri or Lentilactobacillus parafarraginis; of the genus Leuconostoc, such as Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, or Leuconostoc pseudomesenteroides; of the genus Levilactobacillus, such as Levilactobacillus brevis; of the genus Levilactobacillus, such as Levilactobacillus brevis; of the genus Ligilactobacillus, such as Ligilactobacillus animalis, Ligilactobacillus aviaries or Ligilactobacillus salivarius; of the genus Limosilactobacillus, such as Limosilactobacillus fermentum, Limosilactobacillus mucosae, Limosilactobacillus panis, Limosilactobacillus pontis or Limosilactobacillus reuteri; of the genus Loigolactobacillus, such as Loigolactobacillus coryniformis; of the genus Microbacterium, such as Microbacterium imperial; of the genus Oenococcus, such as Oenococcus oeni; of the genus Pediococcus, such as Pediococcus acidilactici, Pediococcus parvulus or Pediococcus pentosaceus; of the genus Propionibacterium, such as Propionibacterium freudenreichii; of the genus Secundilactobacillus, such as Secundilactobacillus colllnoides; of the genus Streptococcus, such as Streptococcus thermophilus; of the genus Bacillus, such as Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus licheniformis, Bacillus mojavensis, Bacillus paralicheniformis, Bacillus pumilus, Bacillus smithli, Bacillus subtilis, Bacillus vallismortis or Bacillus velezensls; of the genus Clostridium, such as Clostridium tyrobutyricum; of the genus Geobacillus, such as Geobacillus stearothermophilus or Geobacillus thermodenitrificans; of the genus Heyndrlckxia, such as Heyndrickxia coagulans; of the genus Lederbergia, such as Lederbergia lenta; of the genus Lysinibaclllus, such as Lyslnlbacillus fusiformls; of the genus Nlallla, such as Niallia circulans; of the genus Paenibaclllus, such as Paenibacillus illinoisensis; of the genus Parageobacillus, such as Parageobacillus thermoglucosidasius; of the genus Pasteuria, such as Pasteuria nishlzawae; of the genus Prlestia, such as Priestia flexa or Priestia megaterium; or of the genus Shouchella, such as Shouchella clausil; or the host cell is selected from yeasts, preferably of the genus Cyberlindnera, such as Cyberlindnera jadinii; of the genus Debaryomyces, such as Debaryomyces hansenii; of the genus Hanseniaspora, such as Hansenlaspora uvarum; of the genus Kluyveromyces, such as Kluyveromyces lactis or Kluyveromyces marxianus; of the genus Komagataella, such as Komagataella pastoris or Komagataella phaffii; of the genus Llmtongozyma, such as Limtongozyma cylindracea; of the genus Ogataea, such as Ogataea angusta or Ogataea polymorpha; of the genus Phaffia, such as Phaffia rhodozyma; of the genus Saccharomyces, such as Saccharomyces bayanus, Saccharomyces cerevisiae or Saccharomyces pastorianus; of the genus Schizosaccharomyces, such as Schizosaccharomyces pombe; of the genus Wickerhamomyces, such as Wickerhamomyces anomalus; of the genus Yarrowia, such as Yarrowia lipolytica; or of the genus Zygosaccharomyces, such as Zygosaccharomyces rouxiiand / or

[0036] (2) the polypeptide having levansucrase activity is heterologously expressed in said host cell; and / or

[0037] (3) step (a) includes a cultivation step that is preferably carried out in the absence of a selection antibiotic; and / or

[0038] (4) step (b) is carried out using a crude cell extract of the host cells of step (a); and / or

[0039] (5) the substrate is a combination of cellobiose and sucrose.

[0040] In some embodiments, step (a) comprises the steps of: i) optionally preculturing the host cells in a suitable preculture medium; ii) culturing the host cells in a suitable main culture medium; and Hi) obtaining a crude cell extract of the host cell culture of ii).

[0041] In various such embodiments, the crude cell extract is obtained by lysis of the cells to obtain a lysed cell composition and removal of any solid components from the lysed cell composition to obtain the crude cell extract. In some embodiments, the crude cell extract contains the polypeptide having levansucrase activity with an activity of at least 70.000, preferably at least 80.0000, more preferably at least 90.000 units per litre culture medium.

[0042] In some embodiments, the preculturing is performed at a temperature of about 20 to 55 °C, preferably of about 25 to 50 °C, more preferably of about 30 to 45 °C; and / or the preculturing is performed for a period of < 15 hours, preferably < 7 hours, more preferably < 6 hours, even more preferably < 5 hours.

[0043] In some embodiments, the main culturing is performed at a temperature of about 20 to 55 °C, preferably of about 25 to 50 °C, more preferably of about 30 to 45 °C; and / or the main culturing is performed for a period of < 15 hours, preferably < 10 hours, more preferably < 7 hours, even more preferably < 5 hours.

[0044] In some embodiments, the total culturing period is about 30 hours, preferably < 17 hours, more preferably < 15 hours, even more preferably < 12 hours, still more preferably < 10 hours.

[0045] In some embodiments, the method does not comprise sterilization of the preculture medium and / or the main culture medium.

[0046] In various embodiments, the preculture medium and / or the main culture medium does not comprise one or more antibiotics.

[0047] In some embodiments, the amount of CBF generated from about 300 litres of a solution containing about 80 to about 200 g / L, preferably about 100 to about 180 g / L, more preferably about 140 to about 170 g / L sucrose, is at least about 20 kg, preferably at least 25 kg, more preferably at least about 30 kg.

[0048] In some embodiments, the concentration of fructosylation side products other than CBF in the final product is less than 5% relative to the total amount of CBF obtained.

[0049] In some embodiments, no fructosylation product is generated in addition to CBF.

[0050] BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 shows a graphic evaluation of kinetics of the levansucrase-conveyed synthesis of celloblofructose. Depicted are the concentrations of the reactants sucrose (A) and cellobiose (V) as well as reaction products glucose (n), fructose (•) and cellobiofructose (♦) quantified by high-performance liquid chromatography.

[0052] Figure 2 shows growth curves of probiotic lactobacilli and commensal microorganisms associated with a healthy gut microbiota. Depicted are changes in biomass of various microbial cultures measured at a wavelength of 600 nm. The depicted microbial strains were supplemented with glucose (♦), water (•), cellobiose (o) and cellobiofructose (▼). Figure 3 shows growth curves of probiotic bifidobacteria. Depicted are changes in biomass of various bifidobacteria! cultures measured at a wavelength of 600 nm. The depicted bifidobacteria were supplemented with glucose (♦), water (•), cellobiose (o) and cellobiofructose (▼).

[0053] Figure 4 depicts the plasmid map of the overexpression plasmid pET-19b(+)_levSKK2i.

[0054] Figure 5 depicts mean blood glucose level [mg / dl] of male animals, sorted by groups (high dose and low dose: n=10, positive control and vehicle: n=5) and measurement period (pre: 30 min before application, 15, 30, 45, 60,120 min after application). Means and standard deviations of each group are given.

[0055] Figure 6 depicts results of the toxicological assessment of cellobiofructose (Example 5); **** = p < 0,005.

[0056] DETAILED DESCRIPTION OF THE INVENTION

[0057] Before describing in detail exemplary embodiments of the present invention, definitions which are important for understanding the present invention are given.

[0058] As used in this specification and in the appended claims, the singular forms of "a” and "an" also Include the respective plurals unless the context clearly dictates otherwise.

[0059] In the context of the present invention, the terms “about” and “approximately” denote an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates a deviation from the indicated numerical value of ±10 %, preferably ±5 %, more preferably ±2 %.

[0060] It is to be understood that the term “comprising” is not limiting. For the purposes of the present invention the term “consisting of’ is considered to be a preferred embodiment of the term “comprising of’. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is meant to also encompass a group which preferably consists of these embodiments only.

[0061] As used herein the term “does not comprise” or “free of’ means in the context that a composition of the present invention is free of a specific compound or group of compounds, which may be combined under a collective term, that the composition does not comprise said compound or group of compounds in an amount of more than 0.5 % by weight, based on the total weight of the composition. Furthermore, it is preferred that a composition according to the present invention does not comprise said compounds or group of compounds in an amount of more than 0.1 % by weight, preferably the composition does not comprise said compounds or group of compounds at all.

[0062] It is to be understood that this Invention is not limited to the particular methodology, protocols, reagents etc. described herein as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention that will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0063] When referring to compositions and the weight percent of the therein comprised ingredients it is to be understood that according to the present invention the overall amount of ingredients does not exceed 100 % (± 1% due to rounding).

[0064] The term “prebiotic”, as used herein, is generally understood to refer to substrates, that are selectively utilized by host microorganisms conferring a health benefit. The selective utilization is generally understood to mediate increased bacterial growth and / or activity of a host’s microbiota.

[0065] The term “probiotic”, as used herein, refers to the properties of living microorganisms that exert beneficial effects on the host's health, such as when administered in adequate amounts.

[0066] The term “microbiota”, as used herein, is generally understood to refer to a collective of microbes present in a host’s anatomical locations including the mouth, esophagus, stomach, small intestine, large intestine, caecum, colon, rectum, and various others, such as the skin, nasal cavaties, lungs, ear, and vagina.

[0067] The term “microbiome”, as used herein, is generally understood to refer to a micro-ecosystem encompassing the microbiota occupying it, which is integrated in macro-ecosystems including eukaryotic hosts and crucial for their functioning and health.

[0068] The term “modifying” or “modulating”, as herein in the context of the microbiota and / or the microbiome, is to be construed in its broadest interpretation to mean a change in the representation of microbes or microbial activity in a bacterial niche of a subject. The change may be an increase or a decrease in the presence of a particular species, genus, family, order, class, or phylum. The change may also be an increase or a decrease in the activity of an organism or a component of an organism, such as a bacterial enzyme, a bacterial antigen, a bacterial signaling molecule, or a bacterial metabolite. The change may also be the change in the relative ratio of one bacterial species, genus, family, order, class, phylum, or component in relation to another.

[0069] In the context of the present invention, the term “individual”, “patient”, or “subject” are used interchangeably herein and include any animal, including mammals, including mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and humans. The compounds described herein can be administered to a mammal, such as a human, but can also be administered to other mammals such as an animal in need of veterinary treatment, e.g., domestic animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like). The mammal treated in the methods described herein is desirably a mammal in which treatment of a disorder described herein is desired, such as a human. The terms "treat”, “treating”, and “treatment”, as used herein, are meant to include alleviating or abrogating a disorder, disease, or condition, or one or more of the symptoms associated with the disorder, disease, or condition; or alleviating or eradicating the cause(s) of the disorder, disease, or condition itself.

[0070] The terms “prevent”, “preventing”, and “prevention”, as used herein, are meant to include a method of delaying and / or precluding the onset of a disorder, disease, or condition, and / or its attendant symptoms; barring a subject from acquiring a disorder, disease, or condition; or reducing a subject’s risk of acquiring a disorder, disease, or condition.

[0071] The terms “alleviate” and “alleviating”, as used herein, refer to easing or reducing one or more symptoms (e.g., pain) of a disorder, disease, or condition. The terms can also refer to reducing adverse effects associated with an active ingredient. Sometimes, the beneficial effects that a subject derives from a prophylactic or therapeutic agent do not result in a cure of the disorder, disease, or condition.

[0072] The term “pharmaceutically acceptable”, as used herein, refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0073] As used herein, the term “dietary fiber” means carbohydrate polymers with three or more monomeric units, which are neither digested nor absorbed in the human small intestine and belong to the following categories: a) edible carbohydrate polymers naturally occurring in the food as consumed; b) edible carbohydrate polymers which have been obtained from food raw material by physical, enzymatic or chemical means and which have a beneficial physiological effect demonstrated by generally accepted scientific evidence; c) edible synthetic carbohydrate polymers which have a beneficial physiological effect demonstrated by generally accepted scientific evidence (in accordance with Regulation (EU) No 1169 / 2011 of the European Parliament and of the Council).

[0074] The term “food products”, as used herein, refers to food processed by a manufacturer en route from producer to consumer., including food compositions, which may comprise one or more non-processed food articles, such as fruits and vegetables and / or parts thereof, and further including dietary supplements, and still further including animal feed.

[0075] The term “food product additive”, as used herein, refers to additive components or compositions, which may be used in the manufacture of food products, i.e., in the processing of food, such as meats, fruits, vegetables, eggs, milk, fish, fungi, etc, such as in the preparation of food dishes. For instance, but without imitation, a food product additive may be a prebiotic compound or composition which may include oligosaccharides and / or dietary fiber components to be added to a food product or beverage.

[0076] The term “beverage”, as used herein, refers to a liquid composition, typically an aqueous ingestible composition. Beverages typically contain one or more compounds, such as one or more stabilizers such as acids, antioxidants, bicarbonates or carbonates, colorants, flavoring agents, or mixtures thereof, and a beverage base, generally water. Beverages include, without limitation, juices, juice concentrates, fruit juice extracts, etc. Typically, beverage compositions are provided for direct ingestion, that is for direct consumption by a subject, e.g., a human, but may also be provided in the form of a concentrate to be diluted with, for instance, water prior to consumption.

[0077] The term “nutraceutical”, as used herein, refers to bioactive chemical compounds that have health promoting, disease preventing or medicinal properties and that may be eaten or drunk or otherwise consumed by a subject to provide a beneficial effect to the subject.

[0078] As surprisingly found by the present inventors, cellobiofructose (CBF) can be fully degraded by enzymes of the human intestinal microbiota, mainly those located in the colon, or natural decomposers such as fungi or soil microbes. An accumulation of CBF in natural habitats is thus prevented and its components can readily be reintroduced into biological material cycles.

[0079] Furthermore, the present inventors were able to prove for the first time that CBF, in contrast to cellobiose, stimulates the growth of probiotic bifidobacteria. In addition, it promotes the growth of various lactobacilli and commensal microorganisms associated with a healthy gut microbiota. Thus, CBF has prebiotic properties and a positive effect on the condition and composition of the human intestinal microbiota and associated physiological functions and dysfunctions, respectively.

[0080] In addition, CBF has sweetening properties and thus can be used as a substitute for sugar and / or commonly used sweetener and sugar substituting substances.

[0081] Furthermore, according to Regulation (EU) No 1169 / 2011 of the European Parliament and of the Council, CBF qualifies as a dietary fiber.

[0082] Further still, the high solubility of cellobiofructose enables its seamless incorporation into a wide range of food matrices, including beverages, syrups, baked goods, ice cream, sauces, and low-water-activity products like confectionery, without compromising texture or stability. Additionally, it allows for the creation of highly concentrated solutions, enhancing its versatility for formulating reduced-sugar and fiber-enriched products.

[0083] All these properties make cellobiofructose a superior sugar replacement for innovative, health-focused food applications with reduced sugar and increased fiber content.

[0084] As a result, incorporation of CBF in foods and beverages can provide for food products and beverages having sweet-tasting properties and an improved fiber content. Furthermore, substituting CBF for sugar in foods and beverages can result in food products and beverages having an overall reduced caloric index, and substituting CBF for other sweeteners or sugar substitutes can provide for more healthy food products and beverages. Additionally, CBF possesses swelling capacity and can, upon consumption, relieve or aid in relieving symptoms of constipation. Therefore, in a first aspect, the present invention provides for the use of CBF as a dietary fiber and / or prebiotic component in a food product, food product additive, nutraceutical and / or beverage.

[0085] CBF is a trisaccharide consisting of one cellobiose molecule and one fructose molecule, While the glucoside units are joined by a beta1-4 bond, the fructose moiety is linked to the cellobiose molecule by a beta1-2 bond.

[0086] For instance, but without limitation, a food product, as herein described and defined, may contain CBF in an amount of about 1 to 95 wt.-%, such as in an amount of 1 .0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1 .9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0,

[0087] 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 35, 40, 45, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, or 95 wt.-%, preferably 5 to 90 wt.-%, more preferably 7 to 80 wt.-%, even more preferably 10 to 70 wt.-%, based on the dry weight of the food product.

[0088] Non-limiting examples of food products include breads, dairy products such as yoghurt or cheese, pasta, confectionery, convenience foods, formula, desserts, dips, pastes and spreads, dried foods, dumplings, fast food such as chips or crackers, fermented food compositions, fruit or vegetable pastes, halal food, kosher food, pies, salads, sandwiches, sauces, sausages, snackfoods, soups, stews, dietary supplements, and animal feed.

[0089] A food product additive, as herein defined and described, or nutraceutical, as herein defined and described, may, for instance but without limitation, comprise CBF in an amount of 1 to 99 wt.-%, such as in an amount of 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0,

[0090] 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 35, 40, 45, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 wt.-%, preferably 5 to 95 wt.-%, more preferably 7 to 85 wt.-%, even more preferably 10 to 75 wt.-%, based on the dry weight of the food product additive or nutraceutical.

[0091] Non-limiting examples of food product additive compositions include oligosaccharide compositions, sweetener compositions, dietary fiber compositions, prebiotic and / or probiotic compositions, and nutrient compositions.

[0092] A beverage, as herein defined and described, may, for instance but without limitation, comprise CBF in an amount of about 1 to 50 wt.-%, such as in an amount of 1 .0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1 .6, 1 .7, 1.8, 1 .9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0,

[0093] 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 35, 40, 45, 45, or 50 wt.-%, preferably 2 to 40 wt.-%, more preferably 3 to 30 wt.-%, based on the total weight of the beverage. Non-limiting examples of beverages include non-alcoholic drinks, such as water-based beverages such as soda drinks, fruit juice compositions and juice drinks and smoothies, teas or tea-based drinks, coffee-based drinks, milk-based drinks, soy milk- or other non-cow milk-based drinks; and alcoholic drinks, such as beer, cider, wine, and spirits.

[0094] The inventors have found that, upon consumption, CBF does not influence blood glucose levels of the consumer. In the context of the present invention, the term “does not influence blood glucose levels” means that the respective compound referred to, upon consumption (for example orally as part of a food or beverage product), does not result in elevated blood glucose levels, wherein “elevated blood glucose levels” means blood glucose levels that are at least about 20 %, 15 % or at least 10 % higher than blood glucose levels prior to consumption of the respective compound within 10 minutes to 2 hours, such as within 15 minutes to 1 .5 hours, for instance within 30 minutes to 1 hour, after consumption. Blood glucose levels may be measured by conventional methods and means known in the art, for instance using glucose testing strips, such as human glucose testing strips, for quantitative blood glucose determination using a conventional blood glucose monitoring system, such as the ACCU-CH EK Aviva blood glucose monitoring system.

[0095] The inventors have found that CBF exhibits properties and characteristics for modifying the microbiome and / or the microbiota, for example as tound in the gastrointestinal tract of a subject, e.g., a human or animal, typically mammal. Thus, particularly, the modifying of the microbiome and / or the microbiota within the gastrointestinal tract of a consumer by a food product, food product additive, nutraceutical and / or beverage, as herein defined and described, is possible.

[0096] In particular, it was surprisingly discovered by the present inventors that CBF exhibits properties and characteristics for modifying the growth of beneficial microorganisms, in particular bifidobacteria, lactobacilli, and commensal microorganisms associated with a healthy gut microbiota, such as those found within the gastrointestinal tract of a subject, e.g., a human or animal, typically mammal.

[0097] Thus, the modification of the growth and / or activity of (beneficial) microorganisms, e.g., bifidobacteria, lactobacilli, and commensal microorganisms associated with a healthy gut microbiota, within the gastrointestinal tract of a consumer of a food product, food product additive, nutraceutical and / or beverage, as herein defined and described, becomes possible. More particularly, it was surprisingly discovered by the present inventors that CBF can increase the growth of bifidobacteria, lactobacilli, and commensal microorganisms associated with a healthy gut microbiota, such as those found within the gastrointestinal tract of a human.

[0098] This finding allows the use of CBF for increasing the growth and / or activity of (beneficial) microorganisms, such as bifidobacterial, within the gastrointestinal tract of a consumer of a food product, food product additive, nutraceutical and / or beverage, as described herein. In some such embodiments, CBF does not influence blood glucose levels of the consumer, as herein defined. Non-limiting examples of bifidobacteria that are to be affected accordingly are selected from the group consisting of B. actinocoloniiforme, B. adolescentis, B. aemilianum, B. aerophilum, B. aesculapii, B. amazonense, B. angulatum, B. animalis, B. anseris, B. apousia, B. apri, B. aquikefiri, B. asteroids, B. avesanii, B. biavatii, B. bifidum, B. bohemicum, B. bombi, B. boum, B. breve, B. callimiconis, B. callitrichidarum, B. callitrichos, B. canis, B. castoris, B. catenulatum, B. catulorum, B. cebidarum, B. choerinum, B.choladohabitans, B. choloepi, B. colobi, B. commune, B. cricetid, B.cuniculi, B. dentium, B. dolichotidis, B. erythrocebi, B. eulemuris, B. faecale, B. felsineum, B. gallicum, B. gallinarum, B. globosum, B. goeldii, B. hapali, B. indicum, B. infantis, B. italicum, B. jacchi, B. lemurum, B. leontopitheci, B. longum, B. magnum, B.margollesii, B. merycicum, B. miconis, B. miconisargentati, B. minimum, B. mongoliense, B. moraviense, B. moukalabense, B. myosotis, B. oedipodis, B. olomucense, B. panos, B. parmae, B. pluvialisilvae, B. polysaccharolyticum, B. pongonis, B. porcinum, B. primatium, B. pseudocatenulatum, B. pseudoIongum, B. psychraerophilum, B. pullorum, B. ramosum, B. reuteri, B. rousetti, B. ruminantium, B. saguini, B. saguinibicoloris, B. samirii Duranti, B. santillanense, B. scaligerum, B. scardovii, B. simiarum, B. simiiventris, B. stellenboschense, B. subtile, B. thermacidophilum, B. thermophilum B. tibiigranuli, B. tissieri, B. tsurumiense, B. vansinderenii, B. vespertilionis, and B. xylocopae, preferably B. adolescentis, B. breve, B. infantis, and B. longum.

[0099] Non-limiting examples of lactobacilli that are to be affected accordingly are selected from the group consisting of Lactobacillus acidophilus, Lactobacillus brevis Lactobacillus easel, Lactobacillus easel immunitas, Lactobacillus easel defensis, Lactobacillus easel shirota, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus lactis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus iners, Lactobacillus paracasei, Lactobacillus plantarum, and Lactobacillus salivarius.

[0100] Non-limiting examples of commensal microorganisms that are to be affected accordingly are selected from the group consisting of Lactococcus easel, Lactococcus lactis, Leuconostoc mesenteroides, Oenococcus oeni, Hominimerdicola aceti, Faecalibacterium prausnitzii, Levilactobacillus brevis, Agathobacter rectalis, Phocaeicola vulgatus, Streptococcus salivarius subsp. thermophilus, and Enterococcus faecalis.

[0101] In view of the above, the present invention is, in a further aspect, concerned with the use of cellobiofructose for modifying, preferably increasing the growth and / or activity of probiotic microorganisms, preferably bifidobacteria and lactobacilli. In various embodiments of this aspect, the probiotic microorganisms are comprised in or intended to be comprised in a food product, food product additive, nutraceutical and / or beverage. Alternatively, they may also be part of a consumer’s microbiome. Non-limiting examples of bifidobacteria, and lactobacilli and preferred bifidobacteria, and preferred lactobacilli have been listed herein above. In some embodiments of said further aspect, CBF does not influence blood glucose levels of the consumer, as herein defined.

[0102] Further, in view of the above-described properties of CBF that have been surprisingly discovered by the present inventors, namely its prebiotic properties as well as its dietary fiber properties, another aspect the present invention is concerned with is CBF or a composition comprising CBF for use in the prevention and / or treatment of diseases and disorders that are related to food intake, such as, without limitation, obesity, constipation, high cholesterol, diabetes, hypertension, and / or heart disease. All of these conditions are closely linked to eating habits and / or condition of the microbiome and / or microbiota of a respective subject, factors of which include caloric intake, dietary fiber intake, and overall quality of food with direct consequence for the condition and composition of the microbiome and / or microbiota of said subject.

[0103] In some embodiments of this aspect, cellobiofructose due to its prebiotic properties modifies the microbiome and / or microbiota of the gastrointestinal tract of the consumer, such as modifies, e.g., increases growth and / or activity of beneficial microorganisms, including bifidobacteria, lactobacilli, and commensal microorganisms associated with a healthy gut microbiota, within the gastrointestinal tract of the consumer. A list of exemplary and preferred bifidobacteria and lactobacilli has been provided herein above. In some embodiments of this aspect, CBF does not influence blood glucose levels of the consumer, as herein defined.

[0104] Although CBF may be used, i.e., consumed, in pure form, a more consumer-friendly and appetizing form may be as component or ingredient in a composition, which may be selected from food products, food product additives, nutraceuticals, beverages, and pharmaceutical compositions. Thus, in some embodiments, the composition comprising CBF is a food product, food product additive, nutraceutical, beverage composition, or a pharmaceutical composition.

[0105] For instance, but without limitation, if the said composition is a food product, it may comprise CBF in an amount of about 1 to 95 wt.-%, 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0,

[0106] 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 35, 40, 45, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, or 95 wt.-%, preferably 5 to 90 wt.-%, more preferably 7 to 80 wt.-%, even more preferably 10 to 70 wt.-%, based on the dry weight of the food product.

[0107] If said composition is a food product additive or a nutraceutical, for instance but without limitation, CBF may be present in said composition in an amount of about 1 to 99 wt.-%, such as in an amount of 1.0, 1.1, 1.2,

[0108] 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0,

[0109] 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0,

[0110] 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 35, 40, 45, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 wt.-%, preferably 5 to 95 wt.-%, more preferably 7 to 85 wt.-%, even more preferably 10 to 75 wt.-%, based on the dry weight of the food product additive or the nutraceutical, respectively.

[0111] If said composition is a beverage, it may comprise CBF in an amount of, for instance but without limitation, about 1 to 50 wt.-%, such as in an amount of 1.0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3,

[0112] 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0,

[0113] 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 35, 40, 45, 45, or 50 wt.-%, preferably 2 to 40 wt.-%, more preferably 3 to 30 wt.-%, based on the total weight of the beverage. If said composition is a pharmaceutical composition, it may comprise CBF in an amount of, for instance but without limitation, about 1 to 99 wt.-%, such as in an amount of 1 .0, 1.1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1 .7, 1 .8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 35, 40, 45, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99 wt.-%, preferably 5 to 95 wt.-%, more preferably 7 to 85 wt.- %, even more preferably 10 to 75 wt.-%, based on the dry weight of the pharmaceutical composition.

[0114] In addition to CBF, further ingredients and components typical for the respective type of composition may be present, such as, for instance but without limitation, in the case of a food product, a food product additive, a nutraceutical or a beverage other types of carbohydrates, fats, proteins, vitamins, minerals, water, etc., or mixtures thereof, and in the case of a pharmaceutical composition one or more pharmaceutically acceptable excipients and optionally other types of active agents or medicaments.

[0115] Non-limiting examples of food products, food product additives, nutraceuticals, and beverages have been listed herein above, and all embodiments described herein above apply mutatis mutandis, insofar applicable.

[0116] In view of the microbiome and / or microbiota-modifying properties of CBF as surprisingly discovered by the present inventors, a further aspect of the present invention resides in a method for modifying, preferably increasing the growth and / or activity of probiotic microorganisms, such as bifidobacteria and lactobacilli, comprising the step of providing CBF as a substrate for the microorganisms. In some embodiments, the method is an in vitro method. In some other embodiments, the method is an in situ method. In this aspect of the invention, CBF may be used in any form including those described above and may be present in such a form or composition in an amount of about 0.1 to 99 wt.-%, such as in an amount of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1 , 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 35, 40, 45, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 wt.-%, preferably 1 to 80 wt.-%, more preferably 5 to 70 wt.-%, even more preferably 10 to 65 wt.-%, based on the total weight of said form or composition.

[0117] Additional components of such a composition may be selected from, for instance but without limitation, additional carbohydrates, such as glucose, sucrose, and cellobiose, buffering agents such as sodium phosphate, vitamins, antioxidants, water, and mixtures thereof.

[0118] Non-limiting examples of types of bifidobacteria and lactobacilli have been listed above, and all embodiments described herein above apply mutatis mutandis, insofar applicable.

[0119] In the following, a brief overview of the various advantages correlated with addition of cellobiofructose in various types of food products, food product additives, neutraceuticals, and beverages is provided: Beverages (Functional Drinks, Juices, and Smoothies):

[0120] The high solubility of cellobiofructose ensures complete dissolution, preventing sedimentation or cloudiness. Additionally, it can enhance mouthfeel and sweetness while contributing dietary fiber for added health benefits, without altering the product's clarity or viscosity.

[0121] Dairy Products (Yogurts, Kefir, and Flavored Milks):

[0122] Cellobiofructose acts as a prebiotic, supporting gut health by promoting beneficial gut bacteria. Its high solubility aids in smooth blending, avoiding grittiness often associated with other fibers, and enhances the creamy texture.

[0123] Low-Sugar Confectionery (Gummies, Hard Candies, and Chews):

[0124] The high solubility of cellobiofructose enables the creation of fiber-rich formulations with a desirable sweetness and stable texture. Its incorporation reduces the need for high-sugar content, supporting reduced-calorie product development while maintaining pleasant chewability or crunch.

[0125] Bakery Items (Cakes, Muffins, and Cookies):

[0126] Cellobiofructose can improve moisture retention, extending shelf life and enhancing the soft texture of baked goods. Its functional fiber content also supports health claims in fiber-enriched bakery products.

[0127] Cereal Bars and Snacks:

[0128] Cellobiofructose can act as a binder in granola or protein bars, delivering both functionality and sweetness. Its high solubility ensures homogenous distribution, avoiding clumping or uneven textures.

[0129] Sugar-Free or Reduced-Sugar Syrups and Sauces:

[0130] Due to its solubility and sweetness, cellobiofructose serves as an excellent sugar substitute, enabling the formulation of syrups with reduced sugar content while maintaining viscosity and flavor balance.

[0131] Frozen Desserts (Ice Cream and Sorbets):

[0132] Cellobiofructose can improve the texture and scoopability of frozen desserts by preventing large ice crystal formation, thanks to its hygroscopic nature, while adding sweetness and dietary fiber.

[0133] Meal Replacements and Nutritional Shakes:

[0134] Cellobiofructose enhances solubility and dispersibility in powdered formulations, ensuring smooth, lump- free reconstitution. Additionally, it provides a fiber source that contributes to satiety and digestive health.

[0135] Savory Products (Soups, Sauces, and Marinades):

[0136] The high solubility of cellobiofructose allows for easy integration into liquid-based savory applications, contributing fiber enrichment without altering taste profiles or texture.

[0137] Summary of unique functional properties of cellobiofructose in food: enhanced solubility: allows for high-concentration solutions, enabling more efficient product development in both liquid and solid formulations. natural sweetness: contributes mild sweetness, reducing the need for artificial sweeteners while maintaining a clean label. prebiotic effects: promotes gut health, adding functional health benefits that are increasingly demanded by consumers. stability: performs well under various pH and temperature conditions, making it suitable for a broad spectrum of food processes.

[0138] CBF can be prepared, for example, according to the process described by S. HESTRIN and G. AVIGAD (Biochem. J., 69, 388-398, 1958) by the action of a levansucrase (Enzyme Code E.C. 2.4.1.10) on a substrate solution containing sucrose and cellobiose.

[0139] However, state-of-the-art production methods, as the one referenced above, suffer from the following limitations: a) the yield of biotechnologically produced levansucrase is to small and does not allow for the economic production of fructosylated compounds, such as CBF; b) microbial production of levansucrase requires laborious purifying and isolating of the enzyme, resulting in overall increased production costs; c) in addition to the primary fructosylation product CBF various other sucrose-based byproducts, such as kestoses and nystoses, are generated, complicating and hampering purification of CBF.

[0140] The invention thus also aims at providing a method for the production of CBF overcoming one or more of the above-outlined limitations of state-of-the-art production methods.

[0141] Consequently, in a further aspect, the present invention is concerned with a method for the production of cellobiofructose (CBF), comprising

[0142] (a) expressing a polypeptide having levansucrase activity in a suitable host cell; and

[0143] (b) contacting said polypeptide having levansucrase activity with a suitable substrate under conditions that allow the enzymatic production of CBF; wherein said polypeptide having levansucrase activity comprises or consists of a polypeptide having at least 80 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 .

[0144] In some embodiments, said polypeptide having levansucrase activity comprises or consists of a polypeptide having at least 81 , 82, 83, 84, 85, 86, 87, 88, 98, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 .

[0145] In the context of the present invention, the term “polypeptide having levansucrase activity” refers to a polypeptide catalyzing the same biochemical reactions or conversions as the enzyme levansucrase (Enzyme Code E.C. 2.4.1.10). Particularly, in the context of the present invention, a polypeptide having levansucrase activity is able to catalyze the fructosylation of suitable acceptor molecules, such as cellobiose or gentiobiose, particularly by transferring fructose to such an acceptor molecule, particularly by splitting sucrose into its monomeric subunits, glucose and fructose, and transferring the fructose component to said acceptor molecule, e.g., by transfructosylation. Particularly, in the context of the present invention, an enzyme having levansucrase activity catalyzes a transfructosylation reaction leading to the synthesis of CBF, e.g., by splitting sucrose into its monomeric subunits, glucose and fructose, and transferring the fructose component to cellobiose, yielding cellobiofructose. Such polypeptide having levansucrase activity preferably carries out the above enzymatic reaction relative to other possible enzymatic reactions in the presence of the suitable substrates.

[0146] In the context of the present invention, the term “suitable host cell” refers to a cell, for example a microbial cell, that is able to express a polypeptide having levansucrase activity, as herein defined and described, preferably such that said polypeptide has an activity of at least 70.000, preferably at least 80.0000, more preferably at least 90.000 units per litre culture medium (unit = amount of glucose [pmol] generated per minute). Non-limiting examples of suitable organisms are described herein below.

[0147] A “nucleic acid sequence encoding” a particular polypeptide or protein is a nucleic acid sequence, which is transcribed and translated into said polypeptide or protein when placed under the control of appropriate regulatory sequences.

[0148] Nucleic acid sequences encoding the levansucrase polypeptides disclosed herein may have the nucleic acid sequence set forth in SEQ ID NO: 2. However, the inventive methods also encompass those variants thereof that result in the same polypeptide disclosed herein, with said variants deviating from the nucleic acid sequence of SEQ ID NO:2 by at least one nucleotide. Such variants also cover codon-optimized variants of SEQ ID NO:2 that are specifically adapted for expression in a given host cell (without changing the amino acid sequence of the encoded polypeptide).

[0149] The polypeptides disclosed herein that have less than 100% sequence identity to the polypeptide of SEQ ID NO:1 are typically variants that essentially retain levansucrase activity in that the levansucrase enzymatic activity relative to the polypeptide of SEQ ID NO:1 is reduced by not more than 20%, preferably by not more than 15 %, more preferably by not more than 10 %, such as by not more than 9, 8, 7, 6, 5, 4, 3, 2 or 1 % (under the same conditions).

[0150] The term “sequence identity” between two nucleic acid sequences is understood as meaning the identity of the nucleic acid sequence over the entire sequence length in each case, in a preferred embodiment over the entire expressed sequence length, preferably cDNA, even more preferably over the coding sequence, preferably CDS. The term “sequence identity” in the context of polypeptides is understood as meaning the identity of the amino acids over a specific protein region, preferably over the entire protein length, in particular the identity which is calculated by comparison with the aid of known methods.

[0151] Various sequence comparison programs are particularly contemplated for use in this aspect of the invention. Protein and / or nucleic acid sequence homologies may be evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85(8):2444-2448, 1988; Altschul et al., J. Mol. Biol. 215(3):403-410, 1990; Thompson et al., Nucleic Acids Res. 22(2):4673-4680, 1994; Higgins et al., Methods EnzymoL 266:383-402, 1996; Altschul et al., J. Mol. Biol. 215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272, 1993).

[0152] Identity is often measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wl 53705). Such software matches similar sequences by assigning degrees of homology to various deletions, substitutions and other modifications. Thus, the terms “homology” and “identity” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence over a comparison window or designated region as measured using any number of sequence comparison algorithms or by manual alignment and visual inspection.

[0153] Particularly, the polypeptide having levansucrase activity may comprise or consist of a polypeptide having at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1.

[0154] For instance, said polypeptide having levansucrase activity may consist of a polypeptide having at least 80, 81 , 82, 83, 84, 85, 86, 87, 88, 98, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 .

[0155] Said polypeptide having levansucrase activity may be a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1.

[0156] Said polypeptide having levansucrase activity may be a polypeptide encoded by a nucleic acid sequence having the nucleic acid sequence set forth in SEQ ID NO: 2.

[0157] In various embodiments of the method for the production of CBF, the host cell is selected from yeasts and bacteria, such as gram-negative bacteria or gram-positive bacteria. In various embodiments, the host cell is selected from microorganisms of the order Vibrionales, preferably of the family Vibrionaceae, more preferably of the genus Vibrio and / or gammaproteobacteria and / or gram-negative marine bacteria, most preferably is Vibrio natriegens or a Vibrio natriegens strain selected from Vibrio natriegens Vmax or Vibrio natriegens DSM 759.

[0158] It may be preferred that the host cell be selected from microorganisms assessed by the EFSA (European Food Safety Authority) as safe for humans, animals and the environment and thusly assigned the “Qualified Presumption of Safety” (QPS) status. Thus, in various other embodiments, the host cell is selected from gram-negative bacteria, preferably of the genus Cupriavidus, such as Cupriavidus necator; the genus Gluconobacter, such as Gluconobacter oxydans; the genus Komagataeibacter, such as Komagataeibacter sucrofermentans; the genus Agrobacterium; or the genus Xanthomonas; or the host cell is selected from gram-positive non-sporulating bacteria, preferably of the genus Acidipropionibacterium, such as Acidiproplonibacterium acidipropionici; of the genus Bifidobacterium, such as Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, or Bifidobacterium longum; of the genus Carnobacterium, such as Carnobacterium divergens; of the genus Companilactobacillus, such as Companilactobacillus alimentarius or Companilactobacillus farciminis; of the genus Corynebacterium, such as Corynebacterium ammoniagenes or Corynebacterium glutamicum; of the genus Fructilactobacillus, such as Fructilactobacillus sanfranciscensis; of the genus Lacticaseibacillus, such as Lacticaseibacillus casei, Lacticaseibacillus huelsenbergensis, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lactiplantibacillus argentoratensis, Lactiplantibacillus paraplantarum, Lactiplantibacillus pentosus or Lactiplantibacillus plantarum; of the genus Lactobacillus, such as Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii or Lactobacillus kefiranofaciens; of the genus Lactococcus, such as Lactococcus lactis; of the genus Lapidilactobacillus, such as Lapidilactobacillus dextrinicus, Latilactobacillus curvatus or Latilactobacillus sakei; of the genus Lentilactobacillus, such as Lentilactobacillus buchneri, Lentilactobacillus diolivorans, Lentilactobacillus hilgardii, Lentilactobacillus kefiri or Lentilactobacillus parafarraginis; of the genus Leuconostoc, such as Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, or Leuconostoc pseudomesenteroides; of the genus Levilactobacillus, such as Levilactobacillus brevis; of the genus Levilactobacillus, such as Levilactobacillus brevis; of the genus Ligilactobacillus, such as Ligilactobacillus animalis, Ligilactobacillus aviaries or Ligilactobacillus salivarius; of the genus Limosilactobacillus, such as Limosilactobacillus fermentum, Limosilactobacillus mucosae, Limosilactobacillus panis, Limosilactobacillus pontis or Limosilactobacillus reuteri; of the genus Loigolactobacillus, such as Loigolactobacillus coryniformis; of the genus Microbacterium, such as Microbacterium imperial; of the genus Oenococcus, such as Oenococcus oeni; of the genus Pediococcus, such as Pediococcus acidilactici, Pediococcus parvulus or Pediococcus pentosaceus; of the genus Propionibacterium, such as Propionibacterium freudenreichii; of the genus Secundilactobacillus, such as Secundilactobacillus collinoides; of the genus Streptococcus, such as Streptococcus thermophilus; of the genus Bacillus, such as Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus licheniformis, Bacillus mojavensis, Bacillus paralicheniformis, Bacillus pumilus, Bacillus smithii, Bacillus subtilis, Bacillus vallismortis or Bacillus velezensis; of the genus Clostridium, such as Clostridium tyrobutyricum; of the genus Geobacillus, such as Geobacillus stearothermophilus or Geobacillus thermodenitrificans; of the genus Heyndrickxia, such as Heyndrickxia coagulans; of the genus Lederbergia, such as Lederbergia lenta; of the genus Lysinibacillus, such as Lysinibacillus fusiformis; of the genus Niallia, such as Niallia circulans; of the genus Paenibacillus, such as Paenibacillus illinoisensis; of the genus Parageobacillus, such as Parageobacillus thermoglucosidasius; of the genus Pasteuria, such as Pasteuria nishizawae; of the genus Priestia, such as Priestia flexa or Priestia megaterium; or of the genus Shouchella, such as Shouchella clausii; or the host cell is selected from yeasts, preferably of the genus Cyberlindnera, such as Cyberlindnera jadinii; of the genus Debaryomyces, such as Debaryomyces hansenii; of the genus Hanseniaspora, such as Hanseniaspora uvarum; of the genus Kluyveromyces, such as Kluyveromyces lactis or Kluyveromyces marxianus; of the genus Komagataella, such as Komagataella pastoris or Komagataella phaffii; of the genus Limtongozyma, such as Limtongozyma cylindracea; of the genus Ogataea, such as Ogataea angusta or Ogataea polymorpha; of the genus Phaffia, such as Phaffia rhodozyma; of the genus Saccharomyces, such as Saccharomyces bayanus, Saccharomyces cerevisiae or Saccharomyces pastorianus; of the genus Schizosaccharomyces, such as Schizosaccharomyces pombe; of the genus Wickerhamomyces, such as Wickerhamomyces anomalus; of the genus Yarrowia, such as Yarrowia lipolytica; or of the genus Zygosaccharomyces, such as Zygosaccharomyces rouxii.

[0159] In various embodiments, the polypeptide having levansucrase activity is heterologously expressed in said host cell.

[0160] Said polypeptide having levansucrase activity may be expressed in said host cell, as herein defined, by means of an expression plasmid. Expression plasmids suitable for the purposes of the present invention are generally known in the art, and as a non-limiting example of such suitable expression plasmids, the expression plasmid pET-19b(+)_levSKK21 may be mentioned, a 6926 bp long plasmid, wherein the gene coding for the polypeptide having levansucrase activity is under the control of the T7 promoter and the T7 terminator (Fig. 4). Thus, in some embodiments, the polypeptide having levansucrase activity, as herein described and defined, is expressed in a suitable host cell, as herein described and defined, via an expression plasmid in which the nucleic acid sequence encoding for the polypeptide is under control of the T7 promoter and, optionally, T7 terminator, such as, without limitation, the expression plasmid pET- 19b(+)_levSKK21.

[0161] Alternatively or additionally, integration of the levansucrase coding sequence into the host genome may occur.

[0162] Alternatively or additionally, in some embodiments, step (a) includes a cultivation step that is preferably carried out in the absence of a selection antibiotic.

[0163] Alternatively or additionally, in some embodiments, step (b) is carried out using a crude cell extract of the host cells of step (a).

[0164] Alternatively or additionally, in some embodiments, the substrate is a combination of cellobiose and sucrose.

[0165] In some embodiments, the method for production of CBF is characterized in that:

[0166] (1) the host cell is selected from microorganisms of the order Vibrionales, preferably of the family Vibrionaceae, more preferably of the genus Vibrio and / or gammaproteobacteria and / or gramnegative marine bacteria, most preferably is Vibrio natriegens or a Vibrio natriegens strain selected from Vibrio natriegens Vmax or Vibrio natriegens DSM 759; or the host cell is selected from gramnegative bacteria, preferably of the genus Cupriavidus, such as Cupriavidus necator; the genus Gluconobacter, such as Gluconobacter oxydans; the genus Komagataeibacter, such as Komagataeibacter sucrofermentans; the genus Agrobacterium; or the genus Xanthomonas; or the host cell is selected from gram-positive non-sporulating bacteria, preferably of the genus Acidiproplonibacterium, such as Acidipropionibacterium acidipropionici; of the genus Bifidobacterium, such as Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, or Bifidobacterium longum; of the genus Carnobacterium, such as Carnobacterium divergens; of the genus Companilactobacillus, such as Companilactobacillus alimentarius or Companilactobacillus farciminis; of the genus Corynebacterium, such as Corynebacterium ammoniagenes or Corynebacterium glutamicum; of the genus Fructilactobacillus, such as Fructilactobacillus sanfranciscensis; of the genus Lacticaseibacillus, such as Lacticaseibacillus casei, Lacticaseibacillus huelsenbergensis, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lactiplantibacillus argentoratensis, Lactiplantibacillus paraplantarum, Lactiplantibacillus pentosus or Lactiplantibacillus plantarum; of the genus Lactobacillus, such as Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii or Lactobacillus kefiranofaciens; of the genus Lactococcus, such as Lactococcus lactis; of the genus Lapidilactobacillus, such as Lapidilactobacillus dextrinlcus, Latilactobacillus curvatus or Latilactobacillus sakei; of the genus Lentilactobacillus, such as Lentilactobacillus buchneri, Lentilactobacillus diolivorans, Lentilactobacillus hilgardii, Lentilactobacillus kefiri or Lentilactobacillus parafarraginis; of the genus Leuconostoc, such as Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, or Leuconostoc pseudomesenteroides; of the genus Levilactobacillus, such as Levilactobacillus brevis; of the genus Levilactobacillus, such as Levilactobacillus brevis; of the genus Ligilactobacillus, such as Ligilactobacillus animalis, Ligilactobacillus aviaries or Ligilactobacillus salivarius; of the genus Limosilactobacillus, such as Limosilactobacillus fermentum, Limosilactobacillus mucosae, Limosilactobacillus panis, Limosilactobacillus pontis or Limosilactobacillus reuteri; of the genus Loigolactobacillus, such as Loigolactobacillus coryniformis; of the genus Microbacterium, such as Microbacterium imperial; of the genus Oenococcus, such as Oenococcus oeni; of the genus Pediococcus, such as Pediococcus acidilactici, Pediococcus parvulus or Pediococcus pentosaceus; of the genus Propionibacterium, such as Propionibacterium freudenreichii; of the genus Secundilactobacillus, such as Secundilactobacillus colllnoides; of the genus Streptococcus, such as Streptococcus thermophilus; of the genus Bacillus, such as Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus licheniformis, Bacillus mojavensis, Bacillus paralicheniformis, Bacillus pumilus, Bacillus smithii, Bacillus subtilis, Bacillus vallismortis or Bacillus velezensis; of the genus Clostridium, such as Clostridium tyrobutyricum; of the genus Geobacillus, such as Geobacillus stearothermophilus or Geobacillus thermodenitrificans; of the genus Heyndrlckxia, such as Heyndrickxia coagulans; of the genus Lederbergia, such as Lederbergia lenta; of the genus Lysinibaclllus, such as Lyslnlbacillus fusiformls; of the genus Nlallla, such as Niallia circulans; of the genus Paenibaclllus, such as Paenibacillus illinoisensis; of the genus Parageobacillus, such as Parageobacillus thermoglucosidasius; of the genus Pasteuria, such as Pasteuria nishlzawae; of the genus Priestia, such as Priestia flexa or Priestia megaterium; or of the genus Shouchella, such as Shouchella clausii; or the host cell is selected from yeasts, preferably of the genus Cyberlindnera, such as Cyberlindnera jadinii; of the genus Debaryomyces, such as Debaryomyces hansenii; of the genus Hanseniaspora, such as Hanseniaspora uvarum; of the genus Kluyveromyces, such as Kluyveromyces lactis or Kluyveromyces marxianus; of the genus Komagataella, such as Komagataella pastoris or Komagataella phaffii; of the genus Limtongozyma, such as Limtongozyma cylindracea; of the genus Ogataea, such as Ogataea angusta or Ogataea polymorpha; of the genus Phaffia, such as Phaffia rhodozyma; of the genus Saccharomyces, such as Saccharomyces bayanus, Saccharomyces cerevisiae or Saccharomyces pastorianus; of the genus Schizosaccharomyces, such as Schizosaccharomyces pombe; of the genus Wickerhamomyces, such as Wickerhamomyces anomalus; of the genus Yarrowia, such as Yarrowia lipolytica; or of the genus Zygosaccharomyces, such as Zygosaccharomyces rouxii; and

[0167] (2) the polypeptide having levansucrase activity is heterologously expressed in said host cell; and

[0168] (3) step (a) includes a cultivation step that is preferably carried out in the absence of a selection antibiotic; and

[0169] (4) step (b) is carried out using a crude cell extract of the host cells of step (a); and

[0170] (5) the substrate is a combination of cellobiose and sucrose or cellobiose and fructose.

[0171] It may be advantageous, if step (a) comprises the following steps: i) optionally preculturing the host cells in a suitable preculture medium; ii) culturing the host cells in a suitable main culture medium; and iii) obtaining a crude cell extract of the host cell culture of ii).

[0172] For instance, it may be advantageous if step (a) comprises the following steps: i) preculturing the host cells in a suitable preculture medium; ii) culturing the host cells in a suitable main culture medium; and iii) obtaining a crude cell extract of the host cell culture of ii).

[0173] In order to facilitate purification of the final product, i.e., CBF, and / or improve overall performance of the polypeptide having levansucrase activity, the crude cell extract may be obtained by lysis of the cells to obtain a lysed cell composition and removal of any solid components from the lysed cell composition to obtain the crude cell extract, such as by filtration or centrifugation and decantation.

[0174] Insofar part of the method, the preculturing may advantageously be performed at a temperature of about 20 to 55 °C, such as at a temperature of 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36,37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, or 55 °C, preferably at a temperature of about 25 to 50 °C, more preferably at a temperature of about 30 to 45 °C.

[0175] Alternatively or additionally, the preculturing may be performed for a period of about < 15 hours, such as 15, 14.5, 14, 13.5, 13, 12.5, 12, 11.5, 11 , 10.5, 10, 9.5, 9, 8.5, 8, or 7.5 hours, preferably < 7 hours, such as 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1 .5, 1 .0, or 0.5 hours, more preferably about < 6 hours, even more preferably about < 5 hours, such as about 4.5, 4, or 3.5 hours.

[0176] The main culturing may, for instance but without limitation, be advantageously performed at a temperature of about 20 to 55 °C, such as at a temperature of 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36,37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, or 55 °C, preferably at a temperature of about 25 to 50 °C, more preferably at a temperature of about 30 to 45 °C.

[0177] For instance, but without limitation, the main culturing may be performed for a period of about < 15 hours, such as 15, 14.5, 14, 13.5, 13, 12.5, 12, 11.5, 11 , 10.5, or 10, preferably < 10 hours, such as 9.5, 9.0, 8.5, 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, or 0.5 hours, more preferably about < 7 hours, even more preferably about < 5 hours, such as about 4.5, 4, or 3.5 hours.

[0178] The total culturing period may be in the range of about 50, 45, 40, 35 or about 30 hours, preferably < 30 hours, such as about 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, or 17 hours, preferably < 17 hours, more preferably < 15 hours, even more preferably < 12 hours, still more preferably < 10 hours.

[0179] The culturing times indicated may vary depending on the type and / or volume of culturing. For instance, but without limitation, it may be necessary to carry out a preculture cascade, particularly with regard to very large fermentation volumes, such that longer preculturing and / or main culturing times might be required.

[0180] Given the high amounts of polypeptide having levansucrase activity, as herein defined and described, generated in the production method of the present invention, said method requiring comparably short culturing times, sterilization of the culture media is not mandatory. Therefore, it is possible that the method does not comprise sterilization of the preculture medium and / or the main culture medium and is carried out with non-sterilized media. Therefore, in some embodiments of these methods, the preculture medium and / or the main culture medium is not sterilized prior to use.

[0181] Furthermore, for the above reasons, employment of antibiotics is not required. Indeed, as surprisingly discovered by the present inventors, production and yield of the polypeptide having levansucrase activity, as herein defined and described, is improved in such methods according to the present invention that do not comprise employment of one or more antibiotics, compared to control methods, i.e., methods according to the present invention that do comprise employment of one or more antibiotics.

[0182] Consequently, in some embodiments, the preculture medium and / or the main culture medium does not comprise one or more antibiotics. In this context, the antibiotics referred to are those that are typically used to exert selection pressure for the cells that contain the expression plasmid so that growth of cells without the expression plasmid is suppressed.

[0183] The effects observed in the context of antibiotic use were found to be particularly pronounced using a polypeptide having levansucrase activity consisting of a polypeptide having at least 80, 81 , 82, 83, 84, 85, 86, 87, 88, 98, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 such as a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 and / or a polypeptide encoded by a nucleic acid sequence having the nucleic acid sequence set forth in SEQ ID NO: 2.

[0184] Even more pronounced were these surprisingly discovered additional improvements in terms of production and yield of said polypeptide having levansucrase activity when further selecting a host cell from the group of the order Vibrionales, more preferably of the family Vibrionaceae, preferably of the genus Vibrio, and / or from the group of gram-negative marine bacteria, preferably Vibrio natriegens. Still more pronounced were said additional improvement in terms of production and yield of said polypeptide having levansucrase activity when selecting a host cell from the group of the order Vibrionales, more preferably of the family Vibrionaceae, preferably of the genus Vibrio, and / or from the group of gram-negative marine bacteria, preferably Vibrio natriegens, wherein said polypeptide is expressed in said organism via the expression plasmid pET-19b(+)_levSKK21 .

[0185] In some embodiments of such methods, neither the preculture medium nor the main culture medium is sterilized prior to use, and / or neither the preculture medium nor main culture medium comprises any antibiotics.

[0186] Particularly, in some embodiments, neither the preculture medium nor the main culture medium is sterilized prior to use, and neither the preculture medium nor main culture medium comprises any antibiotics.

[0187] Methods for obtaining a crude cell extract from a microbial culture are generally known in the art. IN particular embodiments, the crude cell extract contains the polypeptide having levansucrase activity, as herein defined and described, for example in such an amount that it exhibits a specific activity (Vmax) of at least 70.000, preferably at least 80.0000, more preferably at least 90.000 units per litre culture medium, such as an activity of 70.000, 75.000, 80.000, 85.000, 90.000, 95.000, 100.000, or 110.000 units per litre culture medium. The activity values reflect the maximum conversion rate Vmax, measured at physiological conditions (30 °C and pH 7) and a substrate concentration of at least 500 mM. The activity values were quantified by discontinuous sampling of a corresponding reaction mixture and subsequent HPLC analysis, as described in Hovels et al. (Hovels, M., Kosciow, K., Kniewel, J., Jakob, F., & Deppenmeier, U. (2020). High yield production of levan-type fructans by Gluconobacter japonicus LMG 1417. International Journal of Biological Macromolecules, 164, 295-303).

[0188] Due to such high enzymatic activity, the resultant crude cell extract can be used as is, i.e. without further purification of the polypeptide having levansucrase activity.

[0189] For the generation of CBF, said crude cell extract is typically combined with a composition comprising a suitable substrate, such as a combination of sucrose and cellobiose, typically in form of an aqueous solution, allowing the polypeptide having levansucrase activity to catalyze fructosylation of cellobiose to cellobiofructose. In various embodiments, such substrate solutions may contain sucrose in an amount in the range of about 80 to about 200 g / L, such as about 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 g / L, preferably about 100 to about 180 g / L, more preferably about 140 to about 170 g / L sucrose, for instance about 170g IL. Alternatively or additionally, in various embodiments, such substrate solutions may contain cellobiose in an amount in the range of about 80 to about 200 g / L, such as about 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 g / L, preferably about 100 to about 180 g / L, more preferably about 140 to about 170 g / L, such as about 170 g / L. The concentrations of both substrates may be about the same, preferably essentially the same.

[0190] For instance, but without limitation, said composition comprising a combination of sucrose and cellobiose, in addition to a base such as water, for instance tap water or deionized water, comprise one or more additional components, such as, for instance but without limitation, buffers and stabilizing agents, antioxidants, etc. The pH of such a composition may be, for instance but without limitation, within the range of about 5 to 10, such as about 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10, preferably within the range of about 6.5 to 8, such as about 7 or 7.5.

[0191] The amount of CBF generated using the production method of the present invention starting with about 300 litres of sucrose- and cellobiose-containing solution as the substrate, can be at least about 20 kg, preferably at least 25 kg, more preferably at least about 30 kg, wherein the amount of sucrose and cellobiose in said starting solution is about 170 g / L each.

[0192] CBF thus produced can be isolated and purified by means generally known in the art, such as in a preparatory way using, for example, chromatography on activated carbon powder and / or ion-exchange chromatography.

[0193] A major advantage of the methods of the present invention is that essentially no fructosylation product is generated in addition to CBF, if the only acceptor molecule for the fructosylation reaction is cellobiose.

[0194] Thus, in certain embodiments, the method is also characterized in that the concentration of fructosylation side products other than CBF in the final product is less than 5 %, preferably less than 4 %, more preferably less than 3 %, more preferably less than 2 %, such as about 1.5, 1.0, 0.5, 0.4, 0.3, 0.2, 0.1 , or 0.05 %, relative to the total amount of CBF obtained. In some embodiments, no fructosylation product is generated in addition to CBF.

[0195] Analysis of the product formed in accordance with the present invention can be carried out for example by HPLC inverse phase on a Nucleosil C18 column, using for instance a mixture of water and methanol as the eluent, and / or NMR after isolation.

[0196] In the following, particular embodiments of the present invention are described:

[0197] Embodiment 1 : Use of cellobiofructose (CBF) as a dietary fiber and / or prebiotic component in food products, food product additives, nutraceuticals and / or beverages.

[0198] Embodiment 2: The use according to embodiment 1 , wherein cellobiofructose is comprised in the food products in an amount of about 1 to 95 wt.-%, preferably 5 to 90 wt.-%, more preferably 7 to 80 wt.-%, even more preferably 10 to 70 wt.-%, based on the dry weight of the food products; and / or in the food product additives or nutraceuticals in an amount of about 1 to 99 wt.-%, preferably 5 to 95 wt.-%, more preferably 7 to 85 wt.-%, even more preferably 10 to 75 wt.-%, based on the dry weight of the food product additive or nutraceutical; and / or in the beverages in an amount of about 1 to 50 wt.-%, preferably 2 to 40 wt.-%, more preferably 3 to 30 wt.-%, based on the total weight of the beverage.

[0199] Embodiment 3: The use according to embodiment 1 or embodiment 2, wherein cellobiofructose modifies the microbiome and / or microbiota of the gastrointestinal tract of the consumer; and / or cellobiofructose does not influence blood glucose levels of the consumer.

[0200] Embodiment 4: The use according to any one of embodiments 1 to 3, wherein cellobiofructose increases the growth and / or activity of beneficial microorganisms, preferably bifidobacteria, lactobacilli, and commensal microorganisms associated with a healthy gut microbiota, within the gastrointestinal tract of the consumer.

[0201] Embodiment 5: Use of cellobiofructose for increasing the growth and / or activity of probiotic microorganisms, preferably bifidobacteria and lactobacilli.

[0202] Embodiment 6: The use of embodiment 5, wherein the probiotic microorganisms are comprised in or intended to be comprised in food products, food product additives, nutraceuticals and / or beverages.

[0203] Embodiment 7: The use according to any one of embodiments 1 to 6, wherein the microorganisms are bifidobacteria selected from the group consisting of B. actinocoloniiforme, B. adolescentis, B. lactobacilli selected from the group consisting of Lactobacillus acidophilus, Lactobacillus brevis Lactobacillus casei, Lactobacillus casei immunitas, Lactobacillus casei defensis, Lactobacillus casei shirota, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus lactis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus iners, Lactobacillus paracasei, Lactobacillus plantarum, and Lactobacillus salivarius-, and / or commensal microorganisms selected from the group consisting of Lactococcus casei, Lactococcus lactis, Leuconostoc mesenteroides, Oenococcus oeni, Hominimerdicola aceti, Faecalibacterium prausnitzii, Levilactobacillus brevis, Agathobacter rectalis, Phocaeicola vulgatus, Streptococcus salivarius subsp. thermophilus, and Enterococcus faecalis. Embodiment 8: Cellobiofructose (CBF) or composition comprising CBF for use in the prevention and / or treatment of obesity, constipation, high cholesterol, diabetes, hypertension, and / or heart disease.

[0204] Embodiment 9: The composition for use according to embodiment 8, wherein the composition is a food product, food product additive, nutraceutical and / or beverage or a pharmaceutical composition.

[0205] Embodiment 10: A method for increasing the growth and / or activity of probiotic microorganisms, preferably bifidobacteria or lactobacilli, comprising the step of providing cellobiofructose (CBF) as substrate for the microorganisms.

[0206] Embodiment 11 : Method for the production of cellobiofructose (CBF), comprising

[0207] (a) expressing a polypeptide having levansucrase activity in a suitable host cell; and

[0208] (b) contacting said polypeptide having levansucrase activity with a suitable substrate under conditions that allow the enzymatic production of CBF; wherein said polypeptide having levansucrase activity comprises or consists of a polypeptide having at least 80 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 .

[0209] Embodiment 12: The method of embodiment 11 , wherein

[0210] (1) the host cell is selected from microorganisms of the order Vibrionales, preferably of the family Vibrionaceae, more preferably of the genus Vibrio and / or gammaproteobacteria and / or gramnegative marine bacteria, most preferably is Vibrio natriegens or a Vibrio natriegens strain selected from Vibrio natriegens Vmax or Vibrio natriegens DSM 759; or the host cell is selected from gramnegative bacteria, preferably of the genus Cupriavidus, such as Cupriavidus necator; the genus Gluconobacter, such as Gluconobacter oxydans; the genus Komagataeibacter, such as Komagataeibacter sucrofermentans; the genus Agrobacterium; or the genus Xanthomonas; or the host cell is selected from gram-positive non-sporulating bacteria, preferably of the genus Acidipropionibacterium, such as Acidipropionibacterium acidipropionici; of the genus Bifidobacterium, such as Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, or Bifidobacterium longum; of the genus Carnobacterium, such as Carnobacterium divergens; of the genus Companilactobacillus, such as Companilactobacillus alimentarius or Companilactobacillus farciminis; of the genus Corynebacterium, such as Corynebacterium ammoniagenes or Corynebacterium glutamicum; of the genus Fructilactobacillus, such as Fructilactobacillus sanfranciscensis; of the genus Lacticaseibacillus, such as Lacticaseibacillus casei, Lacticaseibacillus huelsenbergensis, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lactiplantibacillus argentoratensis, Lactiplantibacillus paraplantarum, Lactiplantibacillus pentosus or Lactiplantibacillus plantarum; of the genus Lactobacillus, such as Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii or Lactobacillus kefiranofaciens; of the genus Lactococcus, such as Lactococcus lactis; of the genus Lapidilactobacillus, such as Lapidilactobacillus dextrinicus, Latilactobacillus curvatus or Latilactobacillus sakei; of the genus Lentilactobacillus, such as Lentilactobacillus buchneri, Lentilactobacillus diolivorans, Lentilactobacillus hilgardii, Lentilactobacillus kefiri or Lentilactobacillus parafarraginis; of the genus Leuconostoc, such as Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, or Leuconostoc pseudomesenteroides; of the genus Levilactobacillus, such as Levilactobacillus brevis; of the genus Levilactobacillus, such as Levilactobacillus brevis; of the genus Ligilactobacillus, such as Ligilactobacillus animalis, Ligilactobacillus aviaries or Ligilactobacillus salivarius; of the genus Limosilactobacillus, such as Limosilactobacillus fermentum, Limosilactobacillus mucosae, Limosilactobacillus panis, Limosilactobacillus pontis or Limosilactobacillus reuteri; of the genus Loigolactobacillus, such as Loigolactobacillus coryniformis; of the genus Microbacterium, such as Microbacterium imperial; of the genus Oenococcus, such as Oenococcus oeni; of the genus Pediococcus, such as Pediococcus acidilactici, Pediococcus parvulus or Pediococcus pentosaceus; of the genus Propionibacterium, such as Propionibacterium freudenreichii; of the genus Secundilactobacillus, such as Secundilactobacillus colllnoides; of the genus Streptococcus, such as Streptococcus thermophilus; of the genus Bacillus, such as Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus licheniformis, Bacillus mojavensis, Bacillus paralicheniformis, Bacillus pumilus, Bacillus smithli, Bacillus subtilis, Bacillus vallismortis or Bacillus velezensls; of the genus Clostridium, such as Clostridium tyrobutyricum; of the genus Geobacillus, such as Geobacillus stearothermophilus or Geobacillus thermodenitrificans; of the genus Heyndrlckxia, such as Heyndrickxia coagulans; of the genus Lederbergia, such as Lederbergia lenta; of the genus Lysinibaclllus, such as Lyslnlbacillus fusiformls; of the genus Nlallla, such as Niallia circulans; of the genus Paenibaclllus, such as Paenibacillus illinoisensis; of the genus Parageobacillus, such as Parageobacillus thermoglucosidasius; of the genus Pasteuria, such as Pasteuria nishlzawae; of the genus Prlestia, such as Priestia flexa or Priestia megaterium; or of the genus Shouchella, such as Shouchella clausil; or the host cell is selected from yeasts, preferably of the genus Cyberlindnera, such as Cyberlindnera jadinii; of the genus Debaryomyces, such as Debaryomyces hansenii; of the genus Hanseniaspora, such as Hansenlaspora uvarum; of the genus Kluyveromyces, such as Kluyveromyces lactis or Kluyveromyces marxianus; of the genus Komagataella, such as Komagataella pastoris or Komagataella phaffii; of the genus Llmtongozyma, such as Limtongozyma cylindracea; of the genus Ogataea, such as Ogataea angusta or Ogataea polymorpha; of the genus Phaffia, such as Phaffia rhodozyma; of the genus Saccharomyces, such as Saccharomyces bayanus, Saccharomyces cerevisiae or Saccharomyces pastorianus; of the genus Schizosaccharomyces, such as Schizosaccharomyces pombe; of the genus Wickerhamomyces, such as Wickerhamomyces anomalus; of the genus Yarrowia, such as Yarrowia lipolytica; or of the genus Zygosaccharomyces, such as Zygosaccharomyces rouxiiand / or

[0211] (2) the polypeptide having levansucrase activity is heterologously expressed in said host cell; and / or

[0212] (3) step (a) includes a cultivation step that is preferably carried out in the absence of a selection antibiotic; and / or

[0213] (4) step (b) is carried out using a crude cell extract of the host cells of step (a); and / or

[0214] (5) the substrate is a combination of cellobiose and sucrose or cellobiose and fructose.

[0215] Embodiment 13: The method according to embodiment 11 or 12, wherein step (a) comprising the steps of: i) optionally preculturing the host cells in a suitable preculture medium; ii) culturing the host cells in a suitable main culture medium; and iii) obtaining a crude cell extract of the host cell culture of ii).

[0216] Embodiment 14: The method according to embodiment 12 or 13, wherein the crude cell extract is obtained by lysis of the cells to obtain a lysed cell composition and removal of any solid components from the lysed cell composition to obtain the crude cell extract.

[0217] Embodiment 15: The method according to any one of embodiments 13 to 14, wherein

[0218] (1) the preculturing is performed at a temperature of about 20 to 55 °C, preferably of about 25 to 50 °C, more preferably of about 30 to 45 °C, and / or for a period of < 15 hours, preferably < 7 hours, more preferably < 6 hours, even more preferably

[0219] < 5 hours; and / or

[0220] (2) the main culturing is performed at a temperature of about 20 to 55 °C, preferably of about 25 to 50 °C, more preferably of about 30 to 45 °C, and / or for a period of < 15 hours, preferably < 10 hours, more preferably < 7 hours, even more preferably

[0221] < 5 hours; and / or

[0222] (3) the total culturing period is about 30 hours, preferably < 17 hours, more preferably < 15 hours, even more preferably < 12 hours, still more preferably < 10 hours.

[0223] Embodiment 16: The method according to any one of embodiments 11 to 15, wherein

[0224] (1) the preculture medium and / or the main culture medium is not sterilized prior to use; and / or

[0225] (2) the preculture medium and / or the main culture medium does not comprise one or more antibiotics.

[0226] Embodiment 17: The method according to any one of embodiments 11 to 16, wherein the amount of CBF generated from about 300 litres of a solution containing about 170 g / L sucrose and about 170 g / L cellobiose, is at least about 20 kg, preferably at least 25 kg, more preferably at least about 30 kg.

[0227] Embodiment 18: The method according to any one of embodiments 11 to 17, wherein the concentration of fructosylation side products other than CBF in the final product is less than 5% relative to the total amount of CBF obtained.

[0228] In the following, non-limiting examples that illustrate various aspects of the present invention are described. Examples

[0229] Example 1 : Production of levansucrase LGVSKK2I in V. natriegens

[0230] A suitable culture medium is inoculated with the genetically optimized strain V. natriegens pET19b(+)_ / evSKK2 . The pre-culture, which is carried out on a 50 ml scale in an Erlenmeyer flask at 37 °C and a shaking speed of 350 rpm, takes four hours. This is followed by inoculation of the main culture, which is carried out in a 1 L scale under the same conditions. During the five-hour main culture, the recombinant levansucrase LevSKK2i is produced and accumulates in the Vibrio cells. After completion of the main culture, the cells are harvested and disrupted using a suitable method. After filtration and the associated separation of insoluble cell debris, a crude cell extract containing large quantities of the heterologously produced Levansucrase LevSKK2i is available. This extract can be used for the subsequent synthesis of CBF. The entire process therefore takes about ten hours. The levansucrase activity yield obtained during this time amounts to 100.000 units per litre of culture. The activity yield generated by the process according to the present invention is significantly improved compared to state-of-the-art methods for the production of CBS.

[0231] Example 2: Production of CBF

[0232] An amount of crude cell extract obtained in Example 1 containing levansucrase LevSKK2i with an activity of 300 units / L culture volume, corresponding to 1 mg of isolated enzyme, was mixed with an aqueous solution containing sucrose (170 g / L), cellobiose (170 g / L), and sodium phosphate buffer pH 7 (0.3 g / L). The reaction is then maintained at 30 °C for 24 hours.

[0233] The amount of enzyme generated in Example 1 is sufficient to convert 300 litres of the respective sucrose- and cellobiose-containing process solution, which yields a total cellobiofructose amount of about 30 kg.

[0234] Example 3: Evaluation of prebiotic properties

[0235] After enzymatic production, cellobiofructose was purified using activated carbon and employed in growth experiments as a substrate for health-promoting bifidobacteria, lactobacilli, and commensal microorganisms associated with a healthy gut microbiota. The experiments revealed that cellobiofructose stimulates the growth of a broad range of bifidobacteria, lactobacilli and several commensal gut microbes. Escherichia coli, a microbe associated with intestinal disorders, is unable to metabolize cellobiofructose. The results are depicted in Fig. 2 and Fig. 3. Unlike cellobiose, cellobiofructose promotes the growth of probiotic bifidobacteria. This observation is unique and has not previously been described in the literature. The intensity of the biomass increase indicates that the organisms are able to specifically hydrolyze the fructose unit attached to cellobiose by the levansucrase and use it to build biomass.

[0236] Example 4: Evaluation of pharmacokinetics (blood glucose level study)

[0237] General study design

[0238] The test item cellobiofructose was administered once by oral gavage to 20 male rats in two dose groups at the respective dose levels of 5 g / kg and 2 g / kg bodyweight, at a volume of 10 mL / kg bodyweight. A further group of 5 male animals was treated with a positive control solution (20% glucose solution). Additionally, 5 male animals received water (10 mL / kg bodyweight) as vehicle control via the same route.

[0239] Table 1: Dose groups and applied dosages

[0240] The individual dose volumes were calculated from the latest actual animal body. The day of application was indicated as day 1 . During the in-life phase, viability and general clinical signs were monitored. Before and after the application, blood samples from all animals were collected to acquire data on blood glucose levels (pre, 15, 30, 45, 60 and 120 min after application). Subsequently, all animals were euthanized in a CO2- enriched atmosphere.

[0241] Monitoring

[0242] The following parameters were observed and documented during the in-life period:

[0243] Viability / mortality Daily

[0244] General clinical signs / behavior Daily

[0245] Body weight Once before application start (d1)

[0246] Blood sampling (Blood glucose Pre (30 min before application), 15, 30, 45, 60, measurement) 120 min after application

[0247] Viability, general clinical signs, and behavior

[0248] Cage-side observations to detect signs of illness or reactions to treatment, moribund animals or fatalities were conducted at least once daily throughout the in-life phase up to the day of necropsy. Data was recorded on prepared checklists.

[0249] Blood sampling

[0250] On day 1 of the study, blood glucose levels were determined pre-dose (approx. 30 min before application) as well as 15 min, 30 min, 45 min, 60 min and 120 min after application. Therefore, approximately 20 pl blood per time point was drawn from the tail vein of all animals and applied to human glucose testing strips for the quantitative blood glucose determination using the ACCU-CHEK Aviva blood glucose monitoring system.

[0251] To establish a reliable baseline glucose level before measurement, all animals were fasted overnight before administration start. Until the measurement was completed all animals were fasted. After blood glucose measurement the animals received food ad libitum until the collection of individual faeces sample was completed.

[0252] Results No test item related abnormalities regarding general clinical signs were observed in any of the experimental animals. No animal of the experimental groups died or had to be euthanized during the whole in-life phase of this study.

[0253] Inductive statistical analysis revealed that the mean blood glucose levels from for the first measurement time point of this study (30 min before application) ranged from 93,0 mg / dl (high dose) to 100,2 mg / dl (vehicle) with no significant changes between the male experimental animals of the different treatment groups.

[0254] After application, all male animals treated with the 20% glucose solution acting as positive control showed a significant increase in the mean blood glucose levels forwarding from 15 min (142,2 mg / dl) after application, reaching its peak point with 166,8 mg / dl after 45 min after application until the end of the measurement, 120 min after application (111 ,4 mg / dl) compared to the vehicle control group (103,4 mg / dl, 104,2 mg / dl and 91 mg / dl, respectively) (Fig. 5). In comparison, the high and low dose groups showed only slight, but no significant changes for all timepoints compared to the vehicle control group (Fig. 5).

[0255] These results confirm that cellobiofructose is only minimally absorbed and metabolized in the upper intestines and thus align with the physiological behavior expected of dietary fibers, as defined by the European Union's regulatory framework. By resisting enzymatic digestion in the small intestine, cellobiofructose fulfils a key requirement for dietary fiber classification. These findings underscore its suitability as a functional ingredient for the development of healthier food products, reinforcing its value as a non-glycemic, fiber-based sweetener.

[0256] Example 5: Evaluation of cell toxicity

[0257] For toxicological assessment, cellobiofructose was purified by preparative HPLC employing hydrophilic- interaction-chromatography. Per run, 4.5 mL of a cellobiofructose-containing solution produced in accordance with Example 2 were injected into a preparative AZURA HPLC-system (Knauer GmbH), which consisted of the high-pressure-gradient pump P 6.1 L, the preparative autosampler AS 6.1 L equipped with a 10 mL sample loop, and the preparative refractive index detector RID 2.1 L. Fractionation was achieved by the 16-port MultiPos Valve V 4.1 controlled by the valve drive VU 4.1. Data evaluation and automated handling of the valve drive was accomplished by ClarityChrom® 8.2.3 (Knauer GmbH) using thresholds of < 15 mV (solvent recycling), > 15 - 40 (waste), and > 40 (fraction collection). Separation was performed using the main column Eurospher I1 100-5 NH2 250x20mm (Knauer GmbH) combined with the pre-column Eurospher II 100-5 NH2 50x20mm at 22 °C. The mobile phase (60 % [v / v] acetonitrile) was applied in isocratic mode at a flow rate of 20 mL min-1. The purified cellobiofructose was processed and dried by evaporation and subsequent lyophilization as described in Pohlentz et al. (Pohlentz, J. C., Gallala, N., Kosciow, K., & Hovels, M. (2022). Growth behavior of probiotic microorganisms on levan-and inulin-based fructans. Journal of Functional Foods, 99, 105343.).

[0258] To validate cell viability of HT-29 cells in the presence or absence of cellobiofructose, the MTT assay was performed. Therefore, HT-29 cells (10,000 / well) were plated in a 96-well plate and supplemented with 200 L DMEM High glucose medium containing 10 % fetal bovine serum (FBS) and Antibiotic-Antimycotic. Using six biological replicates, cells were supplemented with FWpure (negative control), bortezomib (BTZ; positive control), and aqueous solutions of 1 mM; 10 mM; 20 mM of cellobiofructose. Following incubation at 37 °C for 18 hours in a humidified CO2 (5 %) / air (95 %) atmosphere (referred to as 5 % CO2 atmosphere), the medium was removed, and HT-29 cells were washed with PBS. 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) was solved in sterile DMEM high glucose medium and added to each well at a concentration of 0.5 mg mL-1 . After one hour of incubation at 37 °C in a humidified 5 % CO2 atmosphere, MTT was removed, and formazan crystals were resolved. Therefore, 100 pL of solubilizing solution (99.5 % isopropanol, 0.4 % 1 M HCI, and 0.1 % Triton X-100) were added and incubated at 37 °C until formazan crystals disappeared. The absorbance was measured at 570 nm using a Tecan Infinite 200 M Plex plate reader (Tecan Group AG). The MTT assay showed no impaired cell viability for HT-29 cells in the presence of cellobiofructose, demonstrating the non-cytotoxic nature of cellobiofructose. No significant reduction in cell viability was observed across all tested concentrations. The mean values and standard deviations of the relative cell viability compared to the negative control are shown in Fig. 6. Statistical validation was performed by ANOVA using GrapdPad Prism. **** = p < 0,005.

[0259] These findings demonstrate the safety profile of cellobiofructose, supporting its application as a safe fiber in functional foods and other consumer products.

[0260] Example 6: Evaluation of solubility of cellobiofructose

[0261] In order to assess the solubility of cellobiofructose, the trisaccharide was produced in accordance with Example 2 and purified in accordance with Example 5. The resulting dry powder was dissolved in increasing concentrations in H2Opure at 20 °C, revealing maximal solubility of 400 ± 20 g / L for cellobiofructose. This solubility exceeds that of many other dietary fibers and even that of the substrate used for enzymatic synthesis, cellobiose, which at 140 g / l has a limited water solubility.

Claims

CLAIMS1 . Use of cellobiofructose (CBF) as a dietary fiber and / or prebiotic component in food products, food product additives, nutraceuticals and / or beverages.

2. The use according to claim 1 , wherein cellobiofructose is comprised in the food products in an amount of about 1 to 95 wt.-%, preferably 5 to 90 wt.-%, more preferably 7 to 80 wt.-%, even more preferably 10 to 70 wt.-%, based on the dry weight of the food products; and / or in the food product additives or nutraceuticals in an amount of about 1 to 99 wt.-%, preferably 5 to 95 wt.-%, more preferably 7 to 85 wt.-%, even more preferably 10 to 75 wt.-%, based on the dry weight of the food product additive or nutraceutical; and / or in the beverages in an amount of about 1 to 50 wt.-%, preferably 2 to 40 wt.-%, more preferably 3 to 30 wt.-%, based on the total weight of the beverage.

3. The use according to claim 1 or claim 2, wherein cellobiofructose modifies the microbiome and / or microbiota of the gastrointestinal tract of the consumer; and / or cellobiofructose does not influence blood glucose levels of the consumer.

4. The use according to any one of claims 1 to 3, wherein cellobiofructose increases the growth and / or activity of beneficial microorganisms, preferably bifidobacteria, lactobacilli, and commensal microorganisms associated with a healthy gut microbiota, within the gastrointestinal tract of the consumer.

5. Use of cellobiofructose for increasing the growth and / or activity of probiotic microorganisms, preferably bifidobacteria and lactobacilli.

6. The use of claim 5, wherein the probiotic microorganisms are comprised in or intended to be comprised in food products, food product additives, nutraceuticals and / or beverages.

7. The use according to any one of claims 1 to 6, wherein the microorganisms are bifidobacteria selected from the group consisting of B. actinocoloniiforme, B. adolescentis, B. aemilianum, B. aerophilum, B. aesculapii, B. amazonense, B. angulatum, B. animalis, B. anseris, B. apousia, B. apri, B. aquikefiri, B. asteroids, B. avesanii, B. biavatii, B. bifidum, B. bohemicum, B. bombi, B. boum, B. breve, B. callimiconis, B. callitrichidarum, B. callitrichos, B. canis, B. castoris, B. catenulatum, B. catulorum, B. cebidarum, B. choerinum, B.choladohabitans, B. choloepi, B. colobi, B. commune, B. cricetid, B.cuniculi, B. dentium, B. dolichotidis, B. erythrocebi, B. eulemuris, B. faecale, B. felsineum, B. gallicum, B. gallinarum, B. globosum, B. goeldii, B. hapali, B. indicum, B. infantis, B. italicum, B. jacchi, B. lemurum, B. leontopitheci, B. longum, B. magnum, B.margollesii, B. merycicum, B. miconis, B. miconisargentati, B. minimum, B. mongoliense, B. moraviense, B. moukalabense, B. myosotis, B. oedipodis, B. olomucense, B. panos, B. parmae, B. pluvialisilvae, B. polysaccharolyticum, B. pongonis, B. porcinum, B. primatium, B.lactobacilli selected from the group consisting of Lactobacillus acidophilus, Lactobacillus brevis Lactobacillus casei, Lactobacillus casei immunitas, Lactobacillus casei defensis, Lactobacillus casei shirota, Lactobacillus crispatus, Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus lactis, Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus iners, Lactobacillus paracasei, Lactobacillus plantarum, and Lactobacillus salivarius-, and / or commensal microorganisms selected from the group consisting of Lactococcus casei, Lactococcus lactis, Leuconostoc mesenteroides, Oenococcus oeni, Hominimerdicola aceti, Faecalibacterium prausnitzii, Levilactobacillus brevis, Agathobacter rectalis, Phocaeicola vulgatus, Streptococcus salivarius subsp. thermophilus, and Enterococcus faecalis.

8. Cellobiofructose (CBF) or composition comprising CBF for use in the prevention and / or treatment of obesity, constipation, high cholesterol, diabetes, hypertension, and / or heart disease.

9. The composition for use according to claim 8, wherein the composition is a food product, food product additive, nutraceutical and / or beverage or a pharmaceutical composition.

10. A method for increasing the growth and / or activity of probiotic microorganisms, preferably bifidobacteria or lactobacilli, comprising the step of providing cellobiofructose (CBF) as substrate for the microorganisms.11 . Method for the production of cellobiofructose (CBF), comprising(a) expressing a polypeptide having levansucrase activity in a suitable host cell; and(b) contacting said polypeptide having levansucrase activity with a suitable substrate under conditions that allow the enzymatic production of CBF; wherein said polypeptide having levansucrase activity comprises or consists of a polypeptide having at least 80 % sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 .

12. The method of claim 11 , wherein(1) the host cell is selected from microorganisms of the order Vibrionales, preferably of the family Vibrionaceae, more preferably of the genus Vibrio and / or gammaproteobacteria and / or gramnegative marine bacteria, most preferably is Vibrio natriegens or a Vibrio natriegens strain selected from Vibrio natriegens Vmax or Vibrio natriegens DSM 759; or the host cell is selected from gramnegative bacteria, preferably of the genus Cupriavidus, such as Cupriavidus necator; the genus Gluconobacter, such as Gluconobacter oxydans; the genus Komagataeibacter, such as Komagataeibacter sucrofermentans; the genus Agrobacterium; or the genus Xanthomonas; or the host cell is selected from gram-positive non-sporulating bacteria, preferably of the genus Acidipropionibacterium, such as Acidipropionibacterium acidipropionici; of the genusBifidobacterium, such as Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium breve, or Bifidobacterium longum; of the genus Carnobacterium, such as Carnobacterium divergens; of the genus Companilactobacillus, such as Companilactobacillus alimentarius or Companilactobacillus farciminls; of the genus Corynebacterium, such as Corynebacterium ammoniagenes or Corynebacterium glutamicum; of the genus Fructilactobacillus, such as Fructilactobacillus sanfranciscensis; of the genus Lacticaseibacillus, such as Lacticaseibacillus casei, Lacticaseibacillus huelsenbergensis, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lactiplantibacillus argentoratensis, Lactiplantibacillus paraplantarum, Lactiplantibacillus pentosus or Lactiplantibacillus plantarum; of the genus Lactobacillus, such as Lactobacillus acidophilus, Lactobacillus amylolyticus, Lactobacillus amylovorus, Lactobacillus crispatus, Lactobacillus delbrueckii, Lactobacillus gallinarum, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii or Lactobacillus kefiranofaciens; of the genus Lactococcus, such as Lactococcus lactis; of the genus Lapidilactobacillus, such as Lapidilactobacillus dextrinlcus, Latilactobacillus curvatus or Latilactobacillus sakei; of the genus Lentilactobacillus, such as Lentilactobacillus buchneri, Lentilactobacillus diolivorans, Lentilactobacillus hilgardii, Lentilactobacillus kefiri or Lentilactobacillus parafarraginis; of the genus Leuconostoc, such as Leuconostoc citreum, Leuconostoc lactis, Leuconostoc mesenteroides, or Leuconostoc pseudomesenteroides; of the genus Levilactobacillus, such as Levilactobacillus brevis; of the genus Levilactobacillus, such as Levilactobacillus brevis; of the genus Ligilactobacillus, such as Ligilactobacillus animalis, Ligilactobacillus aviaries or Ligilactobacillus salivarius; of the genus Limosilactobacillus, such as Limosilactobacillus fermentum, Limosilactobacillus mucosae, Limosilactobacillus panis, Limosilactobacillus pontis or Limosilactobacillus reuteri; of the genus Loigolactobacillus, such as Loigolactobacillus coryniformis; of the genus Microbacterium, such as Microbacterium imperial; of the genus Oenococcus, such as Oenococcus oeni; of the genus Pediococcus, such as Pediococcus acidilactici, Pediococcus parvulus or Pediococcus pentosaceus; of the genus Propionibacterium, such as Propionibacterium freudenreichii; of the genus Secundilactobacillus, such as Secundilactobacillus colllnoides; of the genus Streptococcus, such as Streptococcus thermophilus; of the genus Bacillus, such as Bacillus amyloliquefaciens, Bacillus atrophaeus, Bacillus licheniformis, Bacillus mojavensis, Bacillus paralicheniformis, Bacillus pumilus, Bacillus smithii, Bacillus subtilis, Bacillus vallismortis or Bacillus velezensis; of the genus Clostridium, such as Clostridium tyrobutyricum; of the genus Geobacillus, such as Geobacillus stearothermophilus or Geobacillus thermodenitrificans; of the genus Heyndrlckxia, such as Heyndrickxia coagulans; of the genus Lederbergia, such as Lederbergia lenta; of the genus Lysinibacillus, such as Lyslnlbacillus fusiformls; of the genus Nlallla, such as Niallia circulans; of the genus Paenibaclllus, such as Paenibacillus illinoisensis; of the genus Parageobacillus, such as Parageobacillus thermoglucosidasius; of the genus Pasteuria, such as Pasteuria nishizawae; of the genus Priestia, such as Priestia flexa or Priestia megaterium; or of the genus Shouchella, such as Shouchella clausii; or the host cell is selected from yeasts, preferably of the genus Cyberlindnera, such as Cyberlindnera jadinii; of the genus Debaryomyces, such as Debaryomyces hansenii; of the genus Hanseniaspora, such as Hanseniaspora uvarum; of the genus Kluyveromyces, such as Kluyveromyces lactis or Kluyveromyces marxianus; of the genus Komagataella, such asKomagataella pastoris or Komagataella phaffii; of the genus Limtongozyma, such as Limtongozyma cylindracea; of the genus Ogataea, such as Ogataea angusta or Ogataea polymorpha; of the genus Phaffia, such as Phaffia rhodozyma; of the genus Saccharomyces, such as Saccharomyces bayanus, Saccharomyces cerevisiae or Saccharomyces pastorianus; of the genus Schizosaccharomyces, such as Schizosaccharomyces pombe; of the genus Wickerhamomyces, such as Wickerhamomyces anomalus; of the genus Yarrowia, such as Yarrowia lipolytica; or of the genus Zygosaccharomyces, such as Zygosaccharomyces rouxii; and / or(2) the polypeptide having levansucrase activity is heterologously expressed in said host cell; and / or(3) step (a) includes a cultivation step that is preferably carried out in the absence of a selection antibiotic; and / or(4) step (b) is carried out using a crude cell extract of the host cells of step (a); and / or(5) the substrate is a combination of cellobiose and sucrose or cellobiose and fructose.

13. The method according to claim 11 or 12, wherein step (a) comprising the steps of: i) optionally preculturing the host cells in a suitable preculture medium; ii) culturing the host cells in a suitable main culture medium; and iii) obtaining a crude cell extract of the host cell culture of ii).

14. The method according to claim 12 or 13, wherein the crude cell extract is obtained by lysis of the cells to obtain a lysed cell composition and removal of any solid components from the lysed cell composition to obtain the crude cell extract.

15. The method according to any one of claims 11 to 14, wherein(1) the preculture medium and / or the main culture medium is not sterilized prior to use; and / or(2) the preculture medium and / or the main culture medium does not comprise one or more antibiotics; and / or(3) the amount of CBF generated from about 300 litres of a solution containing about 170 g / L sucrose and about 170 g / L cellobiose, is at least about 20 kg, preferably at least 25 kg, more preferably at least about 30 kg; and / or(4) the concentration of fructosylation side products other than CBF in the final product is less than 5% relative to the total amount of CBF obtained.