阅读说明：本技术 制备木葡聚糖-寡糖的方法 (Process for preparing xyloglucan-oligosaccharides ) 是由 S·格劳布纳 V·兹韦尔洛夫 W·施瓦茨 P·科恩贝格尔 B·安德烈森 于 2018-11-16 设计创作，主要内容包括：本发明涉及一种制备寡糖的方法,该寡糖尤其可以用作食物添加剂,以降低食物产品的卡路里含量、使食物产品增甜、提高食物产品的纤维含量、改善食物产品的质地以及刺激肠道微生物组细菌。此外,它们还可以应用于动物饲料或其他应用领域。更具体地,本发明涉及将木葡聚糖多糖高温水解为确定的木葡聚糖寡糖。本发明还涉及用本发明的方法生产的寡糖水解产物,以及涉及所述寡糖水解产物在人类和/或动物营养中作为益生元或其他用途的用途。还提供了新型内切葡聚糖酶,其用于本发明的方法以及其他应用。(The present invention relates to a process for the preparation of oligosaccharides, which are particularly useful as food additives to reduce the calorie content of food products, to sweeten food products, to increase the fiber content of food products, to improve the texture of food products and to stimulate the intestinal microbiome bacteria. Furthermore, they can also be used in animal feed or other fields of application. More specifically, the present invention relates to the pyrohydrolysis of xyloglucan polysaccharides into defined xyloglucan oligosaccharides. The invention also relates to the oligosaccharide hydrolysate produced by the method of the invention and to the use of the oligosaccharide hydrolysate as a prebiotic or for other use in human and/or animal nutrition. Also provided are novel endoglucanases useful in the methods of the invention, as well as other applications.)

1. A method of producing xyloglucan oligosaccharides (XGOS) from a xyloglucan source, the method comprising enzymatically hydrolysing xyloglucan at a temperature above 50 ℃ with an enzyme exhibiting xyloglucanase activity at a temperature above 50 ℃, characterised in that the enzyme exhibits an end product inhibition constant (Ki) equal to or above 5 mM.

2. The method according to claim 1, wherein the xyloglucan source is tamarind, piper nigrum, rapeseed, apple, blueberry, olive or other xyloglucan sources containing fractions thereof, such as tamarind kernel powder, defatted tamarind kernel powder, tamarind seeds and other sources of seeds and cell walls.

3. The method according to claim 1 or 2, wherein the enzyme is an endoglucanase, preferably selected from GH families 5, 9, 12, 16, 44 or 74.

4. The method of any one of the preceding claims, wherein the enzyme hybridizes to a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO: 4 has at least 75% sequence identity.

5. The method according to any one of the preceding claims, wherein the enzyme is recombinantly produced in a host cell, and wherein the host cell does not exhibit endogenous xyloglucanase activity towards a xyloglucan source.

6. The method according to any one of the preceding claims, comprising the steps of:

under conditions favoring the hydrolysis of said polysaccharide by xyloglucanase to form a xyloglucan-derived polysaccharide hydrolysate solution, contacting the xyloglucan-derived polysaccharide with a nucleic acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO: 4 in an aqueous solution.

7. The method of any one of the preceding claims, further comprising one or more of the steps of:

removing the solids;

removal of proteins and salts, for example by ion exchange chromatography;

removal of the colorant, for example by ultrafiltration or nanofiltration; and

recovering from said solution a xyloglucan-derived polysaccharide hydrolysate.

8. The method according to any one of the preceding claims, wherein the enzyme is present in an amount of 0.05% (w/w) or less of the xyloglucan source, and/or wherein the final amount of xyloglucan source used is 100g/l or higher.

9. The process according to any one of the preceding claims, wherein a xyloglucan hydrolysate is produced comprising, consisting essentially of, or consisting of a mixture of DP7 to DP9 XGOS.

10. Use of a hydrolysate comprising a mixture of DP7 to DP9XGOS produced with the method according to any one of the preceding claims for the production of a food product, animal feed product or other product.

11. A product comprising a DP7 to DP9XGOS mixture produced with the method of any one of claims 1 to 9.

12. An enzyme having endoglucanase activity, comprising, consisting essentially of, or consisting of a polypeptide that hybridizes to a polypeptide according to SEQ ID NO: 2. 3 or 4, provided that the endoglucanase is not SEQ ID NO: 1.

13. A nucleic acid molecule comprising a nucleic acid sequence encoding the endoglucanase of claim 12.

14. Use of an enzyme according to claim 12 for the production of a mixture of XGOS from a xyloglucan source, wherein the mixture comprises, consists essentially of, or consists of DP7 to DP9 XGOS.

Technical Field

The present invention relates to a process for the preparation of oligosaccharides, which are particularly useful as food additives to reduce the calorie content of food products, to sweeten food products, to increase the fiber content of food products, to improve the texture of food products and to stimulate the intestinal microbiome bacteria. Furthermore, they can also be used in animal feed or other fields of application. More specifically, the present invention relates to the pyrohydrolysis of xyloglucan (xyloglucan) polysaccharides into defined xyloglucan oligosaccharides (xyloglucan oligosaccharides). The invention also relates to the oligosaccharide hydrolysate produced by the method of the invention and to the use of the oligosaccharide hydrolysate as a prebiotic or for other use in human and/or animal nutrition. Also provided are novel endoglucanases useful in the methods of the invention, as well as other applications.

Background

In general, fats, oils, starches, carbohydrates, sugars, and products derived therefrom are widely used in processing foods. These ingredients have vital functional significance in terms of the appearance, taste, mouthfeel and other organoleptic qualities of the food. However, they can be metabolized by the body during passage through the intestinal tract, thus greatly increasing the calorie content of such foods. Recently, consumers are becoming increasingly health conscious. Thus, many individuals attempt to minimize the intake of high calorie foods and foods containing high fat levels. Consumers demand traditionally processed foods in reduced calorie and low fat forms. By adding non-digestible oligosaccharides, the amount of sugar and fat can be reduced without loss of the sensory quality of the processed food.

Regulatory agencies such as European Agency for Food Safety (EFSA Journal,2010) and American Food Agency (FDA) (https:// www.accessdata.fda.gov/scripts/Interactive Nutrition models/products/diet _ Fiber. pdf) recognize the importance of daily intake of Dietary fiber and recommend 25 grams per day as an average recommended intake per day for an adult. However, studies have shown that the average dietary fiber intake is currently about 18 grams per day (Hoy and Goldman, 2014). Accordingly, there is an increasing demand for food additives to functionally replace the calorie/fat imparting content of processed foods by increasing the fiber content without adversely affecting the organoleptic quality. The present invention solves this problem to provide an additive which meets the above requirements and overcomes the obstacles of the prior art. Patent application WO1991011112a1 describes a process for enzymatic hydrolysis of xyloglucan to oligosaccharides at a temperature between 45-50 ℃ with enzymes added in high concentrations. Furthermore, in addition to the desired oligosaccharides with a high degree of polymerization (DP 7-9), the process also produces more than 15% of small oligosaccharides and monomeric sugars, which must be removed in a second downstream process step. The present invention carefully selects the enzyme and due to the high temperature avoids the production of smaller oligosaccharides and monomeric sugars and improves the process efficiency.

Disclosure of Invention

The invention solves this problem by the method of claim 1. It has been unexpectedly found that the high temperature hydrolysis of xyloglucan sources, such as tamarind (tamarind) kernel powder, provides a low calorie food additive that can be used to replace the calorie-imparting content of currently processed foods while increasing fiber content without adversely affecting organoleptic quality. It was further unexpectedly found that the use of a high temperature step and an enzyme with a high temperature profile simplified the xyloglucan-polysaccharide hydrolysis, improved the overall performance and avoided additional purification steps. The process of the invention can be carried out with only one enzyme, since a carefully selected specific enzyme exhibits xyloglucanase activity at temperatures above 50 ℃ which is sufficient for complete hydrolysis of xyloglucan-polysaccharides even at high substrate amounts of 700g/l or more.

The enzyme may be, for example, an endoglucanase, for example selected from endoglucanases belonging to Glycoside Hydrolase (GH) families 5, 9, 12, 16, 44 or 74.

The invention also provides a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1 to 4 having at least 75% sequence identity. The enzyme suitably exhibits xyloglucanase activity at temperatures above 50 ℃.

The invention also provides a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1 to 4, which exhibits xyloglucanase activity in the presence of a high concentration of DP7-DP 9.

The invention also provides a xyloglucan hydrolysate, which is produced by the method of the invention. The xyloglucan hydrolysate is characterized in that it essentially comprises a mixture of DP7 to DP9 xyloglucan-oligosaccharides (XGOS).

The invention also relates to the use of said xyloglucan hydrolysate for the production of food products. The invention also relates to the use of the xyloglucan hydrolysate and food products comprising the same for human nutrition.

The invention also relates to the use of said xyloglucan hydrolysate for the production of a feed product. The invention also relates to the use of the xyloglucan hydrolysate and feed products comprising the same for animal nutrition.

Xyloglucan hydrolysate produced by the process of the invention and food products comprising the same are also provided for use in improving human health, such as lowering blood glucose levels after a meal as a satiety agent or reducing calories in food.

Detailed Description

In a preferred embodiment, the present invention provides a method of producing xyloglucan-oligosaccharides (XGOS) from a xyloglucan source, the method comprising enzymatically hydrolyzing xyloglucan at a temperature above 50 ℃ with an enzyme that exhibits xyloglucanase activity at a temperature above 50 ℃.

Preferred xyloglucan sources are by-products of food product production, most preferably a product stream comprising tamarind (i.e. a product derived from tamarind), comprising fractions thereof such as tamarind kernel powder, defatted tamarind kernel powder or tamarind seed.

Alternative xyloglucan sources according to the invention are for example pepper grass, rapeseed, apple, bilberry, blueberry and olive, which contain their fractions, in particular seeds and cell walls, or any other source containing a significant xyloglucan polysaccharide content. Significant content means that the xyloglucan content is higher than 5%. The carbohydrate hydrolysates of these xyloglucans carried out with the process described herein mainly comprise oligosaccharides with a Degree of Polymerization (DP) between 6 and 11.

Tamarind xyloglucan hydrolysate is unique in that it comprises mainly (typically > 65% 70%, preferably > 75%, more preferably > 80% or > 85%, most preferably > 90% or > 95%) oligosaccharides with a Degree of Polymerization (DP) between 7 and 9, unlike other carbohydrate hydrolysates.

Tamarind polysaccharide is obtained from the seed of the tamarind tree, Tamarindus indica (sappannaceae, Fabaceae), a common forest tree, possibly originating in tropical africa, widely cultivated in tropical and subtropical arid regions (mainly south and east asia, south america, mediterranean), mainly in india, burma, bangladesh and srilanka. Tamarind forms an unbreakable legume fruit, a 10-15 cm long pod, containing seeds consisting of about 55% pulp, 34% seeds (containing 65% xyloglucan polysaccharide) and 11% shell and fiber. Tamarind seed is a commercial source of chewing gum and has found many commercial applications including food. In 1988, 300.000 tons of tamarind (Kumar and Bhattacharya,2008) were produced in just India for one year, which were converted to about 100.000 tons of tamarind seed and over 66.000 tons of xyloglucan, respectively.

In a preferred embodiment of the invention, the enzyme exhibiting xyloglucanase activity is an endoglucanase. More preferably, the endoglucanase has an activity profile above 50 ℃. This has the advantage that xyloglucan polysaccharides, such as tamarind polysaccharides, are efficiently hydrolysed to produce an oligosaccharide mixture comprising DP7, DP8 and DP9 oligosaccharides. Other advantages of high temperature are the increased solubility of the xyloglucan source and the avoidance of microbial contamination.

Xyloglucan is a polysaccharide consisting of a β -glucan backbone consisting of cellotetraose units (four β -1, 4-linked D-glucopyranose residues), whereby the first three glycosidic residues are linked to the C1 position of the α -D-xylopyranoside (xylopyranosidic) residue by glycosidic bonds at C6, viewed from the non-reducing end of the sugar chain. The second and/or third xylose residue may again be linked at position C2 to the C1 position of a beta-D-galactopyranoside (galctopyranosidic) residue. The fourth glycoside residue is unmodified.

Hydrolysis of tamarind xyloglucan gives the oligosaccharide DP 7: XXXG, DP 8: XXLG and XLXG and DP 9: XLLG wherein G represents an unmodified backbone glucose residue, X represents a backbone glucose residue modified with xylose, and L represents a backbone glucose residue modified with xylose and galactose; the letter code naming convention is according to Fry et al, (1993), as summarized in figure 1. In some cases, tamarind xyloglucan is rare and more common in xyloglucan of other plant origin, the side chains are slightly different and may give different products than those described in figure 1: in most cases, one of the xylose residues in each tetramer may be lost at the third glucose residue, leading to differences in the composition of the resulting oligosaccharides, depending on the hydrolytic preference of the splitting enzyme; the galactoside residue may be replaced by an alpha-L-arabinofuranoside (arabinofuranoside) residue, or further modified with a different sugar residue, such as another galactoside residue or an L-fucopyranoside (fucopyranoside) residue; backbone glycoside residues may also be modified with α -L-arabinofuranoside (arabinofuranosidic) residues in place of xylose; xylose residues may also be linked in the beta-configuration. Typically, the sugar residue is O-acetylated. Furthermore, it is rare in tamarind xyloglucan and xyloglucans of other origin that one of the three xylose-modified glucose backbone residues may be modified at position C2, in addition to the alpha-L-arabinoside residue (Vincken et al, 1996).

Xyloglucan hydrolyses are few; however, hydrolysis is carried out by a variety of enzyme types with different cleavage specificities, almost all of which are at the reducing end of the unmodified glycoside residue. The side chains in xyloglucan protect the polymer to some extent from rapid degradation due to the lack of side chain cleavage activity in the environment and the need for unique activity for each different side chain saccharide.

The glycosidic linkages within xyloglucan polymers can be cleaved by certain hydrolases. This enzyme, called hydrolase, cleaves glycosidic bonds by the addition of water molecules in a reaction that can be written as: A-B + H₂O → A-OH + B-H, wherein A and B are sugar molecules. These hydrolases are specific for the type of sugar and the type of linkage involved, so that the β -1, 4-linkage between two glucose molecules is cleaved by an enzyme with β -1, 4-glucanase activity. When the enzyme statistically cleaves any β -1, 4-glycosidic bond within a long polymeric molecule, such as present in the backbone of xyloglucan, the enzyme is referred to as an endoglucanase (e.g., a cellulase). Endoglucanases are generally active only when two or more consecutive glucose residues in the polymer are unsubstituted (such as in cellulose). However, only every 4 th residue of the tamarind xyloglucan backbone is unsubstituted, and therefore most endoglucanases (such as those in most commercial cellulase preparations) are not active against tamarind xyloglucan. Those specific endoglucanases (mainly in glycoside hydrolase families GH5 and GH 9) which have activity towards tamarind xyloglucan have to be selected experimentally; these enzymes degrade the β -1, 4-glycosidic bond on either side of an unsubstituted glycosidic residue, even if another glucose residue is substituted. In addition, specific endoglucanases comprised in some other hydrolase families (e.g. GH12, GH16, GH44 or GH74) are capable of degrading xyloglucan.

In addition to enzymatic activity towards xyloglucan, suitable enzymes achieve complete hydrolysis even at high substrate levels of 70% (wt/vol) or higher, resulting in high oligosaccharide concentrations, characterized by a favorable inhibition profile of the final product. Wong et al,2010, characterized a GH5 endoglucanase that was linearly non-competitively inhibited by XXXG oligosaccharides with a Ki of 1.46 ± 0.13 mM. Enzymes with more suitable final product inhibition profiles Ki of 5mM or higher can be found in the GH family comprising GH5, GH9, GH12, GH16, GH44 and GH 74.

In a preferred embodiment of the process of the invention, the high temperature conversion of xyloglucan (such as tamarind polysaccharide) to a hydrolysate mainly comprising DP7-DP9 oligosaccharides is performed by the action of an endoglucanase selected from the group consisting of hydrolytic exoenzymes of bacterial or fungal origin. The enzymatic hydrolysis may be carried out in an aqueous solution containing the tamarind polysaccharide in a wide range of polysaccharide concentrations. Thus, the reaction can be carried out by dissolving the tamarind polysaccharide in an aqueous solution at a concentration of about 1% by weight to a concentration limited only by the solubility of the polysaccharide and the viscosity of the solution.

Suitable endoglucanases may be selected from a wide variety of bacterial and fungal sources. It may also be derived from metagenomic DNA. The genes for the selected endoglucanases were cloned and expressed in a suitable test expression system comprising e.coli (e.coli), Bacillus sp, and thermus thermophilus (thermatherophilus). The respective enzymes were then tested for the desired hydrolysis of the tamarind polysaccharide, resulting in (preferably) DP7-DP9 oligosaccharides. Preferred endoglucanase genes are preferably selected and isolated from DNA obtained from thermophilic microorganisms or metagenomic DNA. Thermophilic microorganisms are those having an optimal growth range above 42 ℃ (Lengeler et al). These genes include thermostable enzymes from the group of bacteria comprising the following genera: thermoacidophilus (Acidothermus), Pyrrolobacillus (Caldicellulosiriptor), Thermoascus (Caldithrix), Clostridium (Clostridium), Deinococcus (Deinococcus), Herbinix, Herbivorax, Ignavibacterium, Lachnocortium, Thermus subsp (Melothermus), Rhodothermus (Rhodothermus), Thermoanaerobacter (Thermanomonas), Thermanovibrobacter, Thermanovabacter, Thermoanaerobacter (Thermoanaerobacter), Thermoanaerobacter (Thermoanaerobora), Thermoanaerobacter (Thermoanaerobacter, and Thermotoga (Thermotoga).

In many cases, extracellularly produced enzymes derived from mesophilic bacteria can have high stability even at high temperatures and are potentially suitable for use in the methods of the invention. Thus, another way to select a suitable endoglucanase is to select an enzyme derived from a mesophilic or a psychrophilic microorganism. Mesophilic microorganisms grow optimally at temperatures between 20 and 42 ℃ whereas psychrophilic microorganisms grow at temperatures below 20 ℃ (Lengeler et al). Such endoglucanases are intended to be encompassed by the present invention.

Furthermore, the enzymatic activity of endoglucanases (e.g., endoglucanases derived from mesophilic bacteria) may be improved towards higher temperatures by genetic engineering. Thus, another way to select a suitable endoglucanase is to select an enzyme derived from a mesophilic or even a psychrophilic microorganism and to improve its enzymatic activity towards higher temperatures by genetic engineering. Such endoglucanases are intended to be encompassed by the present invention.

Endoglucanases belonging to GH family 5, 9, 12, 16, 44 or 74 may be derived from a mesophilic or psychrophilic microorganism, such as a mesophilic or psychrophilic bacterium belonging to a group comprising: acarylochloris, Acetylvibrio (Acetivibrio), Acetobacter (Acetoanalobium), Propionibacterium acidovorans (Acidopropionibacterium), Acidobacterium (Acidobacterium), Actinoplanes (Actinoplanes), Actinosynnema (Actinosynnema), Aeromonas (Aeromonas), Agrobacterium (Agrobacterium), Alcaligenes (Alcaligenes), Acidovorax (Algoriegus), Alicyclobacillus (Alicyclobacillus), AriVibrio (Aliivibrio), Alcaligenes (Alisporium), Alcaligenes (Alcaligenes), Alkaliphenylbacillus, Alkluyveromyces (Alcaligenes), Acetobacter (Acetobacter), Anacardia (Anacardia), Anacardiaceae (Acetobacter), Acetobacter (Acetobacter), Anacardiaceae (Acetobacter), Anacardia (Anaeromonas), Anacardia (Anacardia), Anacardia (Acetobacter), non-corynebacterium (Asticcacaculis), Autonicicoccus, Aeromonas (Aureimonas), Bacillus (Bacillus), Bacteroides (Bacteroides), Barnesiella, Bdellovibrio (Bdellovibrio), Leishmania (Beutenbergia), Bifidobacterium (Bifidobacterium), Blastochlorella (Blastochloromyces), Blastococcus (Blastococcus), Blauutia (Blastotia), Bordetella (Bosea), Chromophthora (Bradyrhizobium), Brevundimonas (Brevundimonas), Brucella (Brucella), Burkholderia (Butylia), Burkholderia (Burkholderia), Vibrio (Butyribrio), Euglena (Caldariomyces), Microstreptoverticillium (Caulispora), Corynebacterium (Caulobacter), Corynebacterium (Cellviculorubium), Cellvibrinomonas (Cellvibracter), Cellvibrinomonas (Clostridia), Chromobacter (Clostridia), Cellviculorubium (Clostridia), Cellvibrium (Cellvibrium), Cellvibrium (Clostrinia (Cellvibrium), Cellvibrium (Clostrinia), cneuibacterium, Rhodomonas (Collinosa), Colowella (Colwellia), Kannella (Conexibacter), enterococcus (Coprococcus), Corallococcus (Corallococcus), Terra (Crinalium), Coronella (Crinermis), Rhodococcus (Croecium), Brevibacterium (Curtobacterium), Bluella (Cyanobium), Bluella (Cyanothrice), Cyclobacterium (Cycleobacterium), Cylindrococcus (Cylindrocmum), Cytophaga (Cytophaga), dehalogena (Dehalobacter), dehalogena (Dehalogenimonas), Desulfurium (Desulfossilium), Desulfoenterococcus (Desulfovomalum), Desulfovibrio (Desulvus), Diculvinio), Dicyanella (Corynebacterium), Dicyanobacter (Dicotyleyanobacter), Dicotyledonepezium (Dichlorobacter), Microbacterium (Escherichia), Microbacterium (Corynebacterium), Microbacterium (Microbacterium), filimonas, Filovimonas (Fimbrioninas), Flammeovirga, Cladosporium (Flaviviridobacter), Flavobacter (Flavobacterium), Flavobacterium (Flavobacterium), Formosa, Frankia (Frankia), Freuonymus (Freunella), Fomitothrix (Frondibians), Fuerstia, Clostridium (Fusobacterium), Geotrichum (Geilerinema), Gemmitinosa, Glaciicola, Myxobacter (Gloeobacter), Gloeocapsa (Gloeocappa), Gluconobacter (Glutaminobacter), Gordonia (Gordonia), Phanerium (Gramelalla), Granulella, granular Myxococcus (Granulococcus), Greensis (Grignard), Jambucilaria, Jatropha (Hypocrea), Hypocrea (Hypocrea), bacterospora (Kibdelosporangium), zoococcus (Kineococcus), Kiritimatinib, Norlicia (Kitasatospora), Klebsiella (Klebsiella), Korea (Kribella), Kutzneria, Labilithrix, Humulum (Lacimicrobium), Lactococcus (Lactococcus), Lacinia, Leadterella, Lysinella (Leifsonia), Lorentzia (Lenzea), Leptothrix (Leptongbya), Cillum (Leptospira), Listeria (Listeria), Microbacterium (Lutibacterium), Lysobacter (Lysobacter), Mahella (Maribacterium), Maricilus, Maricola, Margaria, Micrococcus (Mexobacter), Micrococcus (Micrococcus), Micrococcus (Micrococcus), nakamurella, Niabella, Niastella, Nitrospirallum, Nocardia (Nocardiaopsis), Nonomuraea (Nonomuraea), Candida (Nostoc), Neosphingolipid (Novosphingobium), Xanthomonas (Ochrobactrum), Oscillatoria (Oscilaria), Paenarthromobacter, Paenibacillus (Paenibacillus), Paludibacterium, Paludishiaera, Polyporaceae (Pandaea), Pantoea (Pantoea), Parabacterium, Paraerskovia, Pectibacter (Pectibacter), Pediococcus (Pediococcus), Geobacillus (Pedobacterium), Penicillium, Pediococcus (Pediococcus), Pseudomonas (Pseudomonas), Pseudomonas sp), Pseudomonas (Pseudomonas sp), Pseudomonas sp (Pseudomonas sp), Pseudomonas sp (P, Pseudomonas sp (P.sp, Pseudomonas sp (P.sp, Pseudomonas sp, pseudoxanthomonas (Pseudomonas), Pyrolusitum (Psychrofelexus), Laurella (Ralstonia), Raoultella (Raoultella), Rhizobium (Rhizobium), Rhodobacter (Rhodobacter), Rhodococcus (Rhodococcus), Rhodococcus (Rhodovulum), Micrococcus (Rivularia), Rose semilucium (Roseateles), Roseburia (Roseburia), Ruegeria (Ruegeria), Rufibacter ruminis (Rumicola), Ruminococcus (Ruminococcus), Archaeoglobus (Ruminococcus), Saccharomyces (Runella), Saccharophhagus (Saccharothrix), Saccharothrix (Saccharothrix), Halobacterium (Salegeria), Salispora), Haematococcus (Sphingobacter), Sphingobacter (Sphaerothecium), Sphaerothecium (Spinomonas), Spinomonas (Sphaerotheciobolus), Spinomonas (Spinomonas), Sphaerothecium (Spinomonas), Spinomonas (Sphaerotheciobacter), sphingopyxis, spirochetes (Spirochaeta), Spirosoma (Spirosoma), Stackebrandtia, Stationeria (Stanieria), Starkeya, Stenotrophomonas (Stenotrophomonas), Populus (Stigmatella), Streptomyces (Streptomyces), Streptosporangium (Streptomyces), Polypoccus (Synechococcus), Synechocystis (Synechocystis), Syntrophomonas (Syntrophomonas), Tenonella (tannorella), Chrysomycotiana (Teredinibacter), Terrigillus, Thioclava, Thiolapilus, Treponema (Treponema), Verrucosispora (Verrucosispora), Vibrio (Vibrio), Xanthomonas (Xanthomonas), Zungongawawa (Zungawa) and other families comprising genes of Gluconobacter, GH 3632 or GH 3632.

Alternatively, the endoglucanase belonging to GH family 5, 9, 12, 16, 44 or 74 may be derived from a group of eukaryotic cells or organisms, such as eukaryotic cells or organisms belonging to a group of eukaryotic genera comprising: adineta, Agaricus (Agaric), Allium (Allium), Alternaria (Alternaria), Chrysopodium (Alternaria), termitium (Amitermes), Populus (Ampullaria), Anemone (Anoplottermes), Raphius (Aplysia), Arabidopsis (Arabidopsis), Aretaon, Arthrospora (Arthrobotrys), Pseudobambusaea (Arundinaria), Aspergillus (Aspergillus), Phyllostachys (Australia), Australian bamboo (Australia), Australian laminate, Australian Phlebopus, Australian, Avena, Phyllostachys (Bambusia), Banana, Cyclospira (Bemya), Begonia (Bergbaos), Phyllos (Bergbaos), Manutus (Betula), Ananasus (Biopsis), Tribulus (Biophyceae), Camellia (Camellia), Phyllostachys (Califolia), Pelargonium (California), Pelargonium (Camellia), Pelvetia (Camellia), Cymbopogon (Califolius (California), Phyllosa), Phyllochaeta (Califolia (California), Phyllochaeta), Phyllocha (California), Phyllochaeta), taro (Colocasia), Constricta (Conidiophores), Coptotermes (Coptotermes), Corbicula (Corbicula), Cryptodirus (Cryptocercus), Cryptococcus (Cryptococcus), Cucumis (Cucumis), Dendrobium (Dendrobium), Bambusae (Dendrocalamus), Dictyotamus (Dictyotalium), Euphoria (Dimocarpus), Diospyros (Diospyrosos), Eisenia (Eisenia), Gianthus (Entoria), Trichinella (Epidinium), Euastacus (Eucalyptus), Euphorbia (Euphora), Eurycorha, Extatosoma, Ficus (Ficus), Fragaria (Fragaria), Myrothecium (Salmonella), Trichosporoides (Geotrichum), Euonymus (Geotrichum), Neocinnamomum (Geotrichum), Euonymus (Geotrichaeta), Euonymus (Geotrichu (Geotrichum), Euonymus (Geotrichu), Neocinnamomum), Euonymus (Gibberella), Euonymus (Geotrichu), Euonymus (Geotrichum), Euonymus (Gibberella), Phaseolus (Gibberella), Euonymus (Gibberella), Eu, termitomyces (Hospitales), Humicola (Humicola), Hypocrea (Hypocrea), Ilyograpsus, Lactuca (Lactuca), Gliocladium (Leptographa), Leptosphaeria (Leptosphaeria), Leuconostoc (Leucocaracteria), Pleurotus (Leuconostoc), Lilium (Lilium), Carpesium (Limnophilus), Lotus (Lotus), Parameria (Lotus), Paralichthys (Macrophyllum), Ocular (Macropholus), Macrotermitium (Macrotermes), Magnetorum (Magnaporthe), Malus (Malus), Mangifera (Mangifera), Irpex (Rhizophora), Rhododendron (Mastodon), Aureobasidium, Medicago (Medicago), Rhizophora (Rhizophora), Neurospora (Neurospora), neurospora (Neurospora), Nicotiana (Nicotiana), Phanerochaete (Nilapavata), Terminalia (Odontotermes), Cytospora (Oikoleura), Parasilurus (Olyra), Oryza (Oryza), Phyllostachys mexicana (Otatea), Phyllostachys nigra (Oxytenanthes), Panesthia, Panicum (Panicum), Paralichenidae (Paranesarma), Penicillium (Penicillium), Poecium (Pericarpmes), Perinermis (Perinereis), Pyrolus (Perinereis), Alligata (Persea), Perupasmia, Hymenochaetaria (Phakopsora), Phanerochaea (Phanerochaete), Phaseolus (Phaseolus), Phaseolus (Phlomyces), Phyllostachys (Pilus), Populus (Pillus), Populus (Pilus), pyri (Pyrus), Paralichs (Ramulus), Raphanus (Raphanus), Reticulitermes (Reticulitermes), Rhodotorula (Rhodotorula), Rhynchostermes, Rubus (Rubus), Saccharum (Saccharum), Wood (Salganea), Salix (Salix), elderberry (Sambucus), Sasa (Sasa), Scorzonera (Schizostachyum), Sclerotia (Sclerotia), Phyllostachys (Semiarundinaria), Serrenditata, Sesamoides, Phlebia (Sesuvium), Setaria (Setaria), Shibataea (Shibataea), Silenia (Silocola), Wasabia (Sinomenorops) (Sinomenium), Thermois (Thermoides), Trigonoma (Solomonas), Trigonopsis (Sterculia), Trichoderma (Sphacea), Trichoderma (Tuber), Trichoderma (Sterculia), Trichoderma (Sphaerothecium), Trichoderma (Sepiella (Septemloba), Trichoderma (Septemloba (Septemaria), Trichoderma (Septemloba), Trichoderma), urochloa (Urochloa), Ustilago (Ustilago), Vigna (Vigna), Vitis (vitas), Xanthophyllomyces, Zea (Zea) and any other genus comprising and/or expressing the eukaryotic endoglucanase genes of GH5, GH9, GH12, GH16, GH44 or GH74 families.

The bacterial and eukaryotic genera known to date to contain endoglucanase genes are listed in the Carbohydrate-Activeenzymes Database (CAZy) (http:// www.cazy.org).

To identify and isolate DNA sequences encoding endoglucanases, any genomic or metagenomic sequence may be screened using bioinformatics methods to obtain in silico transcribed reading frames having sequential sequence similarity to the amino acid sequence of an endoglucanase from the GH5, GH9, GH12, GH16, GH44 or GH74 families. The base DNA sequence may be amplified in vitro by PCR using a suitable template DNA, or synthesized. The codon usage of the synthetic DNA may be adapted to that of the intended expression host. Alternatively, genomic or metagenomic DNA is fragmented by methods known in the art, ligated into a vector plasmid in a suitable manner following expression signals, transformed into the desired expression host and expressed in a growth culture, or streaked onto a suitable solidified medium on agar plates or in a mixture in a suitable liquid medium. These methods are well known in the art. Colonies on agar plates can be screened for hydrolytic activity by a variety of methods. These methods include, but are not limited to, including the substrate in agar medium, in a layer on top of agar medium prior to cell plating, or in overlay agar spread on top of growing colonies. The substrate is a soluble or insoluble polymeric endoglucanase substrate comprising a derivatized cellulose such as carboxymethylcellulose (CMC), a beta-glucan such as barley beta-glucan, xyloglucan, or a soluble or insoluble chromogenic substrate such as a beta-glucan substrate such as azo-CM-cellulose, azo barley beta-glucan, and azo xyloglucan. After incubation, the clearing zone (clearing zone) of the polymeric substrate is visible by clearing turbidity around the colonies or staining and destaining with a dye such as Congo-red or fluorescent stilbene or umbelliferone derivatives; after incubation, by dissolving the insoluble material, a clearing zone of chromogenic substrate, staining or fluorescence is visible.

Liquid cultures with endoglucanase activity were identified by disrupting the bacterial cells and measuring endoglucanase activity as described below. As is known in the art, endoglucanase-producing cells in culture that exhibit activity may have to be streaked for single cell isolation. The isolated pure cultures were rechecked for activity as described. The endoglucanase gene is isolated from the identified colonies or cultures by methods well known in the art of DNA isolation and PCR amplification.

The presence of endoglucanase activity in a liquid culture, a culture supernatant or an enzyme solution can be determined by various methods, examples of which are listed below. Proteins are detected by antibody-based assays (such as ELISA or Western-Blot), by SDS-PAGE and Coomassie Blue (Coomassie-Blue) or silver staining, or by measuring protein concentration by any suitable method. The enzyme activity can be determined by a number of other methods, such as described in example 2, using a model substrate for endoglucanases comprising CMC, barley- β -glucan and xyloglucan, or a chromogenic substrate comprising a β -1, 4-glycosidic bond.

Endoglucanase activity is characterized by non-progressive (non-processing) hydrolysis at internal bonds within the beta-glucan molecule of cellulose. The hydrolysis points are statistically distributed over the length of the substrate molecules. "non-progressive" means that the next hydrolysis of the enzyme does not proceed in a consecutive manner at adjacent positions; the enzyme falls off the substrate and binds to a different site on the same molecule or another molecule for the next cleavage, thereby generating a new cleavage at any site within the polymer molecule. Endoglucanase hydrolysis activity, also known as endo-mode (endo-mode) cellulase activity, may be determined by a method using soluble polymers comprising β -1, 4-glucan linkages, such as mixed-linkage β -glucans (e.g. β -1,3-1, 4-glucan in barley or lichen) or chemically modified celluloses, such as hydroxyethylcellulose or carboxymethylcellulose (HEC, CMC).

The enzyme activity can be determined quantitatively by the increase in reducing end, i.e.by the generation of a new C1-sugar end, reducing the hemiacetal group. This can be quantified by reducing the developed 3, 5-dinitrosalicylic acid to 3-amino-5-nitro salicylic acid, which can be measured photometrically by absorbance at 540nm (Wood und Bhat 1988). Another method may be to hydrolyze the oligosaccharides produced by the addition of beta-1, 4-glucosidase and quantitate the resulting monomeric glucose in a combined enzyme assay using hexokinase and glucose-6-phosphate dehydrogenase in the presence of ATP and NAD. The reduced NAD (NADH +) was quantified photometrically at 340 nm. Another assay is to use glucose oxidase in combination with a chromogenic assay for hydrogen peroxide formed by peroxidase or by electrochemical measurement. Chromogens such as o-phenylenediamine can be used for photometric detection. Many commercial glucose assays (such as GOD/POD) are available for both methods described in detail.

Another method of determining endoglucanase activity is to reduce viscous polymer solutions, such as from chemically modified cellulose (such as HEC, CMC) or from soluble β -glucans containing β -1, 4-glycosidic linkages (such as barley hybrid-glucan or lichen starch). The use of CMC with a viscometer is a preferred method for detecting specific endoglucanase activity in a mixture having exoglucanase activity. The decrease in viscosity over time was measured. The viscosity decrease is proportional to the cellulase activity and is the most sensitive of the endoglucanase quantification methods. However, the enzyme activity is relative and cannot be expressed in conventional enzyme activity units.

Yet another method for the determination of endoglucanase activity is the release of oligosaccharides from colour-or fluorescence-derived dyed cellulose, cellodextrins and/or cross-linked cellulose derivatives or cellodextrins (such as AZCL-HE-cellulose, azo-alpha-cellulose, azo-Avicel, RedCL-HE-cellulose, 4, 6-O-benzylidene-2-chloro-4-nitrophenyl-beta-3, 1-cellotriosyl-beta-glucopyranoside, 4,6-O- (3-ketobutenyl) -4-nitrophenyl-beta-D-cellopentaglucoside (cello-pentaoside) or 4, 6-O-benzylidene-4-methylumbelliferyl-beta-cellotrioside), as described in McCleary et al (1980) or Manganet al (2016). Such substrates release either dyed oligosaccharides (which are soluble and sometimes differentiated by precipitation of the polymer by the addition of a concentration of ethanol), or chromophore-or fluorophore-containing substrates. The soluble products of the enzymatic reaction can be determined photometrically or fluorometrically. For some assays, it is necessary to add beta-glucosidase. Many commercial endoglucanase assays are available for this method as described in detail.

An increased activity profile with respect to the enzyme activity at temperatures above 50 ℃ can be achieved, for example, by mutagenesis of endoglucanases with a suitable product distribution. Suitably, the endoglucanase derived from a mesophilic or psychrophilic organism is subjected to mutagenesis. The person skilled in the art is aware of the principle techniques for introducing mutations into enzymes to optimize the characteristics of the enzymes. An exemplary mutation is the introduction of a cysteine residue into the amino acid sequence that forms a disulfide bridge to stabilize the enzyme against heat denaturation. In another aspect, thermostability can be achieved by random, site-directed mutagenesis, or directed evolution in which one or more amino acids of the original amino acid sequence are substituted with an amino acid other than the original sequence. In another aspect, deletions or insertions of amino acids, loop regions or protein domains in the original amino acid sequence can be made to increase the thermostability of the enzyme. In another aspect, enzyme thermostability can be achieved by encapsulation, chemical crosslinking of the enzyme, and addition of stabilizing compounds. Such stabilizing compounds are, for example, BSA, glycerol and sorbitol. Other methods of stabilizing the enzyme are chemical cross-linking or any other method which results in a suitable enzyme activity above 50 ℃.

Thus, in a preferred embodiment, the endoglucanase with an enzymatic activity above 50 ℃ used in the method of the invention is derived from a eukaryote or a microorganism, such as a bacterium or a fungus, more preferably a microorganism, most preferably a bacterium.

In another preferred embodiment, the endoglucanase having an enzymatic activity above 50 ℃ used in the method of the invention is derived from a thermophilic microorganism, such as a thermophilic bacterium, or is thermostable by genetic engineering.

According to the present invention, "derivatisation" means the isolation of the endoglucanase of interest from a wild-type organism according to standard techniques well known to the skilled person. "derived" also includes the recombinant production of the endoglucanase of interest in a suitable host or host cell.

In a preferred embodiment, the endoglucanase having enzymatic activity at a temperature above 50 ℃ is characterized by an advantageous oligosaccharide end product inhibition constant (Ki) equal to or above 5 mM. The endoglucanases may be produced in a production host of the prior art. However, in addition to the endoglucanase of interest, some production hosts also express endogenous enzymes, which may exhibit undesirable endogenous xyloglucanase enzyme activity. Such undesirable endogenous enzyme activity may result in enhanced hydrolysis, undesirable hydrolysis by-products, or undesirable transglycosylation of the oligosaccharide. Enhanced hydrolysis or transglycosylation may, for example, result in an increased amount of smaller oligosaccharides, an increased amount of monosaccharides, a more uneven distribution of oligosaccharides or an inappropriate distribution of oligosaccharides, thereby reducing the yield of the target DP7-DP9 XGOS. It is therefore advisable to test the suitability of production hosts to avoid undesired xyloglucan degradation or undesired XGOS profiles, which lead to reduced yields of DP7-DP 9. In a preferred embodiment of the invention, the endoglucanase and the production host are capable of maximizing the production of DP7-DP9XGOS by avoiding the formation of by-products. For industrial XGOS production, it is also preferred to use a stable and comprehensive endoglucanase production process. Most preferably, the endoglucanase used in the method of the invention is recombinantly produced in a bacterial host lacking any endogenous enzyme exhibiting activity towards oligosaccharides, in particular DP7-DP9 XGOS. Thus, contamination by undesired side activities of other enzymes can be effectively excluded.

A preferred method is to express the polypeptide of SEQ ID NO: 1-4.

The most preferred method is to express the polypeptide of SEQ ID NO: 2-4.

Most preferred host cells are prokaryotic cells secreting enzymes. The prokaryotic host cell may be any gram-positive or gram-negative bacterium.

Gram-positive bacteria include, but are not limited to, groups of genera comprising: bacillus (Bacillus), Streptococcus (Streptococcus), Streptomyces (Streptomyces), Staphylococcus (Staphylococcus), Enterococcus (Enterococcus), Lactobacillus (Lactobacillus), Lactococcus (Lactococcus), Clostridium (Clostridium), Geobacillus (Geobacillus), oceanic Bacillus (Oceanobacillus), Paenibacillus (Paenibacillus) and closely related bacteria such as bacteria having > 95% sequence identity in the 16S rRNA gene with the 16S rRNA gene of the above genus.

Gram-negative bacteria include, but are not limited to, the following genera: escherichia (Escherichia), Pseudomonas (Pseudomonas), Salmonella (Salmonella), Campylobacter (Campylobacter), Helicobacter (Helicobacter), Flavobacterium (Flavobacterium), Clostridium (Fusobacterium), Ilyobacter, Neisseria (Neisseria), Thermus (Thermus) and Urea (Ureapasma).

The bacterial host cell may be any Bacillus (Bacillus), Paenibacillus (Paenibacillus) or Geobacillus (Geobacillus) cell. Suitable Bacillus cells for producing endoglucanases for use in the methods of the invention include, but are not limited to, Bacillus alkalophilus (Bacillus alkalophilus), Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus brevis (Bacillus brevis), Bacillus circulans (Bacillus circulans), Bacillus clausii (Bacillus clausii), Bacillus coagulans (Bacillus coagulans), Bacillus firmus (Bacillus firmus), Bacillus lautus (Bacillus lautus), Bacillus lentus (Bacillus lentus), Bacillus licheniformis (Bacillus licheniformis), Bacillus megaterium (Bacillus megaterium), Bacillus pumilus (Bacillus pumilus), Bacillus stearothermophilus (Geobacillus), Bacillus subtilis (Bacillus subtilis) and Bacillus thuringiensis (Bacillus thuringiensis) cells and closely related species such as those having a 16% rRNA sequence identity to the rRNA gene of the above-mentioned species.

The bacterial host cell may also be any Streptococcus (Streptococcus) cell. Suitable Streptococcus cells for producing endoglucanases for use in the methods of the invention include, but are not limited to, Streptococcus equisimilis (Streptococcus equisimilis), Streptococcus pyogenes (Streptococcus pyogenenes), Streptococcus uberis (Streptococcus uberis), and Streptococcus equi subsp.

The bacterial host cell may also be any Streptomyces (Streptomyces) cell. Suitable Streptomyces cells for producing endoglucanases for use in the methods of the invention include, but are not limited to, Streptomyces achromogens (Streptomyces achromogens), Streptomyces avermitilis (Streptomyces avermitilis), Streptomyces coelicolor (Streptomyces coelicolor), Streptomyces griseus (Streptomyces griseus) and Streptomyces lividans (Streptomyces lividans) cells.

The bacterial host cell may also be any Corynebacterium (Corynebacterium) cell. Suitable cells of the genus Corynebacterium for producing the endoglucanase used in the method of the present invention include, but are not limited to, Corynebacterium glutamicum (Corynebacterium glutamicum) cells.

The bacterial host cell may also be any Escherichia (Escherichia) cell. Suitable Escherichia cells for producing the endoglucanases used in the method of the present invention include, but are not limited to, Escherichia coli (Escherichia coli) cells.

The host cell may also be a eukaryotic cell, such as a mammalian, insect, plant or fungal cell.

The fungal host cell may be a yeast (yeast) cell. As used herein, "yeast" includes ascosporogenous yeast (ascosporogenous yeast) (Endomycetales), basidiogenous yeast (basidiosporangiogenous yeast) and yeast (blastomycoles) belonging to the class of the incomplete Fungi (Fungi Imperfect). The group of yeast host cells includes, but is not limited to, Candida (Candida), Hansenula (Hansenula), Kluyveromyces (Kluyveromyces), Pichia (Pichia), Saccharomyces (Saccharomyces), Schizosaccharomyces (Schizosaccharomyces), or Yarrowia (Yarrowia) cells.

In a preferred embodiment, the yeast host cell is selected from the group consisting of: saccharomyces carlsbergensis (Saccharomyces carlsbergensis), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Saccharomyces diastaticus (Saccharomyces diastaticus), Saccharomyces douglasii (Saccharomyces douglasii), Saccharomyces kluyveri (Saccharomyces kluyveri), Saccharomyces norbensis (Saccharomyces norbensis) or Saccharomyces ovatus (Saccharomyces oviformis) cells. In another preferred embodiment, the yeast host cell is a Kluyveromyces lactis (Kluyveromyces lactis) cell. In another preferred embodiment, the yeast host cell is a Yarrowia lipolytica (Yarrowia lipolytica) cell.

In another preferred aspect, the fungal host cell is a filamentous fungal (flamentous fungal) cell. "filamentous fungi" include all filamentous forms of the subdivision Eumycota and Oomycota (Oomycota) (as defined by Hawksworth et al, 1995). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth occurs by hyphal elongation, while carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae occurs by budding of a unicellular thallus and carbon catabolism may be fermentative.

In a preferred embodiment, the filamentous fungal host cell is selected from the group consisting of: acremonium (Acremonium), Aspergillus (Aspergillus), Aureobasidium (Aureobasidium), Bjerkandera, Ceriporiopsis (Ceriporiopsis), Chrysosporium (Chrysosporium), Coprinus (Coprinus), Coriolus (Coriolus), Cryptococcus (Cryptococcus), Filibasidium, Fusarium (Fusarium), Humicola (Humicola), Hypocrea (Hypocrea), Magnaporthe oryzae (Magnaporthe), Mucor (Mucor), Myceliophthora (Myceliophthora), Neocallimastix (Neocallimastix), Neurospora (Neurospora), Penicillium (Paecilomyces), Penicillium (Pelamium), Phanerium (Thermoplasma), Thermobacteriobotrys (Thielavia), Thielavia (Thielavia), Trichoderma (Thielavia), Trichoderma).

In a preferred embodiment, the filamentous fungal host cell is selected from the group consisting of: aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cells. In another preferred embodiment, the filamentous fungal host cell is selected from the group consisting of: fusarium bactridioides (Fusarium bactridioides), Fusarium graminearum (Fusarium cerealis), Fusarium crooki (Fusarium crookwellense), Fusarium flavum (Fusarium culmorum), Fusarium graminearum (Fusarium graminearum), Fusarium graminum (Fusarium graminum), Fusarium heterosporum (Fusarium heterosporum), Fusarium negundi (Fusarium negundo), Fusarium oxysporum (Fusarium oxysporum), Fusarium polybranchum (Fusarium reticulatum), Fusarium roseum (Fusarium roseum), Fusarium sambucinum (Fusarium sambucinum), Fusarium sakahrosporium (Fusarium sporotrichioides), Fusarium trichothecioides (Fusarium trichothecioides), Fusarium. In another preferred embodiment, the filamentous fungal host cell is selected from the group consisting of: bjerkandra adausta, Ceriporiopsis horneri (Ceriporiopsis aneria), Ceriporiopsis cerealis (Ceriporiopsis aneria), Ceriporiopsis sphaericus (Ceriporiopsis gilvesii), Ceriporiopsis pomifera (Ceriporiopsis sp.), Ceriporiopsis cerealis (Ceriporiopsis rivularis), Ceriporiopsis microphyllus (Ceriporiopsis subvernalis), Ceripopsis cerasus (Ceriporiopsis subteruptorum), Ceriposporum keratinophilus (Chrysosporium kenyatum), Chrysosporium lucknowense, Chrysosporium trarum (Chrysosporium tropicalis, Chrysosporium roseum), Chrysosporium (Chrysosporium), Chrysosporium trichothecium), Chrysosporium (Phaseolus, Phanerosporium), Chrysosporium fulvum trichothecium, Phanerium thermophilum (Phanerium), Chrysosporium fulvum, Phanerium (Phanerium), Chrysosporium fulvum trichothecium (Phanerium), Phanerium neospora), Phanerium (Phanerium), Phanerium neospora (Phanerium), Phanerochaemorum purpureum), Phanerium (Phanerium), Phanerium purpureum) Phanerium (Phanerium), Phanerium (Phanerium), Phanerochaellum (Phanerium), Phanerium neospora (Phanerium), Phanerochaemorum Phanerium), Phanerium (Phanerium), Phanerochaemorum (Phanerium), Phanerium (Phanerium), thielavia terrestris, Trametes villosa (Trametes villosa), Trametes versicolor (Trametes versicolor), Trichoderma harzianum (Trichoderma harzianum), Trichoderma koningii (Trichoderma koningii), Trichoderma longibrachiatum (Trichoderma longibrachiatum), Trichoderma reesei (Trichoderma reesei), or Trichoderma viride (Trichoderma viride) cells.

In a most preferred embodiment, an endoglucanase is used in the method of the invention, wherein the endoglucanase hybridizes to a polynucleotide having a sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO: 4 has at least 75% sequence identity to the polypeptide of the amino acid sequence of seq id No. 4.

According to SEQ ID NO:1 from Clostridium thermocellum (Clostridium thermocellum).

Among the bacteria with the strongest hemicellulose degrading activity, Herbivorax saccincola SR1 was isolated from a thermophilic forage silage/cow dung biogas reactor. It grows at a temperature of 45-65 ℃, optimally 60 ℃, and it was found to ferment xylan, xyloglucan and crystalline cellulose. Herbivorax saccharoncola is classified as a new family in Ruminococcaceae (Koeck et al.2016). In the genomic sequence of bacteria, sequences belonging to GH family 5 were identified (Pechtl et al, to be published). The reading frame has been cloned into E.coli and the expressed and purified gene product characterized. For the purposes of the present invention, SEQ ID NO: 2 as a suitable enzyme. Thus, in a particularly preferred embodiment, the endoglucanase used in the method of the invention is derived from the newly isolated microorganism Herbivorax saccharococca DSM101079 and consists essentially of a sequence identical to the newly isolated SEQ ID NO: 2 or consists of a polypeptide having at least 75% amino acid sequence identity to a newly isolated polypeptide of SEQ ID NO: 2 has at least 75% amino acid sequence identity.

In another particularly preferred embodiment, the endoglucanase comprises an amino acid sequence identical to SEQ ID NO: 4, a polypeptide having at least 75% amino acid sequence identity to the polypeptide of SEQ ID NO: 4 or consists of a polypeptide having at least 75% amino acid sequence identity to the polypeptide of SEQ ID NO: 4, wherein the dockerin domain is deleted in the endoglucanase, which results in a 7-fold increase in enzyme activity. By deleting the dockerin domain, a polypeptide from SEQ ID NO: 2 is suitably obtained from the polypeptide of SEQ ID NO: 4.

In another particularly preferred embodiment, the endoglucanase comprises an amino acid sequence identical to SEQ ID NO: 3, a polypeptide having at least 75% amino acid sequence identity to the polypeptide of SEQ ID NO: 3 or consists of a polypeptide having at least 75% amino acid sequence identity to the polypeptide of SEQ ID NO: 3, wherein the dockerin domain is deleted in the endoglucanase, which results in a 4-fold increase in enzyme activity. By deleting the dockerin domain, a polypeptide from SEQ ID NO:1 to obtain the polypeptide of SEQ ID NO: 3.

In another embodiment, the process of the invention comprises a further treatment step of the hydrolysate. The hydrolysate can be treated to remove solids, salts and impurities, for example using prior art methods. The resulting oligosaccharide solution is optionally decolorized by activated carbon treatment or any other existing decolorization method. Finally, the purified oligosaccharide mixture, which essentially comprises DP7-DP9XGOS, may be dried by freeze or spray drying or roll-drying (roll-drying) to provide a free-flowing powdered multi-functional food additive, which may be formulated and packaged for distribution, or the purified oligosaccharide mixture may be stored as a concentrated liquid formulation. The remaining blend of DP7-DP9XGOS may replace the metabolizable carbohydrate component of the processed food at a high level of portion without compromising the organoleptic qualities of the resulting reduced-calorie food. Importantly, the use of the mixture DP7-DP9XGOS in food products also allowed the fat content as a flavor carrier to be reduced without affecting flavor intensity.

According to the present invention, a multifunctional food additive is prepared by using high temperature and single enzyme hydrolysis of tamarind endosperm polysaccharide. Further according to the invention tamarind seed polysaccharide is converted in high yield to a food grade hydrolysate using an endoglucanase having an enzymatic activity profile at a temperature above 50 ℃, which hydrolysate is believed to comprise essentially DP7, DP8 and DP9 XGOS. Typically, the endoglucanase has a spectrum of activity at a reaction temperature in the range of 50.5 to about 80 ℃, preferably 55 to 75 ℃, more preferably 55 to 70 ℃, most preferably 55 to 65 ℃.

More preferably, the endoglucanase having an activity profile at a temperature above 50 ℃ selectively hydrolyses the tamarind polysaccharide to produce an oligosaccharide mixture comprising DP7, DP8 and DP9 XGOS. Generally, the enzymatic hydrolysis of tamarind polysaccharides according to the invention continues until most xyloglucan-polysaccharides are hydrolyzed to DP7-DP9 XGOS.

In a preferred embodiment, the final amount of tamarind polysaccharide substrate used in the process of the invention is equal to or greater than 100g/L, 200g/L, 300g/L, 400g/L, 500g/L, 600g/L, 700g/L and 750 g/L.

In another preferred embodiment, in the method of the invention, 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5% of the endoglucanase is used with respect to the xyloglucan substrate.

In another preferred embodiment, more than 25%, 35%, 45%, 55%, 65% and 75% or more of the tamarind polysaccharide substrate is hydrolyzed to produce substantially DP7-DP9 XGOS. In the context of the present invention, "essentially produced" means that the content of DP7-DP9XGOS as a fraction of all oligosaccharides in the hydrolysate is at least 50% or higher, preferably 60% or 70%, more preferably 75%, most preferably 80% or higher.

Further preferably, the reaction time to produce substantially DP7-DP9XGOS is in the range of 72h to 12h or less, more preferably 72h, or 60h, 48h or 36h, most preferably 24h, 12h or less.

In a most preferred embodiment of the method of the invention, the amount of xyloglucan substrate ultimately used is 500g/L or more, the concentration of enzyme is less than 0.05%, and hydrolysis yields substantially at least 65% DP7-DP9XGOS in less than 24 hours.

In a most preferred embodiment, the process for the production of xyloglucan-oligosaccharides (XGOS) from a xyloglucan source according to the invention comprises the steps of:

enzymatically hydrolyzing xyloglucan at a temperature above 50 ℃ with an endoglucanase which exhibits xyloglucanase activity at a temperature above 50 ℃,

the removal of the solids is carried out,

removing the salts from the mixture of the organic acids,

the removal of other impurities is carried out,

optionally decolorizing the hydrolysate,

drying the hydrolysate by e.g. freeze or spray drying or roller drying, or storing the hydrolysate in the form of a concentrated liquid formulation.

The amount of endoglucanase used to achieve the necessary hydrolysis of the tamarind polysaccharide depends on the reaction conditions and the level of activity of the endoglucanase. Under optimal conditions, the endoglucanase may be used in an amount as low as 0.005% wt/wt relative to the tamarind polysaccharide starting material. Generally, the concentration of endoglucanase suitable for producing the hydrolysate ranges from 0.005% to 0.5%, preferably from 0.01% to 0.5%, more preferably from 0.05% to 0.5% wt/wt, relative to the tamarind polysaccharide starting material. Typical amounts of endoglucanase used in the process of the invention are 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5% wt/wt relative to the tamarind polysaccharide starting material. The reaction time may vary depending on the endoglucanase used and the hydrolysis conditions. Typical reaction temperatures range from 50.5 to about 80 ℃, preferably from 55 to 75 ℃, more preferably from 55 to 70 ℃, and most preferably from 55 to 65 ℃.

Thus, in another most preferred embodiment, the process for the production of xyloglucan-oligosaccharides (XGOS) from a xyloglucan source according to the invention comprises the steps of:

enzymatic hydrolysis of tamarind polysaccharide at a temperature in the range of 50.5 to 80 ℃ with an endoglucanase which exhibits xyloglucanase activity at a temperature in the range of 50.5 to 80 ℃;

the removal of the solids is carried out,

removing the salts from the mixture of the organic acids,

the removal of other impurities is carried out,

optionally decolorizing the hydrolysate,

drying the hydrolysate by e.g. freeze or spray drying or roller drying, or storing the hydrolysate in the form of a concentrated liquid formulation.

The steps of an embodiment of a method for producing xyloglucan-oligosaccharides (XGOS) from a xyloglucan source according to the invention are shown in fig. 2.