CN115243698A

CN115243698A - Diarrhea inhibitor

Info

Publication number: CN115243698A
Application number: CN202180019812.9A
Authority: CN
Inventors: 小林宏治; 船田茂行; 山田胜成
Original assignee: Toray Industries Inc
Current assignee: Toray Industries Inc
Priority date: 2020-03-13
Filing date: 2021-03-12
Publication date: 2022-10-25
Also published as: WO2021182607A1; JPWO2021182607A1

Abstract

The oligosaccharide composition derived from alkali-pretreated bagasse has an excellent diarrhea-inhibiting effect and can be used as an active ingredient of a diarrhea-inhibiting agent.

Description

Diarrhea inhibitor

Technical Field

The present invention relates to a diarrhea inhibitor containing, as an active ingredient, an oligosaccharide derived from alkali-pretreated bagasse.

Background

Generally, since the feed for livestock, pets and the like is not subjected to heat treatment or the like, it has problems that the digestibility is low, the feed efficiency is poor, and symptoms such as diarrhea are caused. Therefore, it is considered that the addition of antibiotics to livestock feed helps efficient production of livestock products by preventing diarrhea and infection of livestock and, therefore, antibiotics have been used. On the other hand, there is also a fear that drug-resistant bacteria appear in livestock by using antibiotics and spread to the environment, spreading horizontally to humans. Therefore, in europe, the addition of antibiotics to livestock feeds for the purpose of growth promotion is prohibited. Due to such circumstances, useful substances instead of antibiotics are expected.

Xylooligosaccharides are a generic term for oligosaccharides in which a plurality of xylose units are bonded to each other via β -glycosidic bonds. Xylooligosaccharides are also used as a raw material for functional foods because they exhibit an excellent intestinal function and the like (non-patent document 1).

Xylooligosaccharides can be obtained by hydrolyzing xylan contained in cellulosic biomass, and as a method for hydrolysis, a method of performing hydrothermal treatment (non-patent document 2), a method of performing acid hydrolysis (non-patent document 3), and a method of performing enzyme treatment (patent document 1) are known.

On the other hand, various kinds of biomass are used as a cellulosic biomass to be a raw material of xylo-oligosaccharide, and the biomass is produced from pulp and cob of corn (patent document 2).

Documents of the prior art

Patent document

Patent document 1: japanese patent laid-open publication No. 2019-195303

Patent document 2: japanese patent laid-open No. 2000-333692

Non-patent document

Non-patent document 1: ayyappan AA et al, compre.Rev.food.Sci.food saf.10,2-16 (2011)

Non-patent document 2: patricia M et al, ind.Crops.prod.62, 460-465 (2014)

Non-patent document 3: ozlem A et al, carbohydr. Res.344, 660-666 (2009)

Disclosure of Invention

Problems to be solved by the invention

The purpose of the present invention is to provide a diarrhea inhibitor having an excellent diarrhea-inhibiting effect compared to conventional xylooligosaccharides.

Means for solving the problems

The present inventors have conducted intensive studies and, as a result, have found that oligosaccharides derived from alkali-pretreated bagasse have a higher diarrhea-inhibiting effect than commercially available xylooligosaccharides derived from corn cob.

That is, the present invention is as described in the following (1) to (10).

(1) A diarrhea inhibitor contains oligosaccharide composition derived from alkali-pretreated bagasse as effective component.

(2) The diarrhea inhibitor according to (1), wherein the oligosaccharide composition is derived from a hydrolytic enzyme treatment product of alkali-pretreated bagasse.

(3) The diarrhea inhibitor according to (1) or (2), wherein the oligosaccharide is xylooligosaccharide.

(4) The diarrhea inhibitor according to (3), wherein the xylooligosaccharide is a mixture of 1 or more than 2 selected from xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose.

(5) A feed comprising the diarrhea inhibitor of any one of (1) to (4).

(6) A livestock feed comprising the diarrhea inhibitor of any one of (1) to (4).

(7) A growth promoter for animals comprises oligosaccharide composition derived from alkali-pretreated bagasse as effective component.

(8) A diarrhea inhibiting method comprises administering oligosaccharide composition derived from alkali-pretreated bagasse to human or animal except human.

(9) A method for promoting growth of animals comprises applying oligosaccharide composition derived from alkali-pretreated bagasse to animals except human.

(10) A method for improving feed conversion ratio of animals comprises applying oligosaccharide composition derived from alkali-pretreated bagasse to animals except human.

ADVANTAGEOUS EFFECTS OF INVENTION

According to the present invention, the oligosaccharide composition derived from alkaline pretreatment has an excellent diarrhea-suppressing effect and is useful as an active ingredient of a diarrhea-suppressing agent.

Detailed Description

The oligosaccharide composition as an active ingredient of the diarrhea inhibitor of the present invention is obtained by subjecting alkali-pretreated bagasse to a hydrolysis reaction.

In the alkali pretreatment of bagasse, the alkali solution used for the alkali treatment is not particularly limited, but sodium hydroxide, potassium hydroxide, calcium hydroxide, and ammonia may be used. From the viewpoint of low cost and easy handling, sodium hydroxide is preferred. The alkali treatment condition is set to a range of 0.1 to 50 wt%, preferably 1 to 20 wt%, and more preferably 5 to 10 wt% of the bagasse solid content in a mixed state with the aqueous alkali solution. If the bagasse solid content concentration is less than 0.1 wt%, the amount of water used becomes extremely large, which is economically disadvantageous. On the other hand, if the bagasse solid content concentration exceeds 50% by weight, the bagasse may not be soaked with the alkali solution, and the effect of pretreatment may not be sufficiently obtained.

As the amount of alkali to be added in the alkali pretreatment, for example, in the case of using an aqueous sodium hydroxide solution, the amount of sodium hydroxide to be added is in the range of 0.1 to 100% by weight, preferably 1 to 50% by weight, and more preferably 5 to 15% by weight with respect to the solid content of bagasse. If the amount of the alkali added is less than 0.1% by weight, hydrolysis at the later stage may not proceed easily, and a sufficient yield of xylooligosaccharide may not be obtained. On the other hand, if the amount exceeds 100% by weight, the amount of alkali increases, and the amount of acid used for pH adjustment during the hydrolysis reaction increases, which is economically disadvantageous.

The temperature for the alkali treatment is preferably 10 to 200 ℃, and from the viewpoint of the yield of sugars in hydrolysis, it is more preferably 25 to 120 ℃, still more preferably 80 to 100 ℃, and particularly preferably 85 to 100 ℃.

The time of the alkali treatment may be appropriately set depending on the amount of alkali and the like, and is usually about 0.5 to 24 hours.

The alkali pretreatment can be carried out at normal temperature under pressure, but is preferably carried out at normal pressure.

The pretreated product after the alkali pretreatment may be directly subjected to the hydrolysis reaction, but it is preferable to subject the solid to the hydrolysis as a pretreated product by solid-liquid separation before the hydrolysis reaction. As the solid-liquid separation method, known methods such as a centrifugal separation method using a screw decanter, a filtration method such as pressure and suction filtration, and a membrane filtration method such as microfiltration can be used. Further, the solid component of the pretreated bagasse may be washed with pure water before and after the solid-liquid separation. Washing is preferred because enzyme reaction inhibitors such as lignin decomposition products can be further reduced, and the amount of acid required for pH adjustment during hydrolysis reaction can be reduced.

The kind of oligosaccharide contained in the oligosaccharide composition is not particularly limited, but xylooligosaccharide is preferable. The xylooligosaccharide is preferably a structure in which 2 to 6 xylose units are covalently bonded, but is not limited thereto, and may contain a side chain. Xylose is linked to each other by β -glycosidic bonds. They are called xylobiose (2-sugar), xylotriose (3-sugar), xylotetraose (4-sugar), xylopentaose (5-sugar), xylohexaose (6-sugar) depending on the number of xylose units. The effective component of the diarrhea inhibitor may be a mixture of 1 or 2 or more of them, but is more preferably a mixture of 3 or more, more preferably a mixture of 4 or more, and particularly preferably a mixture containing xylobiose, xylotriose, xylotetraose, and xylopentaose.

The oligosaccharide composition as an active ingredient of the diarrhea suppressing agent of the present invention is obtained by hydrolysis reaction of a pretreatment product of alkali-pretreated bagasse, and is preferably obtained by hydrolysis of the pretreatment product of alkali-pretreated bagasse with an enzyme.

The enzyme used for the hydrolysis reaction of the pretreated product of the alkali-pretreated bagasse is not particularly limited, but a cellulase composition is preferably used. The cellulase composition is a mixture of various hydrolases that hydrolyze the glycosidic linkages of beta-1, 4-glucan. Examples of the hydrolase contained in the cellulase composition include cellobiohydrolase, xylanase, endoglucanase, β -glucosidase, β -xylosidase, arabinofuranosidase, xylan esterase, ferulic acid esterase, α -glucuronidase, deacetylated chitinase, mannanase, mannosidase, α -galactosidase, β -galactosidase, and the like. Among the cellulase compositions used in the hydrolysis reaction, a cellulase composition having at least the activities of xylanase, cellobiohydrolase, and β -glucosidase and having substantially no activity of β -xylosidase when hydrolyzing bagasse is preferably used from the viewpoint of xylooligosaccharide production. Furthermore, the source of these enzymatic activities is not particularly limited. Examples of the preparation of such cellulase compositions are described in WO2017/170919 or WO 2017/170917. Further, a culture solution obtained by culturing a microorganism may be used as it is as a cellulase composition, or an enzyme purified from the culture solution or other commercially available enzyme products may be mixed and used in the present invention.

In the case of using a cellulase composition derived from a microorganism, a fungus can be preferably used as the microorganism. Specific examples of the fungi include microorganisms such as Trichoderma (Trichoderma), aspergillus (Aspergillus), cellulomonas (Cellulomonas), clostridium (Chloridium), streptomyces (Streptomyces), humicola (Humicola), acremonium (Acremonium), irpex (Irpex), mucor (Mucor), and Talaromyces (Talaromyces). Among these fungi, trichoderma and Aspergillus are preferable.

Specific examples of Trichoderma fungi include Trichoderma reesei QM9414 (Trichoderma reesei QM 9414), trichoderma reesei QM9123 (Trichoderma reesei QM 9123), trichoderma reesei RutC-30 (Trichoderma reesei RutC-30), trichoderma reesei PC3-7 (Trichoderma reesei PC 3-7), trichoderma reesei CL-847 (Trichoderma reesei CL-847), trichoderma reesei MCG77 (Trichoderma reesei MCG 77), trichoderma reesei MCG80 (Trichoderma reesei MCG 80), and Trichoderma viride QM9123 (Trichoderma viride QM 9123). Among these Trichoderma fungi, trichoderma reesei is preferred. Furthermore, a mutant strain having improved productivity of the cellulase composition by subjecting a fungus producing the cellulase composition to mutagenesis treatment with a mutagen, ultraviolet irradiation or the like, or a mutant strain having reduced β -xylosidase activity may be preferably used.

Specific examples of the fungi belonging to the genus Aspergillus include Aspergillus niger, aspergillus fumigatus, aspergillus aculeatus and Aspergillus terreus.

As the cellulase composition, cellulase compositions derived from 1 of the above fungi can be used, and cellulase compositions derived from a plurality of fungi can be mixed and used. When cellulase compositions derived from various fungi are used, the combination is not particularly limited, and for example, cellulase compositions derived from Trichoderma fungi and cellulase compositions derived from Aspergillus fungi may be used in combination. Specifically, examples of the β -Glucosidase derived from an Aspergillus fungus include "Novozyme188" (binder: 12494\125087012452124742.), "β -gluconase from Aspergillus niger" (Megazyme corporation), "125112481125125401251251252bga" (new york corporation), and the like. The β -glucosidase active component preferably contains the above β -glucosidase active component of the fungus belonging to the genus Aspergillus.

The oligosaccharide composition is obtained as a sugar solution containing oligosaccharides by hydrolysis of an alkaline pretreatment product, but it is preferable to selectively separate or concentrate oligosaccharides from monosaccharides such as glucose and xylose contained in the sugar solution in a subsequent step. The method for separation or concentration is not particularly limited, and membrane separation is preferably used. The type of the membrane used for membrane separation is not particularly limited, but a polyamide separation membrane is preferably used. The molecular weight cut-off of the polyamide separation membrane is also not particularly limited, but it is preferable to use a polyamide separation membrane having a molecular weight cut-off in the range of 200 to 1000. When the filtrate is passed through a separation membrane made of polyamide having a molecular weight cut-off in the range of 300 to 1,000, xylooligosaccharides are selectively concentrated on the non-permeation side.

The sugar solution may be filtered through a separation membrane having a molecular weight cut-off of 2,000 to 100,000 as a preliminary step of filtering the sugar solution through a separation membrane made of polyamide having a molecular weight cut-off of 300 to 1,000. The separation membrane having a molecular weight cut-off of 2,000 to 100,000 used in the previous step is not particularly limited as long as it is larger than the separation membrane made of polyamide used later and it transmits xylooligosaccharides represented by xylobiose and does not transmit high molecular weight components such as enzymes, but it is more preferably in the range of 5,000 to 50,000, and still more preferably in the range of 10,000 to 30,000. The method can remove enzyme components and the like used for the production of the sugar solution from a permeate obtained by passing through a separation membrane having a molecular weight cut-off of 2,000 to 100,000, thereby reducing impurities in an oligosaccharide concentrate and reducing the load on a polyamide separation membrane used in a subsequent step. As a material of a separation membrane having a cut-off molecular weight of 2,000 to 100,000, polyethersulfone (PES), polysulfone (PS), polyacrylonitrile (PAN), poly-1, 1-difluoroethylene (PVDF), regenerated cellulose, cellulose ester, sulfonated polysulfone, sulfonated polyethersulfone, polyolefin, polyvinyl alcohol, polymethyl methacrylate, polytetrafluoroethylene, or the like can be used. However, when the sugar solution is prepared by enzymatic treatment of biomass, the enzyme agent may contain an enzyme group involved in the decomposition of cellulose, and regenerated cellulose, and cellulose ester may be decomposed, and therefore, a separation membrane using a synthetic polymer such as PES or PVDF as a raw material is preferably used.

Specific examples of the separation membrane having a cut-off molecular weight of 2,000 to 100,000 include M-, P-, G-and GH (G-10), GK (G-20), GM (G-50), HWS UF, STD UF, VT, MT, ST, SM, MK, MW, LY, BN, BY, manufactured BY Soxhlet chemical industries, ltd. "11245212463125125400.

It is also preferable that the sugar solution is filtered through a microfiltration membrane to remove fine particles before filtration using a separation membrane having a molecular weight cut-off of 2,000 to 100,000. The microfiltration membrane preferably uses a separation membrane having an average pore size of about 0.01 to 10 μm.

The oligosaccharide composition may have a coloration derived from alkali pretreated bagasse. The degree of coloration is not particularly limited, but the absorbance at a wavelength of 420nm measured using a 1cm cuvette diluted so that the total sugar concentration becomes 5g/L is preferably 0.1 to 1.0, more preferably 0.15 to 0.8.

The oligosaccharide composition may contain lignin decomposition products derived from alkali-pretreated bagasse, and specific examples thereof include furan-based compounds such as HMF and furfural, and aromatic compounds such as vanillin, as long as the effects of the present invention are not impaired. Specifically, the amount of the lignin decomposer contained in the oligosaccharide composition is preferably 0.1% by weight or less, more preferably 0.08% by weight or less, further preferably 0.06% by weight or less, further preferably 0.05% by weight or less, further preferably 0.04% by weight or less, further preferably 0.03% by weight or less, and particularly preferably 0.02% by weight or less, relative to the amount of the oligosaccharide.

The saccharide that becomes the main component of the oligosaccharide composition is an oligosaccharide, but may contain monosaccharides such as glucose and xylose derived from alkali-pretreated bagasse. The total sugar concentration in the composition is not particularly limited, but is preferably 20% by weight or more, more preferably 25% by weight or more, even more preferably 30% by weight or more, even more preferably 35% by weight or more, and particularly preferably 40% by weight or more, from the viewpoint of transportation.

The content of the oligosaccharide in the oligosaccharide composition is not particularly limited, but is preferably 10% by weight or more, more preferably 15% by weight or more, even more preferably 20% by weight or more, even more preferably 25% by weight or more, and particularly preferably 0% by weight or more, from the viewpoint of storage and transportation.

The oligosaccharide composition may contain substances derived from alkali-pretreated bagasse other than the above substances, and the degree of the above substances is not particularly limited, but the absorbance at a wavelength of 280nm measured using a 1cm cuvette diluted so that the total sugar concentration becomes 5g/L is preferably 0.2 to 1.0, and more preferably 0.3 to 0.8.

The oligosaccharide composition may be concentrated under reduced pressure or concentrated by evaporation, if necessary, and may be in the form of a liquid or powder. The powdered oligosaccharide can be produced by a known powdering method such as a spray drying method using a liquid oligosaccharide.

The diarrhea inhibitor of the present invention can obtain a diarrhea-inhibiting effect by administering to a human or an animal other than a human, but is preferably administered to an animal other than a human. The method of administration is not particularly limited, and the composition can be used as an ingredient or additive contained in food or feed.

When the diarrhea inhibitor of the present invention is administered to a human or an animal other than a human, the amount of the oligosaccharide to be administered is not particularly limited, but the oligosaccharide is preferably added to a food or feed in an amount of 50 to 500ppm, more preferably 100 to 400ppm, even more preferably 100 to 300ppm, and even more preferably 100 to 200ppm. The mixed feed is a feed prepared by mixing necessary nutritional components (protein and energy), and the components are not particularly limited.

When the diarrhea inhibitor of the present invention is administered to an animal other than a human, the animal to which the diarrhea inhibitor is administered is not particularly limited, but is preferably a livestock, more preferably a pig, a chicken or a layer chicken, further preferably a pig or a chicken, and particularly preferably a pig.

The incidence of diarrhea is reduced by using the diarrhea inhibitor of the present invention. The frequency of occurrence of diarrhea is represented by the following formula (1) in the case of livestock, for example. The degree of reduction in the frequency of diarrhea caused by the diarrhea inhibitor of the present invention is not particularly limited, but is preferably 0.2% or more, more preferably 0.3% or more, still more preferably 0.4% or more, yet more preferably 0.5% or more, yet more preferably 0.7% or more, yet more preferably 0.8% or more, yet more preferably 1% or more, yet more preferably 2% or more, yet more preferably 3% or more, yet more preferably 4% or more, and particularly preferably 5% or more.

(total of heads with diarrhea symptoms/day)/(total heads x days) × 100 · · formula (1).

Oligosaccharides derived from alkaline pretreatment are preferably used as growth promoters because they have a feed conversion ratio-improving effect when used in animals other than humans, preferably livestock. The feed conversion ratio is an amount of feed kg required to increase the weight of an animal by 1 kg. The improvement of the feed conversion ratio means that the feed conversion ratio is decreased. When the diarrhea inhibitor of the present invention is used, the feed conversion ratio is decreased as compared with the case of a control to which the diarrhea inhibitor of the present invention is not added, and a commercially available xylooligosaccharide composition derived from corn cob is added. The degree of decrease in the feed conversion ratio is not particularly limited as long as it is decreased, but is preferably decreased by 0.01 or more, more preferably 0.015 or more, still more preferably 0.02 or more, still more preferably 0.025 or more, and particularly preferably 0.03 or more.

Examples

The present invention will be described in detail below with reference to examples. However, the present invention is not limited to them.

Reference example 1 method for measuring protein concentration

A commercially available protein concentration measuring reagent (Quick Start Bradford protein assay, bio-Rad) was used. To 250. Mu.L of the protein concentration measuring reagent returned to room temperature, 5. Mu.L of a diluted filamentous fungus-derived cellulase solution was added, and the absorbance at 595nm after leaving at room temperature for 5 minutes was measured by a microplate reader. Protein concentration was calculated against the standard curve using BSA as a standard.

Reference example 2 method for measuring beta-xylosidase Activity

A β -xylosidase activity assay method specifically performs a reaction in 90 μ L of 50mM acetic buffer containing 1mM p-nitrophenyl- β -xylopyranoside (124716412512510124501252312489125221248312481124721251512597125975. The activity of free 1. Mu. Mol p-nitrophenol per 1 minute was defined as 1U. Blank in 50mM acetic acid buffer solution containing 1mM p-nitrophenyl-beta-xylopyranoside 90. Mu.L was mixed well by adding 2M sodium carbonate 10. Mu.L, followed by reaction at 30 ℃ for 30 minutes by adding 10. Mu.L of enzyme diluent. Then, the increase in absorbance at 405nm was measured.

Reference example 3 method for measuring beta-glucosidase Activity

The β -glucosidase activity assay method specifically performed a reaction in 90 μ L of 50mM acetic buffer containing 1mM p-nitrophenyl- β -glucopyranoside (1247164125125101245012523124891252212483124811247212515, 12597125971253110 min later, and after 10 minutes, 10 μ L of 2M sodium carbonate was added and mixed well to stop the reaction, and an increase in absorbance at 405nm was measured. The activity of free 1. Mu. Mol p-nitrophenol per 1 minute was defined as 1U. Blank to 90. Mu.L of 50mM acetic acid buffer containing 1mM p-nitrophenyl-beta-glucopyranoside was added 10. Mu.L of 2M sodium carbonate and mixed well, followed by addition of 10. Mu.L of enzyme diluent and reaction at 30 ℃ for 30 minutes. Then, the increase in absorbance at 405nm was measured.

Reference example 4 measurement of Cellobiohydrolase Activity

Cellobiohydrolase activity was measured specifically by carrying out a reaction at 30 ℃ in 90. Mu.L of 50mM acetic buffer containing 1mM p-nitrophenyl- β -galactopyranoside (v/v: 1247112464125501251252389125125221248312481124721251259760 min.). The activity of free 1. Mu. Mol p-nitrophenol per 1 minute was defined as 1U. Blank in 50mM acetic acid buffer solution containing 1mM p-nitrophenyl-beta-galactopyranoside 90. Mu.L was mixed well by adding 2M sodium carbonate 10. Mu.L, followed by reaction at 30 ℃ for 30 minutes by adding 10. Mu.L enzyme diluent. Then, the increase in absorbance at 405nm was measured.

Reference example 5 xylanolytic Activity

A solution in which Xylan (Xylan from Birch wood, manufactured by Fluka) was suspended in 50mM sodium acetate buffer (pH 5.0) to a concentration of 1% by weight was used as a substrate solution. To 500. Mu.L of the substrate solution dispensed, 5. Mu.L of the enzyme solution was added, and the mixture was subjected to reaction at 50 ℃ while being rotated and mixed. The reaction time is basically 30 minutes, and is appropriately changed from 10 to 60 minutes depending on the level of the enzyme activity. After the reaction, the tube was centrifuged, and the reducing sugar concentration of the supernatant component was measured by the DNS method. The amount of enzyme that produced 1. Mu. Mol of reducing sugar in 1 minute in the above reaction system was defined as 1U, and the activity value (U/mL) was calculated according to the following formula.

Xylan lytic activity (U/mL) = reducing sugar concentration (g/L) × 1000 × 505 (μ L)/(150.13 × reaction time (min) × 5 (μ L)).

Reference example 6 measurement of sugar concentration

Xylo-oligosaccharide, glucose and xylose were quantitatively analyzed under the following conditions by using high performance liquid chromatography "LaChrom Eite" (product of hitachi, ltd.). Quantitative analysis was performed based on a calibration curve prepared from a standard sample of xylobiose, xylotriose, xylotetraose, xylopentaose, xylohexaose, and glucose or xylose, which are xylooligosaccharides. In the present example, the term "xylooligosaccharide" refers to a xylooligosaccharide having a retention time according to the present method for measuring a sugar concentration, the retention time being identical to that of xylobiose, xylotriose, xylotetraose, xylopentaose, and xylohexaose as standard substances.

Column: KS802, KS803 (Shodex)

Mobile phase: water (I)

The detection method comprises the following steps: RI (Ri)

Flow rate: 0.5 mL/min

Temperature: at 75 deg.c.

Reference example 7 method for measuring concentration of furan-based/aromatic-based Compound

The concentrations of furan-based compounds (HMF, furfural) and aromatic-based compounds (vanillin, etc.) contained in the sugar solution were analyzed by HPLC under the conditions shown below, and quantified by comparison with a standard.

Column: synergi HidroRP 4.6 mm. Times.250 mm (manufactured by Phenomenex)

Mobile phase: acetonitrile-0.1% ₃ PO ₄ (flow rate 1.0 mL/min) detection method: UV (283 nm)

Temperature: at 40 ℃.

Reference example 8 production of alkali-pretreated bagasse

Bagasse 5g was weighed out and heated until it became 105 ℃. The solid fraction of bagasse was calculated based on the change in weight at that time. The value obtained by multiplying bagasse in a moisture state by the solid fraction was defined as the solid content weight. 100g of bagasse was immersed in a sodium hydroxide aqueous solution so that the solid content weight of the bagasse was 8% by weight in a state of being mixed with the sodium hydroxide aqueous solution and the amount of sodium hydroxide added to the solid content of bagasse was 10% by weight, and pretreated at 85 ℃ for 3 hours. After the pretreatment, the solid content was coarsely separated by a strainer, and then washed with pure water until the weight of the bagasse solid content became 5 wt%. Then, solid-liquid separation was performed by centrifugal separation (3,000g, 10 minutes), and the solution component and the solid component were separated. The solid content was washed with pure water and used in the following experiment as a pretreatment.

Reference example 9 preparation of cellulase composition

[ preculture ]

The resulting mixture was added to distilled water in the form of 5% (w/vol) of corn steep liquor, 2% (w/vol) of glucose, 0.37% (w/vol) of ammonium tartrate, 0.14 (w/vol) of ammonium sulfate, 0.2% (w/vol) of monopotassium phosphate, 0.03% (w/vol) of calcium chloride dihydrate, 0.03% (w/vol) of magnesium sulfate heptahydrate, 0.03% (w/vol) of zinc chloride, 0.02% (w/vol) of iron (III) chloride hexahydrate, 0.004% (w/vol) of copper (II) sulfate pentahydrate, 0.0008% (w/vol) of manganese chloride tetrahydrate, 0.0006% (w/vol) of boric acid, and 0.0026% (w/vol) of heptaammonium molybdate tetrahydrate, 100mL of the resulting mixture was charged into a 500mL conical flask equipped with a baffle, and autoclave-sterilized at 121 ℃ for 15 minutes. After cooling down, PE-M and Tween80, which were separately autoclaved at 121 ℃ for 15 minutes, were added to each other at 0.01% (w/vol) as a preculture medium. Trichoderma reesei ATCC66589 (sold by ATCC) was set to 1X 10 in 100mL of the preculture medium ⁵ Each cell was inoculated in a volume of one mL, and the mixture was subjected to shaking culture at 28 ℃ for 72 hours at 180rpm to prepare a preculture (shaking apparatus: BIO-SHAKER BR-40LF, manufactured by TAITEC).

[ formal culture ]

The resulting mixture was added to distilled water in the form of 5% (w/vol) of corn steep liquor, 2% (w/vol) of glucose, 0.37% (w/vol) of cellulose (12450124999% (1247523), 10% (w/vol) of calcium chloride, 0.03% (w/vol) of ammonium tartrate, 0.14% (w/vol) of ammonium sulfate, 0.2% (w/vol) of potassium dihydrogen phosphate, 0.03% (w/vol) of calcium chloride dihydrate, 0.03% (w/vol) of magnesium sulfate heptahydrate, 0.02% (w/vol) of zinc chloride, 0.01% (w/vol) of iron (III) chloride hexahydrate, 0.004% (w/vol) of copper sulfate (II) pentahydrate, 0.0008% (w/vol) of manganese chloride tetrahydrate, 0.0006% (w/vol) of boric acid, and 0.0026% (w/vol) of hexaammonium heptamolybdate tetrahydrate, and 2.5L of the mixture was sterilized in a 5L stirred jar (DPC-2A manufactured by ABLE corporation) at 15 minutes at 121 ℃. After cooling, 0.1% of each of PE-M and Tween80, which were separately autoclaved at 121 ℃ for 15 minutes, was added as a main culture medium. 2.5L of this main culture medium was inoculated with 3-7 250mL of Trichoderma reesei PC previously precultured in the preculture medium. Then, the culture was carried out at 28 ℃ for 87 hours at 300rpm with an aeration rate of 1vvm, and after centrifugation, the supernatant was subjected to membrane filtration (12511125221250912450v, 12473861252259. Beta-glucosidase (Novozyme 188) was added to the prepared culture solution at a protein weight ratio of 1/100 to obtain a cellulase composition.

Reference example 10 preparation of cellulase composition selectively reducing beta-xylosidase Activity

The cellulase composition obtained in reference example 9 was adjusted to pH7.5 to 8.0 by a 1N aqueous solution of sodium hydroxide, diluted with water to a protein concentration of 4g/L, and then incubated at a temperature ranging from 40 to 50 ℃. When the cellobiohydrolase activity, the beta-glucosidase activity, the xylanolytic activity and the beta-xylosidase activity were measured by the methods described in reference examples 2, 3, 4 and 5, the beta-xylosidase activity at the optimum pH for the enzyme activity was decreased to a value (200U/-protein or less) at which the enzyme activity is substantially inactive when hydrolyzing a biomass, and the cellobiohydrolase activity, the beta-glucosidase activity and the xylanolytic activity remained at 60% or more, and thus the beta-xylosidase activity was selectively inactivated.

Reference example 11 production of xylooligosaccharide solution

The alkali-pretreated bagasse of reference example 8 was added so as to make the dry weight 5 wt%, and diluted hydrochloric acid was added to adjust the pH to 7.0. The cellulase composition having a selectively decreased β -xylosidase activity of reference example 9 was added to pretreated bagasse with adjusted pH so as to obtain 8mg protein/g-biomass, and the mixture was rotary-mixed at pH7.0 and 40 ℃ for 8 hours using a hybridization rotor. Then, the reaction mixture was centrifuged to obtain a supernatant, and the supernatant was filtered through a microfiltration membrane (DESAL E series, product of GE W & PT) having an average pore diameter of 0.04. Mu.m at an operation temperature of 25 ℃ at a membrane surface linear velocity of 20 cm/sec using polysulfone.

Next, a heat-resistant ultrafiltration membrane (GE W) having a molecular weight cut-off of 10,000 was used&HWS UF model "DURATHERM" (registered trademark) manufactured by PT Co., ltd, and the material quality: polyethersulfone) was filtered at an operating temperature of 25 ℃ and a linear velocity of the membrane surface of 20 cm/sec while controlling the operating pressure so that the flux became constant at 0.1 m/D. Then, in order to separate xylooligosaccharides from monosaccharides, as a separation membrane, a separation membrane a: DESAL G series GE (G-5) type (membrane material: polyamide composite membrane, molecular weight cut-off 1,000,GE W)&PT product), and filtered. The membrane separation apparatus used "SEPA" (registered trademark) CF-II (GE W)&PT Co., ltd., effective membrane area 140cm ² ) The operation temperature was set at 25 ℃ and the linear velocity of the membrane surface was set at 20 cm/sec, and filtration treatment was carried out under a filtration pressure of 0.5MPa to recover a non-permeated liquid and selectively concentrate xylooligosaccharides. Then, the resulting mixture was concentrated under reduced pressure to produce a xylooligosaccharide solution. The compositions of sugars contained in xylooligosaccharide solutions measured in accordance with reference example 6 are shown in table 1. The xylooligosaccharide content in the xylooligosaccharide solution was 17% by weight.

Further, with respect to the obtained xylooligosaccharide solution, the concentrations of the aromatic compound and the furan compound were measured in accordance with reference example 7, and as a result, the furan compound was not detected with respect to the xylooligosaccharide amount of 0.03 wt%. Further, the total sugar concentration of the resulting xylooligosaccharide solution was adjusted to 5g/L, and the absorbance at wavelengths of 420nm and 280nm measured using a 1cm cuvette was 0.15 and 0.391, respectively.

TABLE 1

Reference example 12 piglet feeding test without oligosaccharide

A feeding test was performed on 18 piglets (male 9 and female 9) weighing about 8kg using a commercially available mixed feed. In the test, piglets with diarrhea symptoms were counted, and in addition, the weight gain and the feed intake were recorded, and the frequency of occurrence of diarrhea and the feed conversion rate after 28 days were calculated. The results of the feeding test are shown in table 2.

Comparative example 1 piglet feeding tests using a commercially available xylooligosaccharide composition (derived from corn cob) a commercially available xylooligosaccharide composition derived from corn cob (short 1250112512489\\\ 1245252124561241253112473, xylooligosaccharide 70L) was added to the commercially available compounded feed used in reference example 12, and the piglet feeding tests were carried out as in reference example 12. The saccharide composition of the present xylooligosaccharide composition measured in reference example 6 was added to 200ppm of xylooligosaccharide based on the amount of xylooligosaccharide added to the mixed feed, as shown in table 1. The results of the feeding test are shown in table 2.

Example 1 piglet feeding trial using xylooligosaccharide composition derived from alkali-pretreated bagasse

A piglet feeding test was carried out in the same manner as in comparative example 1, except that the xylooligosaccharide composition derived from alkali-pretreated bagasse, which was produced in reference example 11, was used. The results of the feeding test are shown in table 2. From these results, it was found that when xylooligosaccharides derived from alkali-pretreated bagasse were used, the occurrence frequency of diarrhea was low and the effect of suppressing diarrhea was excellent as compared with commercially available xylooligosaccharides derived from corn cob. It was further shown that feed conversion was also improved compared to xylo-oligosaccharides derived from corn cob.

TABLE 2

Claims

1. A diarrhea inhibitor contains oligosaccharide composition derived from alkali pretreated bagasse as effective component.

2. The diarrhea inhibitor according to claim 1, wherein the oligosaccharide composition is derived from a hydrolytic enzyme treatment of alkali pretreated bagasse.

3. The diarrhea inhibitor according to claim 1 or 2, wherein the oligosaccharide is a xylooligosaccharide.

4. The diarrhea inhibitor according to claim 3, wherein the xylooligosaccharide is a mixture of 1 or 2 or more selected from xylobiose, xylotriose, xylotetraose, xylopentaose and xylohexaose.

5. A feed containing the diarrhea inhibitor according to any one of claims 1 to 4.

6. A livestock feed containing the diarrhea inhibitor according to any one of claims 1 to 4.

7. A growth promoter for animals comprises oligosaccharide composition derived from alkali pretreated bagasse as effective component.

8. A diarrhea inhibiting method comprises administering oligosaccharide composition derived from alkali-pretreated bagasse to human or animal except human.

9. A method for promoting growth of animals comprises administering an oligosaccharide composition derived from alkali-pretreated bagasse to animals other than human.

10. A method for improving feed conversion ratio of animals comprises applying oligosaccharide composition derived from alkali-pretreated bagasse to animals except human.