TW201231665A - Method for production of xylooligosaccharide with controllable composition by Bacillus halodurans - Google Patents

Abstract

The present invention provides a method for production of xylooligosaccharide with controllable composition by Bacillus halodurans, comprising culturing an alkaliphilic Bacillus halodurans with a plant material to obtain a combination of xylanases, and contacting the combination of xylanases with a xylan material to obtain xylooligosaccharide. The xylooligosaccharide with various compositions was obtained by using different the plant materials. The plant material comprises birchwood, corn-cob, wheat bran, sugarcane bagasse, rice straw, alkaline treatment of wheat bran, or any combination thereof.

Description

‘201231665 VIII. OBJECTS OF THE INVENTION: TECHNICAL FIELD OF THE INVENTION The present invention relates to a method for producing a wood oligosaccharide, and more particularly to a method for producing a ratio of the number of xylose of different xylooligosaccharides. [Prior Art] It is generally considered that the advantages of oligosaccharides are: improving microbial scale in the intestine, reducing the production of spoilage substances in the intestine, improving excessive lipids in the blood, reducing cholesterol, and reducing constipation. 'Preventing rainbow teeth' • Immunization activity and promotion Secret absorption and so on. The xylooligosaccharide is equivalent to low-digestible oligosaccharides, such as fructooligosaccharides, galactose oligosaccharides and isomaltose. The human body does not metabolize but has various functional characteristics. It can nourish Bifidus. Also known as the proliferation of bifidobacteria or bifidobacteria, it is not easy to cause rainbow teeth. Because it can make the probiotics of the intestines (4) heart (4), especially the growth and thinness of F. edodes and lactobacilli, these sugars belong to the beneficial bacteria (four) heart (four), and the bacteria are also known as probiotics, for promotion A substance that grows probiotics. Xylooligosaccharide, also known as xylooligosaccharide or xylooligosaccharide, is a generic term for oligosaccharides which are linked by 2 to 7 monosaccharide xyloses by glycosidic linkages. Xylooligosaccharides are among the oligosaccharides. • The best effect of F. faecalis is 5_2 times that of other oligosaccharides. The product of Xyl〇〇lig〇saccharides is commercially available, and xylooligosaccharides are produced from plant materials such as wheat bran or corn cob (com-cob). The composition of the plant material contains a large amount of xylose (xylan), and the branch on the polyxylose backbone is composed of a polysaccharide such as glucuronic acid. When only one xylan and many branches are present in the composition of an oligosaccharide, only oligosaccharides having a relatively low degree of polymerization can be produced. The industrial production method of wood sugar is obtained by Xylan degradation by polyxyxyase (xylanase), which is a kind of hemicellulose which is abundant in plants, 201231665 The enzyme is capable of degrading the enzymes of the aforementioned xylan. The long-chain form of xylan' can be seen in the stems and tobacco of African feathers and consists only of xylose. In industrial applications, the form of xylan is mainly arabinoxylan, which can be obtained from agricultural by-products such as wheat bran or corn cob, glucuron arabin (glucuron 〇arabin) 〇xylan) Common in softwood, glucuronoxylan is commonly found in hardwood. In the industrial application of sugars containing these xylans, in addition to xylose, arabinose, glucuronic acid, 4-oxomethylglucuronic acid, glucose, Galactose. Xylose and other sugars, whose characteristics vary with the type of plant from which they are derived. The components of xylooligosaccharides in the market are mainly xylobiose, xylotriose and some oligosaccharides with higher degree of polymerization. Among them, xylobiose and xylotriose are the most important components for the activation of human probiotics. In order to reduce the manufacturing cost in the industry, some special ingredients are induced by cheap raw wheat bran or corn cob! L substances (such as Aspergillus; niger, Bacillus habdurans, etc.) secrete such enzymes, and then directly hydrolyze the xylose-rich raw materials such as corn cobs into xylooligosaccharides by a crude enzyme solution. It can be seen from the foregoing that the xylooligosaccharide is a mixture of oligosaccharides having different numbers of xylose, and the functionality of the wood citrus sugar composed of different amounts of xylose is also different. In general, the auxotrophic effect of wood-raising on lactic acid bacteria and Bifidobacterium is as good as that of xylo-oligosaccharides, and the utilization rate of xylo-sugar is gradually decreasing as the number of xylose increases. Recent studies have found that Lactobacillus brevis (kciokc / / / (10) know eWi) like xylobiose (xyl〇bi〇se), Bifidobacterium pubescens (7) prefer wood trisaccharide (xyi〇tri〇se) and wood four Xylotetraose [Moura et al., LWT- Food Science and Technology, 40 (2007) 963-972]. In addition, studies have shown that the use of Phytophthora toxins for wood sugar is best for xylotriose, followed by xylobiose and xylotetraose [Gullonetal & Coffee 56 (2〇〇8) 7482-7487 ], the average wood sugar utilization rate after cultivating young Bifidobacterium is 77%, and the utilization rate of each component is 9〇% for xylo-oligosaccharide, '84% for xylo-oligosaccharide, and 20124665 for k-cell sugar. 83% and pectin (4) openta〇se) are 71〇/〇. In the soil with xylooligosaccharides, the reproduction rate of Bifidobacterium adolescentis is much higher than that of other F. faecalis β i〇ngum S. The monosaccharide xylose that is rich and wealthy, although it has no good wood, can also be digested and utilized by F. In addition, xylose is a vinegar nutrient (gly_trients^ which is one of the ingredients - 'antibacterial and antifungal functions, especially gram-negative bacteria and white streptococci, can also help the growth of probiotics in the intestine. Polyxylose is the most soluble in alkaline environment. At the same time, the pretreatment process of polyxylase in the application usually requires a high temperature process, so it can achieve no loss of activity and alkali resistance under the test environment. High-temperature resistant polyxylase is particularly important. Therefore, if a poly-xylase capable of heat-resistant alkaline is used, and a manufacturing method capable of controlling the proportion of the number of xylose in the xylooligosaccharide, for different uses (for example, In order to solve the problems of the prior art described above, it is an object of the present invention to provide a microorganism capable of controlling the composition of xylooligosaccharides and to apply the microorganisms. The method of controlling the composition of the wood oligo# sugar is particularly related to the susceptibility of the xylooligosaccharide of the controllable composition and its application. To achieve the aforementioned purpose, To provide a controllable composition of xylooligosaccharide-resistant Bacillus licheniformis (5, such as 7/1^/^/〇# (3〇, has been deposited in the food of the consortium in the Republic of China on the 24th of January 1st) The Industrial Development Institute, whose registration number is bcrc 910501. The Bacillus alkaline bacillus secretes a thermostable alkaline polyxylase, which is a polyxylase xyn45 (SEQ ID ΝΟ. :1) or a polyxylase xyn23 (SEQHDN〇.: 2). The present invention provides a method for producing a controllable composition of xylooligosaccharide 5.201231665 from the aforementioned B. alcaligenes, the steps comprising - plant material Cultivating a test-type Bacillus licheniformis from a high-pH value, obtained from the sexual bud, the enzyme combination comprising a xylase Xyn45 (SEQ ID NO: D and - polyxylase) Xyn23 _ still n〇: 2), the enzyme combination and the xylose lung enzyme reaction 'that is obtained - xylooligosaccharide, the xylooligosaccharide comprises selected from the monosaccharide xylose, xylobiose, xylotriose, a group of poly(xylose) having a degree of polymerization of four or more and any combination thereof; the plant material is selected from the group consisting of birch, corn cob, wheat bran, and bran a group consisting of stains, straw, and treated wheat flour; the high pH range is

8~11; the enzyme is combined with the __enzyme solution in the present embodiment, and the supernatant is obtained by centrifugation of the supernatant by the test bacillus, and the supernatant is dialyzed against sulfuric acid and dialysis; The polyxylose raw material is corn cob; the condition of the enzyme reaction is 3〇~8〇, pH4~u; when the plant material is alkali treated wheat noodle, the proportion of the xyloolipid in the oligosaccharide is 32 %the above. When the plant material is corn cob, the proportion of xylobiose in the xylooligosaccharide is 27% or more; on the other hand, when the enzyme reaction time is terminated within 4 hours, the proportion of xylotriose in the xylooligosaccharide is south 8.6% or more. Since the xylooligosaccharide produced by the method of the invention has the advantages of anti-bacterial, anti-infectious bacteria and helping the probiotic growth in the intestine, it has the characteristics of controlling the composition ratio of different xylose, and can be based on xylooligosaccharide The proportion of xylose is matched with different types of lactic acid bacteria to make health foods and foods and beverages, so that each xylooligosaccharide component can be used to the maximum. The embodiments of the present invention are further described in the following description, and the embodiments of the present invention are set forth to illustrate the present invention, and are not intended to limit the scope of the present invention. In the scope of the invention, the scope of protection of the invention is defined by the scope of the appended claims. [Embodiment] The present invention is directed against Bacillus licheniformis (Bac out of us f2a/oc/uransj (registered number BCrc 'IS] 6 201231665

A ' 910501) endo-type xylase _ 〇 - _ 3) to manufacture xylooligosaccharides of different compositions. The 耐 之 纽 杂 _ _ _ _ _ read the reading of the nuclear field to 'can secrete a large number of ship-resistant poly wood _, which mainly has two active proteins, where the SDS-MGE analysis film is 45 coffee and 23 hearts, so respectively called fine 5 (ah is 0.: 1) and ¥ 23 Qing coffee 0.: 2), are all endo-polychlorinase (end〇-xyl_es). Xyn45 and xyn23 belong to the first and the u family of glycosyl hydrolases, respectively, and the tenth family of glycolytic enzymes have a large molecular weight (> 3 milk) and belong to acidic Pl (low? 1 value). The characteristics of the 帛u family mine are small (30 呦, belonging to the experimental P1 (high PI value), the structure is small and compact. xyn45 and xyn23 have a wide range of pH and For the application range of high temperature, between the thief and the teacher ^

Active, the highest temperature at 7 o'clock is Saki, but H is still 90% of the maximum activity. When xyn45 is at ρΗ 7, the high activity temperature is a wide range of 42. The highest activity temperature is thief. Active, in summary, these two enzymes have more than 5G% 3 in 3G~8GC, the optimal temperature is 72〇c; both enzymes have more than 60% activity at pH 4~11. These two enzymes, either alone or in combination, can catalyze the hydrolysis of polyxylose to a relatively small degree of polymerization (DP), and are therefore the ideal polyxylase for the production of xylooligosaccharides. In the solid paste of the present invention, the contact resistance of the different materials is different, although the ratio of the two kinds of polyxylase in the enzyme combination obtained by the budding bacillus secreting poly-xyloin is different from that of the learning 23, even after _ in the same poly wood The sugar raw material has different composition of xylooligosaccharides, and the xylooligosaccharides having different compositions have different functionalities. Different from the prior art, the use of a single enzyme as a biocatalyst, that is, the invention adjusts the enzyme composition by different inducing materials, A method for producing xylooligosaccharides of different compositions is obtained. The plant material capable of inducing the test of the bacterium to secrete polyxylose can be any material containing polyxylose such as birch, corn cob, wheat noodles, scallop, straw and processed wheat noodles 7 201231665 preferably birch, Alkali-treated wheat bran, corn cob, preferably alkali-treated wheat bran and corn cob. The medium resistant to Bacillus alkaline bacillus usually consists of a basic medium such as Emerson medium or Berg's mineral salts medium plus polyxylose. Xylose is an inducing matrix that induces /w/oc/wrara secretion of polyxylase. The composition of these two media (without xylose) is: Emerson medium consists of 0.55% yeast extract (Yeastextract), 0.5% Digested protein (Peptone), 0.02% magnesium sulfate (MgS04), 0.1% dipotassium hydrogen phosphate (K2HP〇4); the pH was adjusted to 1 以 with 1M sodium hydroxide. The Berger's inorganic salt medium consists of 0.2% sodium nitrate (NaN03), 0.1% yeast extract, 0.05% dipotassium hydrogen phosphate, 0.02% magnesium sulfate with water (MgSO4.7H2O), 0.002% gasification (MnCl), Composition of 0.002% calcium sulfide (CaCl2); the pH was adjusted to 10.5 with 25% sodium carbonate (Na2C03). To incubate the two mediums with 2% birch polydextrose (Birchwood xylan), incubate the Bacillus anabacter, and incubate in a 500 ml Erlenmeyer flask in a 3 rt incubator at 2 〇〇φη for 120 hours. The resulting supernatant was separated and precipitated with ammonium sulfate at 9 〇% saturation. The precipitate after centrifugation at 13,000 rpm was buffered with hydrazine. The resulting enzyme solution was passed through a thin rot (Cel u 8 membranes) After dialysis, protein colloidal electrophoresis (SDS-PAGE) and enzyme activity electrophoresis analysis (zymGgmm), the results are as shown in the first figure 7F where E" represents the sharp medium, "B" represents the Burg's inorganic salt medium, and ''M' stands for the marker. In the figure, it can be seen that the two mediums containing birch polyxylose can be converted to the poly-xylase y 〃 xyn23. However, the use of different media is different in the self-fertilizer content and enzyme activity. 'The protein concentration Μ g/L enzyme activity (Μ Cs measured) is 53 u/mL using the Borg's inorganic salt medium containing birch polyxylose. And using Emerson medium containing wood charcoal The protein concentration is reduced, and the enzyme activity (inhibition measurement) is π 佩. Displaying 8.201231665 shows that the enzyme medium can obtain more enzyme protein and polyxylase activity. Further based on the inflammatory medium, Different poly-xylose raw materials were used: 2% corn cob, wheat noodles and test-processing _ matrix, xylozymes were extracted from the secret buds, and the obtained enzymes were analyzed by electrophoresis. As shown in the second figure, cw and W1 respectively represent the corn sorghum, wheat noodles and the gluten-free glutinous rice bran, which are known as knives and proteins. The results show that the culture using corn cob or wheat noodles The fine material is mainly composed of xyn45, secret white material and active material transfer, but the Qi slave money (four) turn-shaped polymu Linshen', yn23 s has a comparison with the medium of the birch poly-xylose, the income B is more obvious These results show that 'no _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Proportion of the production of 3: corn cob induced the production of poly wood (four) xyn45 and The ratio of xyn23 is 9〇: 1〇; the ratio of polyxylase induced by xylocycline is %: 3〇; the ratio of xylose to xyn23 induced by wheat bran (4): 30; The ratio of the surface-induced polyxylase _5 to xyn23 is 50: 50. Because the composition ratios of the above two kinds of poly (4) _5 are different, the composition ratio of the xylooligosaccharide products obtained from the same poly-xylose raw material is not - As such, the xylose material can be any material containing polyxylose, such as birch, corn cob, wheat flour, bagasse, straw, and processed wheat flour, preferably wheat flour and corn cob, preferably corn cob. The invention is further illustrated by the following examples 1 to 3, which are intended to be illustrative only and not to limit the scope of the invention.

Example 1 A 201231665 Alkali-treated wheat noodle as a material for inducing Alcaligene-resistant Bacillus secreting polyxylase. Preparation of polyxylase: Firstly, a substrate for inducing polymyxylase secretion by Bacillus aeruginosa was prepared to solidify The dough is mixed with 1% sodium hydroxide in a ratio of 1 to 10, and sterilized by 12 cc high pressure for 10 minutes, then washed with pure water to pH 7, and the residue is dried to obtain alkali treated mash. The receiver was placed in a 100 ml Ermen medium (Emerson medium) containing 0.55% yeast extract, 0.5% digested protein, 0.02% sulfuric acid, and 0.1% phosphoric acid. Dipotassium hydrogenate, then adjust the pH to 1 〇. 20/〇 of the above-mentioned alkali-treated wheat bran was added to the entire mixture at 121. The autoclave was sterilized for 3 minutes. After the Erlenmeyer flask was cooled to room temperature, inoculate 10% (v/v) of the resistant Bacillus licheniformis at 37.培养Cultivated incubator|(17〇11)111) culture for 4 to 5 days. After that, the bacterial liquid was at 13, and 113111 was at 4. (: Centrifugation for 30 minutes, the supernatant was precipitated with sulfuric acid at 9〇% saturation, and the precipitate after centrifugation at 13 〇〇〇φΓη was buffered with 0.1 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris_Ha). The solution (pH 7.0) is dissolved back, and after dialysis, it is a crude enzyme solution. The activity per unit volume of the crude enzyme solution is 179 U/mL (measured at 37 ° C). Preparation of polyxose: corn cob powder After treatment with 90% sodium hydroxide (NaOH) at a solid-liquid ratio of 1 to 20 at 90 ° C for 90 minutes, the resulting supernatant was neutralized with acetic acid (acetie acid) to pH 5.0, followed by 3 The volume of 95% is soaked in ethanol for 60 minutes, and the precipitate obtained is xylose (xylan). With this xylose as the matrix, the polyxylose is decomposed under the action of the aforementioned polyxylogase. The xylooligosaccharide is produced. The conditions for the production of xylooligosaccharides by the xylase reaction are: 2% (w/v) of the substrate, 8.95 U/mL of the enzyme, and 反应: pH 8.0. The product is subjected to high performance liquid chromatography. High-perf〇rmanCe liquid chromat〇graphy HpLC analysis The structure of the high performance liquid chromatography is as follows. The chromatographic column (C〇lumn) is Bi〇Rad cdumn Aminex HPX-87H (300mm x 7.8mm); f ^(Column temperature)^ 65〇C ; Detector is refractive index detector (8) detect〇r); mobile phase (Μ〇·phase) is (5) 10 201231665 5 mM Sulfuric acid (H2S〇4); flow rate 0.6 ml/min. The third figure shows the high-performance liquid chromatograph analysis of the water in the matrix before the reaction of polyxylose and polyxylase, and the product in the solution after the reaction. The maps of A to D in the figure represent polyxylose and A spectrum before the reaction of the xylase, the reaction for 4 hours, the reaction for 24 hours, and the reaction for 47 hours. The composition of the product changes with time as shown in the following table. \ **DP>4 xylooligosaccharide xylo-oligosaccharide DPI1 2 xylose total conversion rate (%) 2 h concentration (g/L) 9.97 1.44 3.28 0.13 0.24 15.07 38.1 percentage (%) 66.17 9.58 21.79 0.88 1.59 100 4 h Concentration (g/L) 10.29 1.46 4.65 0.06 0.38 16.83 46.9 Percent (%) 61.14 8.67 27.61 0.34 2.24 100 24 h Concentration (g/L) 10.37 1.05 6.51 0.08 0.73 18.73 56.4 Percent (%) 55.37 5.59 34.76 0.41 .3.88 100 47 h Concentration (g/L) 10.69 1.05 7.04 0.10 0.87 19.74 61.5 Percent (%) 54.17 5.31 35.65 0.49 4.39 100

1 DP1 represents a non-xylose monosaccharide (arabinose, glucose, etc.). 2 DP>4 represents a xylooligosaccharide having a degree of polymerization greater than 4. The water-soluble component of the substrate before the reaction was 7.45 g/L, and all of them were xylooligosaccharides with a degree of polymerization greater than 4, and no xylooligosaccharides having a t-complexity of less than 3 were present. The water-soluble xylooligosaccharide added after the reaction is hydrolyzed by polyxylase, and thus the conversion rate of 24 hours (i.e., the yield of xylooligosaccharide caused by the polyxylase reaction) is calculated to be 56.4%, and 47 hours is 61.5. % 4 includes the water-miscible wood obtained by the reaction a priori treatment. The total Muxi yield is 93 7% at the third hour and 98.7% after 47 hours. The calculation method of the total xylooligosaccharide yield is as follows: 2% (w/v) is equivalent to 2 〇 machine, and the total amount of sugar collected by the wood after reaction for 24 hours and 47 1 is 1S 73讥 and 19%, respectively. 201231665 The total wood oligosaccharide yield was 18.73/2〇*1〇〇%=93 7% at 24 hours, and 19.74/20* 100%=98.7% after 47 hours. The xylose reaction was found to change first, and then the xylo-oligosaccharide concentration was increased first. The xylo-oligosaccharide concentration increased rapidly, and the xylo-trisaccharide decreased in the later stage due to decomposition into xylobiose and xylose. After 24 hours of reaction, the proportion of each oligosaccharide did not change much, indicating that the reaction was slowed down. From this result, it can be seen that the reaction needs to be terminated relatively quickly (2 to 4 hours), although the conversion rate (wood oligosaccharide yield) is lower, but the average is about 42.5%.

Example 1 B The enzyme solution of Example A was used to increase the concentration. The preparation of polyxylase and the preparation of polyxylose were the same as those in Example A. The conditions for the production of xylooligosaccharides by the xylase reaction are: 2% (w/v) of the substrate; 17.5 U/mL of the enzyme's reaction temperature 50 C; pH 8_0. The resulting product was analyzed by high performance liquid chromatography. After 15 hours of reaction, the product composition (concentration and percentage) was as follows: \ **DP>4 xylooligosaccharide xylo-oligosaccharide DPI* xylose total conversion (%) concentration (g/L) 10.73 1.19 6.08 0.18 0.68 14.72 57.1 Percent (%) 56.87 6.33 32.24 0.95 3.62 100 *DP1 stands for non-xylose monosaccharides (arabinose, glucose, etc.). **DP>4 represents a xylooligosaccharide having a degree of polymerization greater than 4. Compared with Example 1, the amount of polyxylase was increased from 8.95 U/mL to 17.5 U/mL, so the reaction rate was faster. The reaction of 15 hours was equivalent to the reaction of Example 1 for 24 hours. In the final product, it was found that in the wood sugar collection with a degree of polymerization of less than 4, xylobiose accounted for the majority (more than 32%), xylose and other monosaccharides were together at most 5%, and xylotriose was between 5 and 6%. Example 2 12 201231665 Using corn cob as a material for inducing polymyxylase secreted by Bacillus aeruginosa

Preparation of polyxylase: In a 250 ml Erlenmeyer flask, put into 100 ml of Emerson medium containing 0.55% yeast extract, 〇50/0 digested protein, 0.02. /. Sulfuric acid town, and 0.1% dipotassium hydrogenate, adjust the pH to 1〇. After adding 2% corn cob powder, the whole mixture was autoclaved at 121 for 3 minutes. After the flask was cooled to room temperature, 10% (v/v) of Bacillus aeruginosa was inoculated and cultured in a 37 °c incubator (170 rpm) for 4 to 5 days. After that, the bacterial solution was centrifuged at 13,000 rpm for 30 minutes at 4 ° C, the supernatant was precipitated with ammonium sulfate under 9 〇% saturation, and the precipitate after centrifugation at 13 〇〇〇 φιη was 0.1 mM tris (hydroxyl thiol). The amino decane hydrochloride buffer solution (ρΗ 7·0) is dissolved back, and after dialysis, it is a crude enzyme solution. The activity per unit volume of this crude enzyme solution was 128 U/mL (measured at 37 ° C). The preparation of the xylose was the same as in Example A. The conditions for the production of xylooligosaccharides by the xylase reaction were as follows: the amount of the substrate was 2% (w/v); the amount of polyxylase was 17.5 U/mL; and the reaction temperature was 5 (TC; ρΗ8·0. The obtained product was in a high-performance liquid layer. Analysis of the analyzer, the fourth figure shows the water-soluble components in the matrix before the reaction of polyxylose and polyxylase, and the high-performance liquid chromatograph analysis of the products in the solution after 15 hours of reaction, in which the ruthenium is poly-xylose and poly Before the xylase reaction, 'B is a map of polyxylase reaction for 15 hours. After 15 hours of reaction, the product composition (concentration and percentage) is as follows: First experiment **DP>4 xylooligosaccharide xylo-oligosaccharide DPI* Total conversion of xylose (%) Concentration (g/L) 10.23 1.14 4.92 0.36 1.27 17.93 52.4 Percent c%) 57.08 6.34 27.47 2.02 7.10 100 First experiment**DP>4 Wood trisaccharide disaccharide DPI* Total conversion rate of xylose (%) [S] 13 201231665

1.21 6.66 4.95 27.27 0.39 2.13 1.32 7.26 18.16 100 53.6 *DP1 represents a non-xylose monosaccharide (arabinose, glucose, etc.) "DP" 4th generation has a degree of silkiness greater than 4 Liquor oligosaccharides. It can be seen from this example that corn cob culture The enzyme solution obtained by Bacillus alkaline bacillus catalyzes the xylooligosaccharide component obtained by decomposition of polyxylose, the proportion of xylobiose is relatively low (about 27%), and xylose and other monosaccharides are about 9% higher, wood The trisaccharide is maintained at about 7 〇 / 0.

Example 3 The preparation of the alkali-resistant enzyme of the polyxylase and the preparation of the xylose were carried out in the same manner as in the first embodiment. The conditions for the production of xylooligosaccharides by the enzyme reaction were as follows: the amount of the substrate was 2%, w/v; the amount of the enzyme was 17 9 U/mL; the reaction temperature was 5 (TC; the pH values were 9, 10, and 11 respectively. The obtained product was in a high-performance liquid phase. After chromatograph analysis, the product composition (concentration and percentage) is as follows: PH9 concentration (g/L> percentage**DP>4 xylooligosaccharide6.7 57.8 xylobiose 0.8 6.9 xylobiose 3.7 31.9 DPI* 0 0.0 xylose 0.4 3.4 Total 11.6 100 Conversion (%) 36 pH 10 Concentration (g/L) 5.8 0.7 2.6 0 0.3 9.4 25 Percentage 61.7 7.4 27.7 0.0 3.2 100 pH 11 Concentration (g/q 58 0.4 0.9 0 0.1 7.2 14 Percent (% 80.6 5.6 12.5 0.0 1.4 100

*DP1 represents a non-xylose monosaccharide (arabinose, glucose, etc.). MDP > 4 represents a xylooligosaccharide having a degree of polymerization greater than 4. 201231665 Compared with the example-B, the pH value is increased, the reaction speed wheel is slow, and the shame conversion rate is decreased. The money cultivates the chiral leaven. The secret (transformed to xylose) is lower, but the ratio of woody sugar to xylobiose decreases slightly at high pH. This example shows that the enzyme-like filament T is still capable of recording the production of xyloligosaccharides. It can be seen from Example-A that when the wheat noodles used for the treatment were used as the plant material for inducing polyxylase, the count of the county increased (〇~47 tree), and the relationship between xylose and xylobiose increased, respectively, from 1.59 to 4.39. % and 21.79~35.65%, while the proportion of xylotriose gradually decreased from 9.58 in 2 hours to 5.31% in 47 hours; while in the second example, corn cob was used as the plant material for inducing polyxylase. The other conditions in the supplement-B are the same. The comparison of the xylose produced by the corn cob is 7.1G% higher, and the treated wheat bran is only 362%; while the corncob produces a lower proportion of xylobiose of 27.47%. The alkali treated wheat noodles are 32%. Therefore, it is possible to select appropriate (four) to achieve the proportion of xylooligosaccharide which requires the number of xylose, and according to the result of the third embodiment, the enzyme can be catalyzed in an alkaline environment. [Simple description of the diagram] The first figure shows the protein colloid electrophoresis and enzyme activity electrophoresis analysis of Bacillus subtilis-resistant protein in different media. "E" and "B" respectively represent Emerson medium containing birch polyxylose and Berger's inorganic salt medium; "M" is a protein of known molecular weight. The first picture shows different induction substrates. Protein colloidal electrophoresis and enzyme activity electrophoresis analysis of the secretion of Bacillus licheniformis. ''C', 'W' and 'W1' respectively represent Emerson medium containing corncob, wheat bran and alkali-treated wheat bran; "M" is a marker protein of known molecular weight. The second figure is the high-performance liquid chromatograph analysis of the water-soluble component in the matrix before the reaction of the polyxylose and the polyxylase in the first embodiment and the product in the solution after the reaction. A: Polyxylose 201231665 Before reacting with polyxylase, B: polyxylase was reacted for 4 hours, C: polyxylase was reacted for 24 hours, and D: polyxylase was reacted for 47 hours. The fourth figure is the water-soluble component of the matrix before the reaction of the polyxylose and the polyxylase in the second embodiment, and the high-performance liquid chromatograph analysis of the product in the solution after reacting for 15 hours. A: B: polyxylase was reacted for 15 hours before the reaction of polyxylose with polyxylase. [Main component symbol description] None

[S] 16 201231665 Sequence Listing <110> National Chung Cheng University <120> Method for producing controllable xylooligosaccharides by Bacillus aeruginosa <160> 2 <210> 1 <211> 396 _ <;212>PRT<213> artificial sequence

<400〉 1

MITLFKKPFVAGLAISLLVGGGLGNVAAAQGGPPKSGVFGEN QKRNDQPFAWQVASLSERYQEQFDIGAAVEPYQLEGRQAQIL KHH YNSLVAEN AMKPVSLQPREGEWNWEGADKIVEF ARKHN MELRFHTLVWHSQVPEWFFIDENGNRMVDETDPEKRKANKQ LLLERMENHIKTVVERYKDDVTSWDVVNEVIDDGGGLRESE WYQITGTDYIKVAFETARKYGGEEAKLYINDYNTEVPSKRDD LYNLVKDLLEQGVPIDGVGHQSHIQIGWPSIEDTRASFEKFTS LGLDNQVTELDMSLYGWPPTGAYTSYDDIPEELFQAQADRYD QLFELYEELS ATISS VTFWGIADNHTWLDDRAREYNNG VGVD APFVFDHNYRVKPAYWGIID

<210> 2 <211> 210 <212>PRT <213> artificial sequence <400〉 2

MFKFVTKVLTVVIAATISFCLSAVPASANTYWQYWTDGGGTV

NATNGPGGNYSVTWRDTGNFVVGKGWEIGSPNRTIHYNAGV

WEPSGNGYLTLYGWTRNQLIEYYVVDNWGTYRPTGTHRGTV

VSDGGTYDIYTTMRYNAPSIDGTQTFQQFWSVRQSKRPTGNN

VSITFSNHVNAWRNAGMNLGSSWSYQVLATEGYQSSGRSNVT

VW

[S]

Claims (1)

201231665 VII. Scope of application for patents: 1. A test-resistant Bacillus licheniformis (5acz7/w5 /za/oi/wrara) with controllable composition of hibiscus and sugar, the registration number is BCRC 910501. 2. The alkaline-resistant Bacillus bacterium according to claim 1, wherein the Bacillus alkaline-resistant Bacillus secretes a thermostable alkaline polyxylase, and the heat-resistant alkaline polyxylase is a xylose Enzyme xyn45 (SEQ ID ΝΟ.: 1) or a polyxylase xyn23 (SEq ID N〇: 2). 3. A method for producing a sugar-receiving plant having a controllable composition by the Bacillus alcaligenes as described in the scope of the patent application, the steps comprising the following steps: (1) cultivating a plant material at a high pH Alcaligene-resistant Bacillus (2) Obtaining an enzyme combination from the ''Bacillus'), the enzyme combination comprising a xylase xyn45 (SEQ ID NO.: 1) and a xylase xyn23 (SEq still N〇: 2); (3) reacting the enzyme with the xylose raw material; and (4) obtaining the xylooligosaccharide, which comprises a monosaccharide xylose, a xylobiose, a xylotriose, a polymerization degree of four or more A group of polyxylose and any combination thereof. 4. The method of claim 3, wherein the step (1) of the plant material is selected from the group consisting of ladder wood jade #麦楚u, straw and processed wheat noodles. 5. The method of claim 3, wherein the high pH range of step (1) is 8 to 11. 6. The method of claim 3, wherein the step of the enzyme combination is the alkali-resistant bacillus, the supernatant-supernatant, and the supernatant is subjected to precipitation by sulfuric acid. 7 such as i ΐ patent! ^ The method described in Item 3, the towel step (3) The poly-xylose raw material is selected from the f-made & straight 5 two wheat noodles 'made, straw and alkali treated wheat noodles The group that makes up. For the method described in item 3 or 7, the t-step is a jade core. 9·^ The method described in item 3, wherein the step of the «reaction secrets is 30~80 C, pH 4~11. When the green matter is the green noodles, the proportion of xylobiose in the woody sugar is 32% or more. U. The method of claim 3, wherein when the plant material is butterfly 201231665, the proportion of xylobiose in the xylooligosaccharide is 27% or more. 12. The method of claim 10, wherein the ratio of xylotriose in the xylooligosaccharide is more than 8.6% when the enzyme reaction time is terminated within 4 hours.