JP2012157350A - Method for producing xylooligosaccharide with controllable composition from alkali-resistant bacillus halodurans - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a xylooligosaccharide with a controllable composition from alkali-resistant Bacillus halodurans.SOLUTION: The method for producing the xylooligosaccharide with a controllable composition from alkali-resistant Bacillus halodurans is provided. In the method, the alkali-resistant Bacillus halodurans is cultured using a plant material to acquire xylanase with alkali resistance and high temperature resistance, and the xylanase and a xylan raw material are made to react with each other to obtain the xylooligosaccharide. A final product of a different xylooligosaccharide composition is obtained utilizing the difference of the plant material. The plant material is a birch material, a cob of corn, wheat bran, sugarcane bagasse, straw, alkali-treated wheat bran, or an optional combination of them.

Description

  The present invention relates to a method for producing a kind of xylooligosaccharide, and more particularly to a production method capable of controlling the composition ratio of a kind of different xylooligosaccharides.

  In general, the advantages of oligosaccharides are to improve the gut microbiota, reduce the incidence of gut rot, improve blood lipid excess, reduce cholesterol, reduce constipation, prevent tooth decay, and enhance immunostimulation Enhance, promote calcium absorption, and so on. Xylooligosaccharides, fructo-oligosaccharides, galactooligosaccharides, malt oligosaccharides, etc. belong to the low digestible oligosaccharides and cannot be metabolized by the human body, but have a variety of functional properties, such as the growth of intestinal bifilus, Hard to cause. For this reason it can grow intestinal probiotics, in particular bifidobacteria and lactobacilli, these oligosaccharides belong to prebiotics and are substances that promote the growth of probiotics.

  Xylooligosaccharide is a general term for oligosaccharides formed by glycosidic bonding of 2 to 7 xyloses. Xylooligosaccharide is one of the oligosaccharides that has the most excellent function of growing bifilus bacteria, and is 5-20 times that of other similar oligosaccharides.

  Currently, xylooligosaccharide products are already on the market, which are produced from plant materials such as wheat bran, corn-cob. A large amount of xylan is contained in the composition of the plant material, and the side chain on the xylan main chain is composed of a saccharide other than xylan, for example, glucuronic acid. When the oligosaccharide composition has only xylan and many side chains, only oligosaccharides with a relatively low degree of polymerization can be produced.

  The industrial production method of xylo-oligosaccharide is obtained by decomposing xylan using xylanase. Xylan is a semi-cellulose present in a large amount in the plant body, and xylanase uses the aforementioned xylan. It is an enzyme that can be decomposed. Long-chain xylan is found in gerbera stems and cigarettes, and is composed solely of xylose. For industrial applications, xylan is predominantly arabinoxylan, which is obtained from agricultural by-products such as bran or corn cobs, and glucuronoarabinoxylan is a softwood. Glucuronoxylan is found in hardwood. Industrially, these xylan-containing saccharides are added to arabinose, glucuronic acid, 4-O-methylglucuronic acid, glucose, and hemi-lactose in addition to xylose. Xylose and other sugars have different characteristics depending on the type of source plant.

  The composition components of commercially available xylo-oligosaccharide products are mainly xylobiose, xylotriose, and oligosaccharides with a relatively high degree of polymerization. Among them, xylobiose and xylotriose are used for probiotics in the human body. It is the most important component to activate. Industrially, in order to reduce the production cost, it is possible to induce specific microorganisms (for example, Aspergillus niger, Bacillus halodurans, etc.) that are convenient raw materials such as bran or corn cobs, and to secrete these enzymes, and then directly to the crude enzyme A raw material rich in xylo-oligosaccharides, such as corn cobs, is hydrolyzed by a catalytic reaction to give xylo-oligosaccharides.

  The aforementioned xylooligosaccharides are low polymerized sugar mixtures having different xylose numbers, and the xylooligosaccharides composed of different xylose numbers have different functionalities. In general, the culture effect of xylooligosaccharides against lactic acid bacteria and bifidobacteria is better for xylooligosaccharides with a small number of xyloses, and the utilization rate of xylooligosaccharides gradually decreases as the number of xyloses increases. According to recent studies, Lactobacillus brevis prefers xylobiose, and Bifidobacterium adolescentis prefers xylotriose and xylotetraose. Prefer (Non-Patent Document 1). Other studies have shown that the availability of Bifidobacterium-adrescentis to xylo-oligosaccharides is best for xylotriose, followed by xylotriose (2) After culturing Bifidobacterium-adrescentis for 24 hours, the total xylo-oligosaccharide utilization rate is 77%, and the utilization rate of each composition is 90% for xylotriose and 84% for xylobiose. Xylotetraose was 83% and xylopentaose was 71%. Bifidobacterium-adrescentis is an environment with xylooligosaccharides, and its reproductive rate is much higher than other Bifidobacterium B. Longum, B. infantis, and B. breve. Xylose in xylo-oligosaccharides is not as effective as xylo-oligosaccharides, but can be digested and utilized by bifilus bacteria. Furthermore, xylose is one of the eight components of glyconutrient (glyconutrient) and has antibacterial and antigonococcal functions, especially against gram-negative and white streptococci, Help the growth of internal probiotics.

  Xylan has the highest solubility in an alkaline environment, and xylanase requires that the pretreatment process carried out during application normally undergoes a high temperature process, and therefore does not lose activity in an alkaline environment, and Clearly, the ability to obtain alkali-resistant, high-temperature resistant xylanases is of utmost importance.

  For this reason, if there is a production method that uses heat-resistant and alkali-resistant xylanase and can control the xylose number composition ratio in the xylooligosaccharide, it can be used for different applications (for example, for the growth of different types of lactic acid bacteria). Can contribute greatly.

Literature name Moura et al., LWT-Food Science and Technology, 40 (2007) 963-972 Literature name Gullon al., J. Agric. Food Chem. 56 (2008) 7482-7487

  In order to solve the above-mentioned problems of the well-known technique, the object of the present invention is to provide a kind of Bacillus halodurans capable of controlling the composition of xylo-oligosaccharides, which was already September 26, 2011. The deposit is CTCCC M 2011330, which is deposited on the day with the CHINA CENTER FOR TYPE CULTURE COLLECTION (CCTCC). This alkali-resistant Bacillus halodulans can secrete two types of heat-resistant and alkali-resistant xylanases, which are xylanase xyn45 (SEQ ID NO.:1) or xylanase xyn23 (SEQ ID NO.:2). The

  The present invention provides a method for producing a xylo-oligosaccharide having a composition controllable from the aforementioned alkali-resistant Bacillus halodulans, wherein the step comprises culturing the alkali-resistant Bacillus halodulans bacterium at a high pH value using plant material, and An enzyme combination is obtained from alkaline Bacillus halodurans, and the enzyme combination includes xylanase xyn45 (SEQ ID NO.:1) and xylanase xyn23 (SEQ ID NO.:2). The enzyme combination and xylan raw material are subjected to an enzyme reaction to obtain xylo-oligosaccharide, and the xylo-oligosaccharide is composed of xylol, xylobiose, xylotriose, xylan having a polymerization degree of 4 or more and any combination thereof. Selected from the group. The plant material is selected from the group consisting of birch, corn cobs, bran, sugarcane bagasse, straw, and alkali-treated bran. The range of the high pH value is 8-11. The enzyme combination is referred to as an enzyme solution in this example, which is obtained by centrifuging the alkali-resistant Bacillus halodurans to obtain a supernatant, precipitating the supernatant with ammonium sulfate, resuspending, and A liquid obtained by dialysis. The xylan raw material is corn cobs. The conditions for the enzyme reaction are 30 to 80 degrees Celsius and pH 4 to 11. When the plant material is treated with alkali, the xylobiose ratio in the xylooligosaccharide is 32% or more. When the plant material is corn cobs, the ratio of xylobiose in the xylooligosaccharide is 27% or more. Further, when the enzyme reaction time ends within 4 hours, the ratio of xylotriose in the xylo-oligosaccharide becomes 8.6% or more.

  The xylooligosaccharide produced by the method of the present invention has the advantage that it can control the composition ratio of different xylose numbers, in addition to the advantage of helping the growth of antibacterial, antifungal and intestinal probiotics, By combining different types of lactic acid bacteria health foods and foods according to the number ratio, the maximum utilization of various xylooligosaccharide components can be achieved.

The results of protein gel electrophoresis and enzyme activity electrophoresis analysis of the secretion of alkali-resistant Bacillus halodurans under different media are shown, E and B are the cultures in the Emerson medium and Berg Mineral salt medium containing birch xylan, M represents a marker of known molecular weight. Figure 2 shows protein gel electrophoresis and enzyme activity electrophoretic analysis results of secretion of alkali-resistant Bacillus halodurans under different induction substrates, C, W and W1 are corn cobs, bran and alkali-treated bran Emerson, respectively. Medium, M represents a marker of known molecular weight. Example 1 shows the HPLC analysis spectra of water-soluble components in the substrate before the reaction of xylan and xylanase in A and the product in the solution after the reaction, A is before the xylan and xylanase reaction, B is 4 hours after the xylanase reaction, C represents 24 hours after xylanase reaction, and D represents 47 hours after xylinase reaction. FIG. 2 is a HPLC analysis spectrum of a water-soluble component in a substrate before the reaction of xylan and xylanase in Example 2 and a product in a solution after 15 hours of reaction, A is before the reaction of xylan and xylanase, and B is after 15 hours of xylanase reaction. On behalf of

  In order to describe in detail the technical contents, structural features, and objects to be achieved of the present invention, examples will be described below. The following descriptions are merely examples of the present invention, and are not intended to limit the scope of the present invention. Any equivalent changes and modifications that can be made based on the claims of the present invention are described below. Shall belong to the scope covered by the rights.

  The present invention produces xylo-oligosaccharides of different compositions by the endo-xylanases of alkali-resistant Bacillus halodurans (deposit code CCTCC M 2011330).

  The alkali-resistant Bacillus halodurans used in the present invention is obtained by screening at a wastewater treatment plant in a pulp factory, and can secrete a large amount of heat-resistant alkali-resistant xylanase, which has two types of active proteins, The locations in the SDS-PAGE analysis gel pieces are 45 kDa and 23 kDa, and are therefore referred to as xyn45 (SEQ ID NO.:1) and xyn23 (SEQ ID NO.:2), respectively, and endoxylanases (endo-xylanases), respectively. ). xyn45 and xyn23y belong to the 10th and 11th family glycosyl hydrolases, respectively. The 10th family glycosyl hydrolases are characterized by a relatively large molecular weight (> 30kD) and an acidic pI value (low pI value). Belonging to, the structure is relatively large and relaxed. The properties of the eleventh family of glycosyl hydrolases are that the molecular weight is relatively small (<30 kD), belongs to the alkaline pI value (high pI value), the structure is relatively small and close. xyn45 and xyn23 have a wide range of pH values and high temperature resistance, and are both active at 70 degrees Celsius and between pH 4-11. xyn23 has a maximum activity of 60 degrees Celsius at pH 7, but the activity at 70 degrees Celsius has 90% of the maximum activity. xyn45 has a very wide temperature range of pH 7 when the pH is 7, the range is 42-75 degrees Celsius, and the highest temperature is 70 degrees Celsius. Taken together, these two enzymes are 30-80 degrees Celsius and both have 50% or more activity and the best temperature is 72 degrees Celsius. These two types of enzymes have an activity of 60% or more at pH 4-11. When these two types of glycosyl hydrolases are used alone or in combination, both can hydrolyze xylan to form oligosaccharides with a relatively low degree of polymerization (DP), which can be used to produce xylooligosaccharides. It is an ideal xylanase capable of

  In an embodiment of the present invention, different plant materials induce xylanase secretion of alkali-resistant Bacillus halodurans, and two different xylanases xyn45 (SEQ ID NO.:1) and xylanase xyn23 (SEQ ID NO.:2) ) Enzyme combinations containing Then, it is made to act on the same xylan raw material, and the xylo-oligosaccharide from which a composition differs is caught. Xylooligosaccharides with different compositions also differ in their functionality, and in the existing technology, different from using a single enzyme as a biocatalyst, i.e., the present invention adjusts the enzyme composition with different inducers to achieve different compositions. This is a production method for obtaining xylooligosaccharides.

  The plant material that induces xylanase secretion of the alkali-resistant Bacillus halodulans is any material containing xylan, such as birch wood, corn cobs, bran, sugarcane bagasse, straw, and alkali-treated bran, preferably Birch wood, alkali treated bran, corn cobs, most preferably alkali treated bran and corn cobs.

  The culture medium of alkali-resistant Bacillus halodulans is usually composed by adding xylan to a basic culture medium such as Emerson medium or Berg's mineral salts medium. Xylan is used as an induction substrate and induces secretion of xylanase from Bacillus halodurans, and the composition of these two types of media (without xylan) is as follows.

Emerson medium consists of 0.55% Yeast extract, 0.5% peptone, 0.02% magnesium sulfate (MgSO ₄ ), 0.1% dipotassium hydrogen phosphate (K ₂ HPO ₄ ) The pH is adjusted to 10 with 1M sodium hydroxide.

Berg Mineral Salt Medium consists of 0.2% sodium nitrate (NaNO ₃ ), 0.1% yeast extract, 0.05% dipotassium hydrogen phosphate, 0.02% magnesium sulfate hydrate (MgSO ₄ · 7H ₂ O), 0.002% manganese chloride (MnCl), and 0.002% calcium chloride (CaCl ₂ ). Further, the pH value is adjusted to 10.5 with 25% sodium carbonate (Na ₂ CO ₃ ).

  2% birchwood xylan is added to these two types of culture medium. After sterilization, alkali-resistant Bacillus halodulans bacteria are cultured, and cultured in a 500 ml Erlenmeyer flask at 37 rpm in a culture box at 200 rpm for 120 hours. The supernatant obtained by centrifugation was precipitated with ammonium sulfate under 90% saturation, and the precipitate after centrifugation at 13000 rpm was re-recovered with 0.1 mM Tris-HCl buffer solution (pH 7.0). After suspending and dialyzing the obtained enzyme solution on a thin film (Cellu Sep membranes), protein gel electrophoresis (SDS-PAGE) and enzyme activity electrophoresis (zymogram) were performed, and the results are shown in FIG. Among them, E represents Emerson medium, B represents Berg Mineral salt medium, and M represents a marker.

  As can be seen from FIG. 1, the medium containing these two kinds of birch xylan induced alkali-resistant Bacillus halodulans bacteria and secreted xylanases xyn45 and xyn23 having enzyme activity. However, the protein content and enzyme activity obtained using different media are different, and when using a Berg Mineral salt medium containing birch xylan, the protein concentration is 0.5 g / L and the enzyme activity (measured at 55 degrees Celsius) is 53 U. / mL. Using an Emerson medium containing birch xylan, the protein concentration was 2.4 g / L and the enzyme activity (measured at 55 degrees Celsius) was 211 U / mL. Clearly, by using the Emerson medium, a relatively large amount of enzyme protein and xylanase activity can be obtained.

  Furthermore, based on Emerson's medium, different xylan raw materials are used, that is, 2% corn cobs, bran and alkali-treated bran are used as induction substrates, and alkali-resistant Bacillus halodurans are cultured to secrete xylanase. The results of protein gel electrophoresis and enzyme activity electrophoresis analysis of the resulting enzyme solution are shown in FIG. Among them, C, W, and W1 represent Emerson's medium containing corn cobs, bran, and alkali-treated bran, respectively, and M is a known molecular weight marker. The results show that xylanase obtained by induction of medium using corn cobs or bran is mainly xyn45, and the protein point and activity of xyn23 are both very unclear, but induced with alkali-treated bran. The xylanases xyn45 and xyn23 thus obtained are both, and the xyn23 obtained is clearer when compared with the aforementioned birch xylan medium. From these results, it was shown that the ratio of xylanase xyn45 and xyn23 secreted by alkali-resistant Bacillus halodurans was significantly different under the induction of different plant materials.

  The generation ratio of xylanases xyn45 and xyn23 secreted by alkali-resistant Bacillus halodurans under the induction of different plant materials is shown below. The ratio of xylanase xyn45 and xyn23 induced by corn cobs was 90:10. The ratio of xylanases xyn45 and xyn23 generated by the induction of birch xylan was 70:30. The ratio of xylanase xyn45 and xyn23 generated by induction with bran was 70:30. The ratio of xylanase xyn45 and xyn23 generated by induction with alkali treated bran was 50:50.

  Due to the difference in the composition ratio between the two xylanases xyn45 and xyn23 described above, the composition ratio of the xylooligosaccharide product obtained by acting on the same xylan raw material is not the same, and the xylan raw material can be any xylan-containing material such as birch, corn Cob, bran, sugarcane bagasse, straw, and alkali-treated bran, preferably bran and corn cobs, most preferably corn cobs.

  EXAMPLES The present invention will be described in more detail with reference to Examples 1 to 3 below, but these examples are for supporting the contents of the specification and are not intended to limit the scope of the present invention.

＊ＤＰ１は非キシロースの単糖（アラビノース、ブドウ糖等）を代表する。
＊＊ＤＰ＞４は重合度が４より大きいキシロオリゴ糖を代表する。
反応前の基質の水可溶成分は７．４５ ｇ／Ｌとされ、いずれも重合度が４より大きいキシロオリゴ糖成分であり、重合度が３より小さいキシロオリゴ糖の存在は無い。反応後に増加する水可溶性キシロオリゴ糖はキシラナーゼが形成する加水分解物とされ、これにより計算して得られた２４時間の転化率（キシラナーゼ反応が形成するキシロオリゴ糖生産率）は５６．４％であり、４７時間では６１．５％である。もし、反応前アルカリ処理アルカリ処理で得られる水可溶性キシロオリゴ糖を包含すると、総キシロオリゴ糖生産率は第２４時間は９３．７％とされ、反応４７時間後には９８．７％となる。
前述の総キシロオリゴ糖生産率計算方式：基質用量２％（ｗ／ｖ）は２０ｇ／Ｌに相当し、２４時間と４７時間反応の後のキシロオリゴ糖総量はそれぞれ１８．７３ｇ／Ｌと１９．７４ｇ／Ｌである。これにより、総キシロオリゴ糖生産率は反応２４時間後には１８．７３／２０×１００％＝９３．７％、反応４８時間後には１９．７４／２０×１００％＝９８．７％である。
キシラナーゼ反応の時間に伴う変化はキシロトリオースがまず生産され、その後、キシロバイオース濃度が急速に増加し、キシロトリオースは分解されてキシロバイオースとキシロースになり後期に濃度は下降する。反応２４時間の後、各オリゴ糖の比率変化は大きくなくなり、反応は緩慢となる。この結果から分かるように、比較的高い比率のキシロトリオースを得ようとすれば、反応は比較的早く（２時間から４時間）終了する必要があり、この時、転化率（キシロオリゴ糖生産率）は少し低いが、平均すると約４２．５％ある。 [Example 1A]
Alkaline-treated bran is used as a material for inducing xylanase secretion of alkali-resistant Bacillus halodurans.
Production of xylanase: First, a substrate that induces secretion of an alkali-resistant Bacillus haloduran xylanase is produced. In other words, a solid-liquid ratio of 1 to 10 was mixed with fuma and 1% sodium hydroxide, sterilized in a high-pressure kettle at 121 degrees Celsius for 10 minutes, then washed with pure water to pH 7, and surplus The bran is treated with alkali by drying. Subsequently, 100 ml of Emerson medium was placed in a 250 ml Erlenmeyer flask. The Emerson medium contains 0.55% yeast extract, 0.5% peptone, 0.02% magnesium sulfate, and 0.1% dipotassium hydrogen phosphate, and is adjusted to a pH of 10. ing. 2% of the above alkali treated bran is added and the whole mixture is sterilized in a high pressure kettle at 121 degrees Celsius for 30 minutes. The Erlenmeyer flask is cooled to room temperature, then inoculated with 10% (v / v) alkali-resistant Bacillus halodurans, and cultured in a 37 ° C. culture box at 170 rpm for 4-5 days. Thereafter, the bacterial solution is centrifuged at 13000 rpm at 4 degrees Celsius for 30 minutes, the supernatant is precipitated with ammonium sulfate under 90% saturation, and the precipitate after centrifugation at 13000 rpm is 0.1 mM Tris-HCl. Resuspend in buffer solution (pH 7.0) to make crude enzyme solution after dialysis. The activity of the crude enzyme solution in a unit volume was 179 U / mL (measured at 37 degrees Celsius).
Xylan production: Corn cob is ground and treated with 15% sodium hydroxide (NaOH) at a solid-liquid ratio of 1:20 at 90 degrees Celsius for 90 minutes, and the resulting supernatant is acetic acid The solution is neutralized with (acetic acid) to adjust the pH to 5.0, and then 3 times the volume of 95% ethanol is added and immersed for 60 minutes, and the resulting precipitate is xylan. Using this xylan as a substrate, under the action of xylanase obtained as described above, xylan is decomposed to generate xylooligosaccharides.
Conditions for producing xylo-oligosaccharides by reaction of xylanase: substrate dose 2% (w / v), enzyme dose 8.95 U / mL, reaction temperature 50 degrees Celsius, pH 8.0. The obtained product was analyzed by HPLC (High-performance liquid chromatography). Among them, the HPLC instrument structure is as follows. The column is a BioRad column Aminex HPX-87H (300 mm × 7.8 mm), the column temperature is 65 degrees Celsius, and the detector is a refractive index detector (RI detector). The mobile phase is 5 mM sulfuric acid (H ₂ SO ₄ ), and the flow rate is 0.6 ml / min.
FIG. 3 shows HPLC analysis spectra of the water-soluble component in the substrate before the xylan and xylanase reaction and the product in the solution after the reaction. In the figure, the spectra of A to D are the reaction time of 4 hours before the reaction of xylan and xylanase, respectively. , Representing the spectrum of 24 hours reaction, 47 hours reaction.
Changes in product composition over time are shown in the table below.

* DP1 represents a non-xylose monosaccharide (arabinose, glucose, etc.).
** DP> 4 represents xylo-oligosaccharides with a degree of polymerization greater than 4.
The water-soluble component of the substrate before the reaction is 7.45 g / L, all of which are xylo-oligosaccharide components having a degree of polymerization of greater than 4, and no xylo-oligosaccharide having a degree of polymerization of less than 3. The water-soluble xylo-oligosaccharide that increases after the reaction is regarded as a hydrolyzate formed by xylanase, and the conversion rate obtained by this calculation (xylo-oligosaccharide production rate formed by xylanase reaction) is 56.4%. , 47 hours, 61.5%. If the water-soluble xylo-oligosaccharide obtained by alkali treatment before the reaction is included, the total xylo-oligosaccharide production rate is 93.7% in the 24th hour and 98.7% after 47 hours of the reaction.
Total xylooligosaccharide production rate calculation method as described above: The substrate dose of 2% (w / v) corresponds to 20 g / L, and the total amount of xylooligosaccharide after reaction for 24 hours and 47 hours is 18.73 g / L and 19.74 g, respectively. / L. Thus, the total xylooligosaccharide production rate is 18.73 / 20 × 100% = 93.7% after 24 hours of reaction and 19.74 / 20 × 100% = 98.7% after 48 hours of reaction.
The change with time of the xylanase reaction is that xylotriose is produced first, then the xylobiose concentration increases rapidly, xylotriose is degraded to xylobiose and xylose, and the concentration decreases later. After 24 hours of reaction, the ratio change of each oligosaccharide does not become large and the reaction becomes slow. As can be seen from this result, if a relatively high ratio of xylotriose is to be obtained, the reaction must be completed relatively quickly (2 to 4 hours). At this time, the conversion rate (xylo-oligosaccharide production rate) ) Is a little low, but on average it is about 42.5%.

＊ＤＰ１は非キシロースの単糖（アラビノース、ブドウ糖等）を代表する。
＊＊ＤＰ＞４は重合度が４より大きいキシロオリゴ糖を代表する。
実施例一Ａと比較すると、キシラナーゼ用量は８．９５Ｕ／ｍＬから１７．５Ｕ／ｍＬにアップする。これにより反応速度は比較的速くなり、反応１５時間後に実施例一Ａの反応２４時間後の結果に相当する結果が得られた。最終製品は重合度が４より小さいキシロオリゴ糖中、キシロバイオースが絶大な部分（３２％以上）を占め、キシロース及びその他の単糖を合わせてもただ５％しかなく、キシロトリオースは５〜６％の間である。 [Example 1B]
A concentration test is performed with the enzyme solution of Example 1A.
Production of xylanase and xylan is the same as Example A.
Conditions for producing xylooligosaccharides by xylanase reaction: substrate dose is 2% (w / v), enzyme dose is 17.5 U / mL, reaction temperature is 50 degrees Celsius, pH 8.0. The obtained product was analyzed by HPLC (High-performance liquid chromatography).
After 15 hours of reaction, the product composition (concentration and percentage) is as follows:

* DP1 represents a non-xylose monosaccharide (arabinose, glucose, etc.).
** DP> 4 represents xylo-oligosaccharides with a degree of polymerization greater than 4.
Compared to Example 1A, the xylanase dose is increased from 8.95 U / mL to 17.5 U / mL. As a result, the reaction rate was relatively high, and a result corresponding to the result after 24 hours of the reaction of Example 1A was obtained after 15 hours of reaction. The final product is composed of xylooligosaccharides with a degree of polymerization of less than 4 and xylobioses accounted for the largest part (over 32%), and only 5% of xylose and other monosaccharides combined. Between 6%.

第２次実験

＊ＤＰ１は非キシロースの単糖（アラビノース、ブドウ糖等）を代表する。
＊＊ＤＰ＞４は重合度が４より大きいキシロオリゴ糖を代表する。
本実施例から分かるように、トウモロコシの穂軸で培養した耐アルカリ性バチルスハロデュランス菌より得られる酵素液でキシランを分解して得られるキシロオリゴ糖成分は、キシロバイオース比率が比較的低く（約２７％）、キシロース及びその他の単糖を合わせると比較的高い約９％であり、キシロトリオースは約７％を維持する。 [Example 2]
Corn cob is used as a material for inducing xylanase secretion of alkali-resistant Bacillus halodurans.
Preparation of xylanase: Place 100 ml of Emerson medium in a 250 ml Erlenmeyer flask. The Emerson medium contains 0.55% yeast extract, 0.5% peptone, 0.02% magnesium sulfate, and 0.1% dipotassium hydrogen phosphate, and is adjusted to a pH of 10. The 2% of the above corn cob powder is added and the entire mixture is sterilized in a high pressure kettle at 121 degrees Celsius for 30 minutes. After the Erlenmeyer flask is cooled to room temperature, it is inoculated with 10% (v / v) alkali-resistant Bacillus halodurans, and cultured in a culture box at 37 degrees Celsius at 170 rpm for 4-5 days. Thereafter, the bacterial solution is centrifuged at 13000 rpm at 4 degrees Celsius for 30 minutes, the supernatant is precipitated with ammonium sulfate under 90% saturation, and the precipitate after centrifugation at 13000 rpm is 0.1 mM Tris- Resuspend in HCl buffer solution (pH 7.0) to make crude enzyme solution after dialysis. The activity of the crude enzyme solution in a unit volume was 128 U / mL (measured at 37 degrees Celsius).
The production of xylan is the same as in Example 1A.
Conditions for producing xylo-oligosaccharides by reaction of xylanase: substrate dose 2% (w / v), xylanase dose 17.5 U / mL, reaction temperature 50 degrees Celsius, pH 8.0. The obtained product was analyzed by HPLC (High-performance liquid chromatography).
FIG. 4 is a HPLC analysis spectrum of the water-soluble component in the substrate before the xylan and xylanase reaction and the product in the solution after the reaction for 15 hours. In the figure, A is before the reaction of xylan and xylanase, and B is the xylanase reaction for 15 hours. It is a later spectrum.
After 15 hours of reaction, the product composition (concentration and percentage) is as follows:
First experiment

Second experiment

* DP1 represents a non-xylose monosaccharide (arabinose, glucose, etc.).
** DP> 4 represents xylo-oligosaccharides with a degree of polymerization greater than 4.
As can be seen from this example, the xylo-oligosaccharide component obtained by decomposing xylan with an enzyme solution obtained from an alkali-resistant Bacillus halodulans cultured on corn cobs has a relatively low xylobiose ratio (about 27 %), Xylose and other monosaccharides together are about 9% which is relatively high and xylotriose maintains about 7%.

＊ＤＰ１は非キシロースの単糖（アラビノース、ブドウ糖等）を代表する。
＊＊ＤＰ＞４は重合度が４より大きいキシロオリゴ糖を代表する。
実施例一Ｂと比較すると、ｐＨ値は高くなり反応速度は遅くなり、これにより転化率は下降する。アルカリ処理したフスマで耐アルカリ性バチルスハロデュランス菌を培養して得られる酵素液は、単糖（主にはキシロース）比率が比較的低いが、キシロバイオースのキシロトリオースに対する比率は高ｐＨ値での反応時に僅かに下降する。本実施例は酵素はアルカリ条件下でもなおキシロオリゴ糖製造の能力を有することを示す。
実施例一Ａから分かるように、アルカリ処理したフスマをキシラナーゼ発生を誘導する植物材料となす時、時間の増加（０〜４７時間）に伴い、キシロースとキシロバイオースの比率は増加し、それぞれ１．５９〜４．３９％及び２１．７９〜３５．６５％であり、キシロトリオースの比率は、２時間後の９．５８から４７時間後の５．３１％に漸減する。実施例二でトウモロコシの穂軸をキシラナーゼ発生を誘導する植物材料となし、実施例一Ｂ中のその他の条件を同じにした結果と比較すると、トウモロコシの穂軸を使用して発生するキシロースの比率は７．１０％と比較的高く、アルカリ処理したフスマではただ３．６２％である。トウモロコシの穂軸を使用して発生するキシロバイオースの比率は比較的低く２７．４７％で、アルカリ処理したフスマは３２．２４％である。ゆえに、必要により適当な材料を選択することで、必要なキシロース数のキシロオリゴ糖組成比率を達成でき、並びに実施例三の結果により、酵素はアルカリ性環境下で反応できることを示す。 [Example 3]
The enzyme solution of Example 1 is used to test the alkali resistance of xylanase.
Enzyme production and xylan production are the same as in Example 1.
Conditions for producing xylo-oligosaccharides by enzymatic reaction: substrate dose 2% w / v, enzyme dose 17.9 U / mL, reaction temperature 50 degrees Celsius, pH values are 9, 10 and 11, respectively. The resulting product is analyzed by HPLC.
After 15 hours of reaction, the product composition (concentration and percentage) is as follows:

* DP1 represents a non-xylose monosaccharide (arabinose, glucose, etc.).
** DP> 4 represents xylo-oligosaccharides with a degree of polymerization greater than 4.
Compared to Example 1B, the pH value becomes higher and the reaction rate becomes slower, thereby lowering the conversion rate. The enzyme solution obtained by culturing alkali-resistant Bacillus halodulans with alkali-treated bran has a relatively low monosaccharide (mainly xylose) ratio, but the ratio of xylobiose to xylotriose has a high pH value. Slightly lowers during the reaction. This example shows that the enzyme is still capable of producing xylo-oligosaccharides under alkaline conditions.
As can be seen from Example 1A, when the alkali-treated bran is used as a plant material that induces the generation of xylanase, the ratio of xylose and xylobiose increases with increasing time (0 to 47 hours). .59-4.39% and 21.79-35.65%, with the xylotriose ratio gradually decreasing from 9.58 after 2 hours to 5.31% after 47 hours. The ratio of xylose generated using corn cobs when compared with the results obtained in Example 2 where the corn cobs are plant materials that induce xylanase generation and the other conditions in Example 1B are the same. Is relatively high at 7.10% and only 3.62% for alkali treated bran. The proportion of xylobiose generated using corn cobs is relatively low at 27.47% and alkali treated bran is 32.24%. Therefore, by selecting an appropriate material as necessary, the composition ratio of xylooligosaccharide having the required number of xylose can be achieved, and the result of Example 3 shows that the enzyme can react in an alkaline environment.

Claims (12)

  Alkali-resistant Bacillus halodurans capable of controlling the composition of the xylooligosaccharide having the deposit code CCTCC M 2011330.   2. The alkali-resistant Bacillus halodulans according to claim 1, wherein the alkali-resistant Bacillus halodulans can secrete two types of heat-resistant and alkali-resistant xylanases, the heat-resistant and alkali-resistant xylanases are xylanase xyn45 (SEQ ID NO.:1) and xylanase An alkali-resistant Bacillus halodulans bacterium characterized in that xyn23 (SEQ ID NO .: 2) is used. The method for producing a xylo-oligosaccharide whose composition can be controlled with an alkali-resistant Bacillus halodulans bacterium according to claim 1, wherein the step comprises:
(1) culturing an alkali-resistant Bacillus halodulans bacterium under a high pH value with a plant material;
(2) obtaining an enzyme combination comprising xylanase xyn45 (SEQ ID NO.:1) and xylanase xyn23 (SEQ ID NO.:2) from the alkali-resistant Bacillus halodurans,
(3) a step of causing an enzyme reaction between the enzyme combination and a xylan raw material;
(4) obtaining a xylooligosaccharide selected from the group consisting of xylose, xylobiose, xylotriose, xylan having a polymerization degree of 4 or more, and any combination thereof,
A method characterized by comprising.   4. The method according to claim 3, wherein the plant material in step (1) is selected from the group consisting of birch wood, corn cob, bran, sugarcane bagasse, straw, and alkali-treated bran. Method.   The method according to claim 3, wherein the range of the high pH value in the step (1) is 8-11.   4. The method of claim 3, wherein the enzyme combination of step (2) comprises centrifuging the alkali-resistant Bacillus halodurans to obtain a supernatant, resuspending the supernatant after precipitation with ammonium sulfate, and A method characterized by being a liquid obtained by dialysis.   4. The method according to claim 3, wherein the xylan raw material in step (3) is selected from the group consisting of birch, corn cob, bran, sugarcane bagasse, straw, and alkali-treated bran.   The method according to claim 3 or 7, wherein the xylan raw material in step (3) is corn cobs.   4. The method according to claim 3, wherein the conditions of the enzyme reaction in the step (3) are 30 to 80 degrees Celsius and pH 4 to 11.   4. The method according to claim 3, wherein when the plant material is an alkali-treated bran, the ratio of xylobiose in the xylooligosaccharide is 32% or more.   4. The method according to claim 3, wherein when the plant material is corn cobs, the ratio of xylobiose in the xylooligosaccharide is 27% or more.   The method according to claim 10, wherein when the time of the enzymatic reaction is finished within 4 hours, the ratio of xylotriose in the xylo-oligosaccharide is 8.6% or more.

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