WO2013053071A1

WO2013053071A1 - Production method for preparing high purity mannan oligosaccharide from enzymolysis of hemicellulose

Info

Publication number: WO2013053071A1
Application number: PCT/CN2011/001686
Authority: WO
Inventors: 黄代勇; 钱金宏
Original assignee: Huang Daiyong; Qian Jimhowell
Priority date: 2011-10-10
Filing date: 2011-10-10
Publication date: 2013-04-18

Abstract

A method for preparing a high purity mannan oligosaccharide from enzymolysis of a hemicellulose having a mannan oligosaccharide, also a product. By using a high-efficiency β-mannanase, BM-II, produced from fermentation of Bacillus subtilis strain TQBm having the deposit number of CCTCC No. M211147, Amorphophallus konjac gum, guar gum, locust bean gum, and Sesbania gum are hydrolyzed into relatively small molecules, then are separated by centrifugation, clarified by flocculation, desalinated, decolorized, and deodorized by using an ion exchange technology, then large molecules in a sugar solution are separated by using combinedly a super filtration technology and a molecular sieve chromatography technology, and finally the high purity mannan oligosaccharide is prepared via concentration and drying. The high purity mannan oligosaccharide acquired via the preparation is of low costs, free of chemical contamination, allows for implementation of industrialized production, significantly increases beneficial bacteria such as human intestinal bifidobacteria, significantly reduces harmful bacteria such as Enterobacter, also provides the effects of reductions in blood sugar and in blood lipids and of immunomodulation, and is a raw material and additive of a medicament for gastrointestinal tract health, for sugar reduction and lipids reduction, for immunomodulation, and for disease treatment and disease prevention.

Description

说明书 Description

醇解半纤维素制备高纯度甘露低聚糖的生产方法技术领域 Method for producing high-purity mannose oligosaccharides by alcoholysis hemicellulose

本发明涉及酶技术和生物化工技术，更确切说是一种酶解含甘露聚糖的半纤维素生成纯度高达 98% (P98)的甘露低聚糖的制备方法及产品，是胃肠道健康、免疫调节、抗病防病药物的新型原料和助剂。背景技术 The invention relates to an enzyme technology and a biochemical technology, and more specifically to a method and a product for preparing a mannan oligosaccharide having a purity of up to 98% (P98) by enzymatic hydrolysis of a mannan-containing hemicellulose, which is a gastrointestinal health , immune regulators, new raw materials and additives for disease-resistant and disease-preventing drugs. Background technique

半纤维素 (Hemi cellulose)是指在植物细胞壁中与纤维素共生、可溶于碱溶液，遇酸后远较纤维素易于水解的那部分植物多糖。半纤维素主要分为三类，即聚木糖类、聚葡萄甘露糖类和聚半乳糖葡萄甘露糖类。本发明所涉及到的半纤维素是聚葡萄甘露糖类 (代表产品是魔芋胶）和聚半乳甘露糖类（代表产品是瓜尔豆胶)，统称甘露聚糖类。 Hemi cellulose refers to the part of plant polysaccharide that is symbiotic with cellulose in the cell wall of plants, soluble in alkaline solution, and is much easier to hydrolyze than cellulose after acid. Hemicellulose is mainly classified into three types, namely, polyxylose, polyglucomannan, and polygalactose grape mannose. The hemicelluloses to which the present invention relates are polyglucomannans (representing products are konjac gum) and polygalactomannoses (representing products are guar gum), collectively referred to as mannans.

半纤维素中的甘露聚糖经酶水解生成低碳链甘露糖杂聚物。已有的文献表明，低碳链甘露糖杂聚物（也称甘露低聚糖或甘露寡糖），能显著增进人体肠道内以双歧杆菌为代表的有益菌的增殖、减少动物肠道病原菌，还能增强免疫力、提高肠薪膜功能等多种特性。最近的报道是，黄代勇等（酶法生产高纯度 P95甘露低聚糖中试研究，食品科技， 2007, 10)，李剑芳等（β _甘露聚糖酶制备魔芋葡甘露低聚糖的研究，食品与发酵工业， 2007， 01 )，张敏等（野皂荚多糖胶制备半乳甘露低聚糖的研究，食品工业斜技， 2008， 09，），王玲玲等（酶解法制备低相对分子质量胡芦巴半乳甘露聚糖及其产物的表征，林产化学与工业， 2009， 29卷（2)，吴长菲等（魔芋葡甘露低聚糖的酶法制备工艺的初步研究，生物技术通报， 2010, 01 )。 The mannan in hemicellulose is enzymatically hydrolyzed to form a low carbon chain mannose heteropolymer. The literature has shown that low-carbon chain mannose heteropolymers (also known as mannose oligosaccharides or mannooligosaccharides) can significantly increase the proliferation of beneficial bacteria represented by bifidobacteria in the human intestinal tract and reduce intestinal pathogens in animals. It can also enhance immunity, improve the function of the intestinal salvage membrane and so on. Recent reports are: Huang Daiyong et al. (Enzymatic production of high-purity P95 mannose oligosaccharides pilot study, Food Science and Technology, 2007, 10), Li Jianfang et al (β-mannanase preparation of konjac glucomannan oligosaccharides, food And Fermentation Industry, 2007, 01), Zhang Min et al. (Study on preparation of galactomannan oligosaccharides from wild saponin polysaccharides, Food Industry, 2008, 09,), Wang Lingling et al. (Enzymatic hydrolysis of low molecular weight cucurbits Characterization of galactomannan and its products, Forest Products Chemistry and Industry, 2009, Vol. 29 (2), Wu Changfei et al. (Preliminary study on enzymatic preparation of konjac glucomannan oligosaccharides, Biotechnology Bulletin, 2010, 01 ).

迄今为止，有关低碳链甘露糖杂聚物的制备方法研究中，或是存在酶活力不高，底物浓度低，成本高，缺乏工业生产的基础；或是水解过程中加碱、加酸，给后处理工艺带来困难，还不利于环保；或是产品的纯度较低，普遍仅为 70〜80%，虽然黄代勇等（2007) 的研究成果中，魔芋甘露低聚糖的纯度达到了 95%，但其糖分子聚合度大至 3〜10糖，由于功能作用的局限性，还只能应用于食品和化工产品中，要应用到医药产品中还有一定的差距。特别是半乳甘露低聚糖研究水平还很低，离工业化还有很大的差距。因此，甘露低聚糖的原料多样性及高纯度制备方法仍然面临很多的技术难题。而这也是本发明创新技术的起点。发明内容 So far, in the research on the preparation method of low-carbon chain mannose heteropolymer, the enzyme activity is not high, the substrate concentration is low, the cost is high, and the basis of industrial production is lacking; or the alkali and acid are added during the hydrolysis process. It brings difficulties to the post-treatment process and is not conducive to environmental protection; or the purity of the product is low, generally only 70~80%. Although the results of Huang Daiyong et al. (2007), the purity of konjac mannose oligosaccharide has reached 95%, but its sugar molecule polymerization degree is as large as 3~10 sugar. Due to the limitation of function, it can only be applied to food and chemical products. There is still a certain gap to be applied to pharmaceutical products. In particular, the research level of galactomannan oligosaccharides is still very low, and there is still a big gap from industrialization. Therefore, Gan The raw material diversity and high-purity preparation methods of oligosaccharides still face many technical problems. This is also the starting point for the innovative technology of the present invention. Summary of the invention

本发明的目的是提供一种以含甘露聚糖的半纤维素为原料，采用保藏号为 CCTCC No : M 211147的枯草芽孢杆菌 TQBm发酵产出的高效能 e -甘露聚糖 S技术、食品生产技术、精细化工技术之***集成生产纯度高达 98% ( P98 ), 分子聚合度为 4〜8糖的甘露低聚糖的方法及产品，该产品可作为胃肠道健康、免疫调节、抗病防病药物的新型原料和助剂。 The object of the present invention is to provide a high-efficiency e-mannan S technology and food production produced by fermentation of Bacillus subtilis TQBm with the preservation number CCTCC No : M 211147 using hemicellulose containing mannan as raw material. The system and technology of fine chemical technology integrate the production method and product of mannose oligosaccharide with purity of up to 98% (P98) and molecular polymerization degree of 4~8 sugar. The product can be used as gastrointestinal health, immune regulation and disease resistance. New raw materials and additives for disease drugs.

本发明是这样实现的，其工艺过程为： The present invention is achieved in such a manner that the process is:

1、酶解转化。加入保藏号为 CCTCC No : Μ 2Π147的枯草芽抱杆菌 TQBm发酵产出的高效能 β -甘露聚糖酶，底物为魔芋胶时，酶解转化的底物浓度为 15〜20%，酶料比为酶：魔芋胶 =lml_: 30g_{; ;} 底物为瓜尔豆胶时，解转化的底物浓度为 10〜15%，酶料比为酶：瓜尔豆胶 =lm 15go ; 降解温度 48〜52'C，搅拌 40〜60r/min， pH 6.5〜7.5, 降解时间： 3〜4小时，降解粘度至 80〜100 mPa*s时停止酶解转化，降解原液半纤维素中甘露聚糖的生物转化率为 85〜95%。 1. Enzymatic hydrolysis. Add the high-performance β-mannanase produced by the fermentation of Bacillus subtilis TQBm with the accession number CCTCC No : Μ 2Π147. When the substrate is konjac, the substrate concentration of the enzymatically transformed substrate is 15~20%. The ratio is enzyme: konjac gum = lml _: 30g _{; when} the substrate is guar gum, the concentration of the substrate to be converted is 10~15%, the ratio of enzyme to material is: guar gum = lm 15go; degradation temperature 48 ~52'C, stirring 40~60r/min, pH 6.5~7.5, degradation time: 3~4 hours, stop enzymatic hydrolysis when degrading viscosity to 80~100 mPa*s, degrading mannan in hemicellulose The bioconversion rate is 85 to 95%.

2、离心分离，将酶解物离心去杂； 2. Centrifugal separation, centrifuging the enzymatic hydrolysate;

3、絮凝、澄清、过滤处理； 3. Flocculation, clarification, filtration treatment;

4、离子交换处理； 4. Ion exchange treatment;

5、超滤和分子筛纯化处理； 5. Ultrafiltration and molecular sieve purification treatment;

6、真空浓缩处理； 6. Vacuum concentration treatment;

7、喷雾干燥处理，产出甘露低聚糖成品。 7. Spray drying treatment to produce finished mannose oligosaccharides.

8、利用液相色谱和质谱对甘露低聚糖进行检测：总糖： 99.5%；功效成份甘露 2 糖〜甘露 10糖： 98.0 %, 其中，甘露 4糖〜甘露 8糖占 85%以上。 8. Detection of mannose oligosaccharides by liquid chromatography and mass spectrometry: total sugar: 99.5%; functional ingredient mannose 2 sugar ~ mannose 10 sugar: 98.0%, wherein, mannose 4 sugar ~ nectar 8 sugar accounted for more than 85%.

本发明采用保藏号为 CCTCC No : M 211147的枯草芽孢杆菌 TQBm发酵产出的高效能 e -甘露聚糖酶技术和食品生产技术、精细化工技术之***集成制备源于魔芋胶和瓜尔豆胶的 P98甘露低聚糖，工艺简单，能耗低，操作方便，产品纯度高，成本可控，无化学污染。 The invention adopts the high-efficiency e-mannanase technology produced by the fermentation of Bacillus subtilis TQBm with the accession number CCTCC No : M 211147, and the system integration of food production technology and fine chemical technology is prepared from konjac gum and guar gum. The P98 mannose oligosaccharide has simple process, low energy consumption, convenient operation, high product purity, controllable cost and no chemical pollution.

本发明所用甘露聚糖的半纤维素包括魔芋胶、瓜尔豆胶、槐豆胶、田菁胶、皂荚胶、胡芦巴胶等。本发明所生产出的产品纯度高，功效成份甘露 4糖〜甘露 8糖占的比重大，具有在同类产品中可唯一应用于医药产品的优势，将大大启发人们拓展低聚糖高端应用范围的新思路，新创造、新前景。具体实施方式 The hemicellulose of the mannan used in the present invention includes konjac gum, guar gum, locust bean gum, celery gum, acacia gum, fenugreek gum and the like. The product produced by the invention has high purity, and the functional ingredient mannose 4 sugar ~ mannose 8 sugar accounts for a large proportion, and has the advantage of being uniquely applied to medical products in the same kind of products, and will greatly inspire people to expand the high-end application range of oligosaccharides. New ideas, new creations, new prospects. detailed description

下面以实施例对本发明进一步说明。 The invention is further illustrated by the following examples.

实施例 1: Example 1:

1、酶解； 1. Enzymatic hydrolysis;

a)加水：在 1000L酶解罐中，按有效容量 700kg计，魔芋胶：水 =20:80 (w/w), 加水 560kg于降解罐中，水的 pH在 6.5〜7.5之间。 a) Add water: In the 1000L hydrolysis tank, according to the effective capacity of 700kg, konjac: water = 20:80 (w / w), add water 560kg in the degradation tank, the pH of the water is between 6.5 ~ 7.5.

b)预热：酶解罐夹套通入蒸汽或热水把罐内的水预热至 50〜52Ό。 b) Preheating: The enzymatic tank jacket is steamed or hot water to preheat the water in the tank to 50~52Ό.

c)加酶：待降解水温稳定后，按酶与底物之比为 1 : 20〜30加入 β -甘露聚糖酶 ΒΜ— II , 并不断搅拌，使其混合均匀，保温 5〜10 min后投料。 c) Add enzyme: After the temperature of the degraded water is stable, add β-mannanase ΒΜ-II according to the ratio of enzyme to substrate 1: 20~30, and stir constantly to make it mix evenly, after 5~10 min incubation Feeding.

d)加料：称取魔芋胶 140kg加入酶解罐中，底物浓度 15〜20%，并充分搅拌。 d) Feeding: Weigh 140 kg of konjac gum into the enzymolysis tank, the substrate concentration is 15~20%, and stir well.

e)酶解：罐内温度保持在 50〜55°C 之间，不断搅拌，酶解 3〜4 小时。在酶解过程中用 NDJ— 1型旋转粘度计，以 12rpm的转速检测 50Ό 时酶解液的粘度，当酶解液粘度为 80〜100 mPa*s时，停止降解，该液体称为 "酶解液原液"。 e) Enzymatic hydrolysis: The temperature in the tank is kept between 50~55 °C, stirring constantly, and enzymatic hydrolysis for 3~4 hours. In the enzymatic hydrolysis process, the viscosity of the enzymatic hydrolysate at 50 Torr was measured with a NDJ-1 type rotational viscometer at 12 rpm. When the viscosity of the enzymatic hydrolysate was 80 to 100 mPa*s, the degradation was stopped. The liquid was called "enzyme." Dissolve stock solution".

2、用离心机过滤除渣得初滤液； 2. Filter the slag to obtain the initial filtrate by using a centrifuge;

3、采用硅藻土过滤和 0. 2 μ m微滤膜进行微滤处理得微滤液； 3, using diatomaceous earth filtration and 0. 2 μ m microfiltration membrane for microfiltration to obtain a microfiltrate;

4、用孔径为截留 3万分子量的聚矾超滤膜作超滤提纯,工作条件为:温度 25~35'C，压力 0.1〜0.12 Mpa，物料流速 300〜350I7h。 4. The ultrafiltration membrane with a pore size of 30,000 molecular weight is used for ultrafiltration purification. The working conditions are: temperature 25~35'C, pressure 0.1~0.12 Mpa, material flow rate 300~350I7h.

[0037] 5、用孔径为截留 6千分子量的聚飒超滤膜作进一步超滤提纯，工作条件为：温度 25~35Γ，压力 0.1〜0.12MPa，物料流速 300〜350L/h。 [0037] 5. Using a polyfluorene ultrafiltration membrane with a pore size of 6,000 molecular weight for further ultrafiltration purification, the working conditions are: temperature 25~35Γ, pressure 0.1~0.12MPa, material flow rate 300~350L/h.

6、采用 D315 大孔树脂一 732 阳离子交换树脂一 717 阴离子交换树脂的树脂组合对降解液进行脱盐、脱色、脱味的提纯处理，取样检测色值和灰分，脱色率为 97%，灰分 0.1%，得离子交换提纯液； 6. Using D315 macroporous resin-732 cation exchange resin-717 anion exchange resin resin combination to demineralize, decolorize and deodorize the degradation solution, sample and detect color value and ash, decolorization rate is 97%, ash 0.1% , obtained ion exchange purification liquid;

7、将离子交换提纯液经真空浓缩、喷雾干燥后得到 P98甘露聚糖 58.3kg，糖粉得率为 41.64% (相对于魔芋胶)。利用液相色谱一质谱联用检测 P98魔芋甘露低聚糖，总糖： ^99.5%,功效成份（甘露 2糖〜甘露 10糖） 98.0%，其中，甘露 4糖〜甘露 8糖占 85% 以上。 7. The ion exchange purification solution was concentrated in a vacuum and spray-dried to obtain 58.3 kg of P98 mannan, and the yield of the sugar powder was 41.64% (relative to konjac gum). Liquid chromatography-mass spectrometry was used to detect P98 konjac mannose oligosaccharides, total sugar: ^99.5%, efficacy component (mannose 2 sugar ~ mannose 10 sugar) 98.0%, of which mannose 4 sugar ~ nectar 8 sugar accounted for more than 85% .

实施例 2: 1、酶解； Example 2: 1. Enzymatic hydrolysis;

a)加水：在 1000L酶解罐中，按有效容量 700kg计，瓜尔豆胶：水 =15:85 (w/w), 加水 595kg于酶解罐中，水的 pH在 6.5〜7.5之间。 a) Add water: In the 1000L enzymatic hydrolysis tank, according to the effective capacity of 700kg, guar gum: water = 15:85 (w / w), add 595kg of water in the enzymatic hydrolysis tank, the pH of the water is between 6.5 ~ 7.5 .

b)预热：酶解罐夹套通入蒸汽或热水把罐内的水预热至 50〜52°C。 b) Preheating: The enzymatic tank jacket is steamed or hot water to preheat the water in the tank to 50~52 °C.

c )加酶：待温度稳定在要求的温度时，按酶与底物之比为 1 : 10〜15加入 -甘露聚糖酶 BM— II。并不断搅拌，使其混合均匀，保温 5〜10分钟后投料。 c) Adding enzyme: When the temperature is stable at the required temperature, the ratio of enzyme to substrate is 1 : 10~15 to add -mannanase BM-II. Stir constantly, mix it evenly, and keep it for 5~10 minutes.

d)加料：称取瓜尔豆胶 105kg加入酶解罐中（底物浓度 15%)，充分搅拌。 d) Feeding: Weigh 105kg of guar gum into the enzymatic hydrolysis tank (substrate concentration 15%), and stir well.

e)酶解：罐内温度保持在 50〜55°C 之间，不断搅泮，酶解 3〜4 小时。在酶解过程中用 NDJ— 1型旋转粘度计，以 12rpm的转速检测 50 时酶解液的粘度，当酶解液粘度为 80〜100 mP_a，s时，停止酶解，该液体称为 "酶解液原液"。 e) Enzymatic hydrolysis: The temperature in the tank is kept between 50 and 55 ° C, and the mixture is continuously stirred and digested for 3 to 4 hours. In the enzymatic hydrolysis process, the viscosity of the enzymatic hydrolysate was measured at a rotation speed of 12 rpm using an NDJ-1 type rotational viscometer. When the viscosity of the enzymatic hydrolyzate was 80 to 100 mP _a , s, the enzymatic hydrolysis was stopped. "Enzymatic hydrolysate stock solution".

以下离心分离、过滤、微滤、超滤提纯、树脂处理提纯、浓缩干燥等工序与实施例 1相同。 The following procedures of centrifugation, filtration, microfiltration, ultrafiltration purification, resin treatment purification, concentration and drying are the same as in the first embodiment.

制备出 P98甘露低聚糖成品 47. 72kg，糖粉得率为 45.45% (相对于瓜尔豆胶）。利用液相色谱一质谱联用检测 P98瓜尔豆胶甘露低聚糖，总糖： 99.5%，功效成份（甘露 2糖〜甘露 10糖） 98%，其中，甘露 4糖〜甘露 8糖占 85%以上。 The finished P98 mannose oligosaccharide was prepared 47. 72kg, and the sugar powder yield was 45.45% (relative to guar gum). Determination of P98 guar gum mannose oligosaccharide by liquid chromatography-mass spectrometry, total sugar: 99.5%, efficacy component (mannose 2 sugar ~ mannose 10 sugar) 98%, of which mannose 4 sugar ~ nectar 8 sugar accounted for 85 %the above.

实施例 3: Example 3:

以槐豆胶，田菁胶，皂荚胶，胡芦巴胶为原料，应用 β -甘露聚糖酶 BM— II制备 P98 甘露低聚糖的方法与实施例 2相同。 The method for preparing P98 mannose oligosaccharide using the β-mannanase BM-II was the same as that of Example 2, using locust bean gum, celery gum, acacia gum and fenugreek gum as raw materials.

功能试验结果 Functional test result

Ρ98甘露低聚糖〈调节人体肠道菌群的功能试验〉 Ρ98 Mannan oligosaccharide <Functional test for regulating human intestinal flora>

体检指标全部正常的 30名成年志愿者试食 Ρ98甘露低糖后的检测结果如下：细菌肠杆菌肠球菌拟杆菌产气荚双杆杆菌乳杆菌内容膜梭菌 The test results of 30 adult volunteers with normal physical examination indicators were as follows: Bacteria Enterobacteriaceae Enterococci Bacteroides Producing a bacterium Abacillus brevis Lactobacillus Content Clostridium

食用前 7.60±0.11 6.03±0.25 8.01±0.08 1.23±0.11 7.11±0.15 6.84±0.17 食品后 7.22±0.11** 6.43±0.22 8.28±0.08* 1.06±0.05 8.88±0.11 ** 8.36±0.16**Before consumption 7.60±0.11 6.03±0.25 8.01±0.08 1.23±0.11 7.11±0.15 6.84±0.17 After food 7.22±0.11** 6.43±0.22 8.28±0.08* 1.06±0.05 8.88±0.11 ** 8.36±0.16**

Ρ值 0.002 0.167 0.028 0.183 0.000 0.000 Depreciation 0.002 0.167 0.028 0.183 0.000 0.000

*与食用前比较 Ρ<0.05 **与食用前比较 Ρ<0.01 *Compared with before consumption Ρ<0.05 **Compared with before consumption Ρ<0.01

结论：受试人群肠道内的菌群数量发生有益的变化：粪便中双歧杆菌、乳杆菌的数量明显增加，差异有极显著性（ Ρ<0.01 )，拟杆菌的数量明显增加，差异有显著性（ Ρ<0.05 Conclusion: There are beneficial changes in the number of bacteria in the intestine of the test population: The number of bifidobacteria and lactobacilli in the feces is significantly increased, the difference is extremely significant (Ρ<0.01), and the number of Bacteroides is significantly increased, the difference is significant. Sex (Ρ<0.05

)，肠杆菌数量明显减少，差异有极显著性（ Ρ<0.01 )。 The number of Enterobacteriaceae was significantly reduced, and the difference was extremely significant (Ρ<0.01).

Ρ98甘露低聚糖〈调节人体血糖血脂功能试验〉项目对照组 P98甘露低聚糖组血糖 (mol/L) 8.98 ± 0.53 6.10 ± 0.94Ρ98 Mannan oligosaccharide <Modulation of blood sugar and blood lipid function test> Blood glucose (mol/L) of P98 mannose oligosaccharide group in the control group 8.98 ± 0.53 6.10 ± 0.94

TG (总甘油三酯） 2.78 ± 0.28 1.14 ± 0.11 TG (total triglyceride) 2.78 ± 0.28 1.14 ± 0.11

TC (总胆固醇） 3.22 ± 0.39 1.98 ± 0.24 TC (total cholesterol) 3.22 ± 0.39 1.98 ± 0.24

HDL-C (高密度脂蛋白胆固醇） 1.41 ± 0.18 1.88 ± 0.21 HDL-C (High Density Lipoprotein Cholesterol) 1.41 ± 0.18 1.88 ± 0.21

SOD (超氧化物歧化酶， ku/L) 115.10 ± 8.49 174.20 ± 10.81 SOD (superoxide dismutase, ku/L) 115.10 ± 8.49 174.20 ± 10.81

LP0 (过氧化脂质， mol/L) 4.32 ± 1.08 2.53 ± 1.03 结论： P98甘露低聚糖有调节血糖血脂的作用较明显。 LP0 (lipid peroxide, mol/L) 4.32 ± 1.08 2.53 ± 1.03 Conclusion: P98 mannose oligosaccharide has a significant effect on regulating blood sugar and blood lipids.

P98甘露低聚糖〈调节人体免疫功能试验〉 P98 Mannan Oligosaccharide <Modulation of Human Immune Function Test>

P98甘露低聚糖聚对体液免疫功能的影响

Effect of P98 Mannan Oligosaccharide on the Immune Function of Body Fluid

*与空白组比较 P<0.05, 表明中高剂量组可显著升高血清溶血素含量。 * P < 0.05 compared with the blank group, indicating that the medium and high dose group can significantly increase the serum hemolysin content.

P98甘露低聚糖对单核一巨噬细胞功能的影响 Effect of P98 Mannan Oligosaccharide on the Function of Mononuclear-Macrophages

*与空白组比较 P<0.05,表明中高剂量组能显著提高吞噬率、吞噬指数和碳廓清指数。 P98甘露低聚糖对 NK细胞活性的影响

* P < 0.05 compared with the blank group, indicating that the middle and high dose groups can significantly increase the phagocytosis rate, phagocytic index and carbon clearance index. Effect of P98 Mannan Oligosaccharide on NK Cell Activity

*与空白组比较 P<0.05，表明中高剂量组能显著增强 NK细胞活性 * Compared with the blank group, P < 0.05, indicating that the medium and high dose group can significantly enhance NK cell activity.

结论： P98甘露低聚糖有调节免疫功能的作用。 Conclusion: P98 mannose oligosaccharide has the function of regulating immune function.

Claims

权利要求书 Claim

1、酶解半纤维素制备高纯度甘露低聚糖的生产方法，其特征在于其工艺过程为： 1 ) 、酶解转化：使用保藏号为 CCTCC No: M 211147芽孢杆菌 TQBra发酵产出的 e - 甘露聚糖酶来酶解含甘露聚糖的半纤维素，底物浓度 10〜20%，酶底比为 l:15〜30(v/w)_; 降解温度 48〜52Ό，搅拌 40〜60r/min， pH 6.5〜7.5，降解时间： 3〜5 小时，降解粘度至 80〜100 mPa-s时停止酶解转化，降解原液半纤维素中甘露聚糖的生物转化率为 85〜 95%；所述含甘露聚糖的半纤维素是指：魔芋胶，瓜尔豆胶，槐豆胶，田菁胶，皂荚胶，胡芦巴胶； 1. A method for producing high-purity mannose oligosaccharides by enzymatic hydrolysis of hemicellulose, characterized in that the process is: 1), enzymatic hydrolysis conversion: e-produced by the fermentation number of CCCCC No: M 211147 Bacillus TQBra - Mannanase to digest the hemicellulose containing mannan, the substrate concentration is 10~20%, the enzyme bottom ratio is 1:15~30 (v/w) _{; the} degradation temperature is 48~52Ό, stirring 40~60r /min, pH 6.5~7.5, degradation time: 3~5 hours, the enzymatic hydrolysis is stopped when the viscosity is degraded to 80~100 mPa-s, and the bioconversion rate of mannan in the degraded liquid hemicellulose is 85~95%; The mannan-containing hemicellulose refers to: konjac gum, guar gum, locust bean gum, celery gum, acacia gum, fenugreek gum;

2)、离心分离，将酶解物离心去杂； 2), centrifuging, centrifuging the enzymatic hydrolysate;

3)、絮凝、澄清、过滤处理； 3), flocculation, clarification, filtration treatment;

4)、离子交换处理； 4), ion exchange treatment;

5)、超滤和分子筛纯化处理； 5), ultrafiltration and molecular sieve purification treatment;

6)、真空浓缩处理； 6), vacuum concentration treatment;

7)、喷雾干燥处理，产出甘露低聚糖成品，经检测：总糖： 99.5%；功效成份甘露 2糖〜甘露 10糖 98.0%，其中，甘露 4糖甘露 8糖占 85%以上。 7), spray drying treatment, the production of mannose oligosaccharide products, after testing: total sugar: 99.5%; efficacy ingredients nectar 2 sugar ~ nectar 10 sugar 98.0%, of which, nectar 4 sugar mannose 8 sugar accounted for more than 85%.

2.根据权利要求 1所述的酶解半纤维素制备高纯度甘露低聚糖的生产方法，其特征在于酶解反应的最佳条件为：底物浓度 15〜20%，酶底比为 l:15〜30(v/w)， pH 7.0, 温度 50°C，时间 3小时，降解原液中半纤维素酶解成甘露聚糖的生物转化率为 85〜95%。 2 . The method for producing high-purity mannose oligosaccharide according to claim 1 , wherein the optimal conditions for the enzymatic hydrolysis reaction are: a substrate concentration of 15 to 20%, and an enzyme bottom ratio of 1 : 15~30 (v/w), pH 7.0, temperature 50 ° C, time 3 hours, the bioconversion rate of hemicellulose hydrolyzed into mannan in the degradation solution was 85~95%.