WO2022062453A1 - 一种高产丁酸的淀粉基膳食纤维及其加工方法 - Google Patents

一种高产丁酸的淀粉基膳食纤维及其加工方法 Download PDF

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WO2022062453A1
WO2022062453A1 PCT/CN2021/096726 CN2021096726W WO2022062453A1 WO 2022062453 A1 WO2022062453 A1 WO 2022062453A1 CN 2021096726 W CN2021096726 W CN 2021096726W WO 2022062453 A1 WO2022062453 A1 WO 2022062453A1
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starch
dietary fiber
butyrate
yield
based dietary
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French (fr)
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缪铭
杨玉琪
赵新琦
张涛
金征宇
季万兰
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江南大学
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
    • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/716—Glucans
    • A61K31/718—Starch or degraded starch, e.g. amylose, amylopectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/10—Laxatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/12—Antidiarrhoeals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B30/00—Preparation of starch, degraded or non-chemically modified starch, amylose, or amylopectin
    • C08B30/12—Degraded, destructured or non-chemically modified starch, e.g. mechanically, enzymatically or by irradiation; Bleaching of starch
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the invention relates to a high-yield butyric acid starch-based dietary fiber and a processing method thereof, belonging to the technical field of modern food nutrition food processing.
  • Starch as the most abundant carbohydrate resource in my country, has a wide range of sources and low price. Using starch as raw material to synthesize starch-based dietary fiber with certain anti-digestive properties is of great significance to the development of the food industry. Compared with starch, starch-based dietary fiber still retains a large amount of undigested parts under the action of various digestive enzymes in the gastrointestinal tract, and has low blood sugar levels, which is beneficial to prevent the prevalence of various chronic diseases.
  • the undigested part is fermented under the action of intestinal microorganisms to produce a large amount of short-chain fatty acids, of which butyric acid can regulate the growth of beneficial intestinal bacteria, maintain the integrity of the colonic mucosa, and reduce diseases such as cancer and ulcerative colon.
  • diseases such as inflammation can effectively maintain the body's intestinal health.
  • starch-based dietary fiber is easier to add to food, it has received widespread attention.
  • the existing starch-based dietary fibers are mainly extracted from crops such as soybean dregs, wheat bran, Jerusalem artichoke and their processing by-products by mechanical methods, chemical methods, enzymatic methods and mixed methods.
  • the existing enzymatic method mainly uses protease, amylase, cellulase, etc. to extract from crops and processing by-products, and the starch-based dietary fiber extracted by the above method not only has low yield, low purity, and poor safety, but also has a preparation process. Cumbersome, high cost, unable to cope with the increasing social demands. Based on the above reasons, it is of great significance for the development of the food industry to provide a convenient, efficient and safe preparation method of high-yield butyrate starch-based dietary fiber.
  • the present invention provides a convenient, efficient and safe preparation method of high-yield butyrate starch-based dietary fiber.
  • the method provided by the invention solves the problems of cumbersome extraction process, low yield, poor safety and high cost of the current starch-based dietary fiber, and its application range can involve functional food, medicine and other fields.
  • epidemiological studies have shown that starch-based dietary fiber As a kind of prebiotic, it can be used alone or in combination with probiotics to play a variety of health care functions, which can reduce intestinal diarrhea, constipation, obesity, and diabetes; at the same time, starch-based dietary fiber can prevent colon cancer , to a certain extent, it treats chronic diseases, and has the reputation of "gut scavenger".
  • the present invention is achieved through the following technical solutions: a method for processing high-yield butyric acid starch-based dietary fiber, using starch as a substrate, and the method specifically comprises the following steps:
  • the heating in step (1) is preferably heating in a boiling water bath.
  • the ⁇ -amylase is a mesophilic commercial ⁇ -amylase;
  • the glycosyltransferase is a transglycosidase derived from a microorganism of the GH70 family, and the ⁇ -amylase
  • the ratio of enzymatic activity to glycosyltransferase is 1:10-5:1.
  • the GH70 family microorganisms include GH70 family fungi and bacteria.
  • the glycosyltransferase is a multifunctional transglycosidase derived from the GH70 family such as Lactobacillus reuteri, Lactobacillus fermentum, Streptococcus thermophilus, and Azotobacter pyogenes.
  • the glycosyltransferase is capable of enzymatically degrading the products of ⁇ -amylase enzymatically degrading starch to generate ⁇ -1,4/6 and ⁇ -1,6-glucose- ⁇ -1 ,4 structure of dietary fiber.
  • the starch is any one of corn starch, potato starch, tapioca starch, rice starch, wheat starch, etc., or ordinary starch, waxy starch, high amylose starch, Any of starch dextrins.
  • step (1) a certain quality of starch is weighed to prepare starch milk with a concentration of 10-30wt%, which means that starch is obtained by dispersing the starch in a buffered saline solution (pH 6.5-7.0). suspension.
  • the buffered saline comprises acetic acid-sodium acetate buffer, disodium hydrogen phosphate-citric acid buffer, citric acid-sodium citrate buffer, potassium dihydrogen phosphate-sodium hydroxide Buffer, Tris-HCl buffer, etc.
  • the membrane molecular weight cut-off of the enzyme-membrane coupling reactor is 10-60 kDa.
  • the "substrate" in the 1-2.5 U/g substrate refers to the mass of starch.
  • the second object of the present invention is to provide the high-yield butyrate starch-based dietary fiber prepared by the above-mentioned processing method.
  • the molecular weight of the high-yielding butyrate-based starch-based dietary fiber is 0.5 ⁇ 10 7 -9.5 ⁇ 10 7 Da.
  • the content of ⁇ -1,6 glycosidic bonds in the high-yielding butyrate starch-based dietary fiber is higher than 20%.
  • the third object of the present invention is to provide a food and a food additive comprising the above-mentioned high-yielding butyrate-based starch-based dietary fiber.
  • the fourth object of the present invention is to provide the above-mentioned processing method of the high-yield butyrate starch-based dietary fiber and the application of the high-yield butyrate starch-based dietary fiber in the fields of food and medicine.
  • the present invention utilizes multifunctional carbohydrase synergistic catalysis-membrane filtration fractionation coupling technology to obtain starch-based dietary fiber with high yield of butyric acid.
  • the method has simple steps, mild reaction conditions, high safety, and realizes continuous and low-cost green production. , and make full use of my country's rich starch resources to improve the quality of modern food.
  • the product of the present invention has outstanding performance of high-yield butyric acid by enteric fermentation, and has more remarkable health-care function, and truly meets the demands of consumers for modern functional food.
  • the starch-based dietary fiber obtained by the invention is not easy to be digested in the stomach and small intestine of the human body, and is easily fermented by microorganisms in the large intestine to produce short-chain fatty acids, especially butyric acid, and the production of butyric acid by enteric fermentation is higher than that of the original substrate. More than 2 times, can regulate human intestinal health.
  • the starch-based dietary fiber prepared by the present invention can be used as a food additive for the processing of various foods, greatly improving the value of the product, and at the same time, it is of great significance for improving people's health level, so it has higher social and economic benefits. effect.
  • Figure 1 1 H NMR chart of the starch-based dietary fiber prepared in Example 1.
  • starch-based dietary fiber was prepared into an aqueous solution of 5 mg/mL, and its molecular weight distribution was measured by HPSEC-MALL-RI system.
  • 1 H NMR test Weigh 25 mg of starch-based dietary fiber into a clean 1.5 mL centrifuge tube, add 0.5 mL of heavy water to dissolve, and then freeze-dried for 24 hours. This was repeated twice to perform heavy water exchange. The sample was dissolved in 0.5 mL of deuterated water, and analyzed by 1 H NMR using a nuclear magnetic resonance spectrometer.
  • starch-based dietary fiber structure Accurately weigh starch-based dietary fiber and dissolve it in 25mM acetic acid-sodium acetate buffer (pH 4.0), configure it into a 2.5mg/mL reaction solution, and add 0.5U of enzyme combination (I : isoamylase, isoprulanase and beta-amylase; II: isoamylase; III: isoamylase and beta-amylase), water bath at 40°C for 24h. Subsequently, the degree of polymerization of the hydrolyzate in the above reaction solution was determined by HPAEC-PAD.
  • enzyme combination I : isoamylase, isoprulanase and beta-amylase
  • II isoamylase
  • III isoamylase and beta-amylase
  • Determination method of butyric acid produced by anaerobic fermentation in vitro use starch-based dietary fiber or starch as carbon source to prepare anaerobic fermentation medium, add fecal diluent according to 10% of the additive amount, and then place it in a 37 °C incubator for anaerobic fermentation During fermentation, samples were taken at different fermentation times to measure the content of butyric acid in the fermentation broth by gas chromatography.
  • Corn starch, corn starch dextrin, high amylose corn starch, potato starch and wheat starch were purchased from Shanghai National Starch Company.
  • Alpha-amylase was purchased from Sigma; Glycosyltransferase, see the literature Microbial Starch-Converting Enzymes: Recent Insights and Perspectives, Comprehensive Reviews in Food Science and Food Safety, 2018, 17(5): 1238-1260, including enzyme gene mining , engineering bacteria construction, fermentation enzymes and other steps.
  • the purity of starch-based dietary fiber in this example is 98.3%, the molecular weight is 1.1 ⁇ 10 7 Da, and the content of ⁇ -1,6 glycosidic bonds is 33%; the content of butyric acid produced by simulated anaerobic fermentation in vitro is the original substrate corn starch 2.7 times.
  • the starch-based dietary fiber in this example has a purity of 96.5%, and the measured molecular weight is 0.6 ⁇ 10 7 Da; the content of ⁇ -1,6 glycosidic bonds is 24%; the content of butyric acid produced by simulated anaerobic fermentation in vitro is the original substrate corn starch 3.1 times that of dextrin.
  • the purity of starch-based dietary fiber in this example is 99.1%, the molecular weight is 2.7 ⁇ 10 7 Da; the content of ⁇ -1,6 glycosidic bonds is 30%; the content of butyric acid produced by simulated anaerobic fermentation in vitro is the original substrate high linear corn 2.7 times that of starch.
  • the starch-based dietary fiber in this example has a purity of 97.2%, and the measured molecular weight is 4.6 ⁇ 10 7 Da; the content of ⁇ -1,6 glycosidic bonds is 25%; the content of butyric acid produced by simulated anaerobic fermentation in vitro is the original substrate wheat starch 2.5 times.
  • the purity of starch-based dietary fiber in this example is 97.8%, the molecular weight is 4.0 ⁇ 10 7 Da; the content of ⁇ -1,6 glycosidic bonds is 29%; the content of butyric acid produced by simulated anaerobic fermentation in vitro is the original substrate potato starch 3.0 times.
  • the purity of starch-based dietary fiber in this example is 99.1%, the molecular weight is 2.7 ⁇ 10 7 Da; the content of ⁇ -1,6 glycosidic bonds is 26%; the content of butyric acid produced by simulated anaerobic fermentation in vitro is the original substrate potato starch 2.8 times.
  • the purity of starch-based dietary fiber in this example is 98.3%, the molecular weight is 1.6 ⁇ 10 7 Da; the content of ⁇ -1,6 glycosidic bonds is 21%; the content of butyric acid produced by simulated anaerobic fermentation in vitro is the original substrate potato starch 2.2 times.
  • the method of the invention processes cereals, tuberous roots, bean starch and ordinary starch, high amylose starch and starch dextrin of various types of starch, and all can obtain molecular weights of 0.5 ⁇ 10 7 -9.5 ⁇ 10 7 Da, ⁇ -1, 6.
  • the prepared starch-based dietary fiber has a purity of 92.2%, a molecular weight of 9.0 ⁇ 10 6 Da; the content of ⁇ -1,6 glycosidic bonds is 18%; the content of butyric acid produced by simulated anaerobic fermentation in vitro is the same as that of the original substrate corn starch. 1.6 times.
  • the prepared starch-based dietary fiber has a purity of 90.5%, a molecular weight of 1.5 ⁇ 10 8 Da; the content of ⁇ -1,6 glycosidic bonds is 14%; the content of butyric acid produced by simulated anaerobic fermentation in vitro is the same as that of the original substrate wheat starch. 1.1 times.
  • the prepared starch-based dietary fiber has a purity of 85.7%, a molecular weight of 2.9 ⁇ 10 8 Da; the content of ⁇ -1,6 glycosidic bonds is 11%; the content of butyric acid produced by simulated anaerobic fermentation in vitro is the same as that of the original substrate potato starch. 0.9 times.
  • Example 1 the consumption of ⁇ -amylase and glycosyltransferase was replaced by 80U and 900U respectively from 400U to prepare corresponding functional sugar products.
  • the performance results of the obtained functional sugar products are shown in Table 1.

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Abstract

一种高产丁酸的淀粉基膳食纤维及其加工方法，以淀粉为原料，通过α-淀粉酶和糖基转移酶组成的复合酶协同催化-膜滤分级藕联技术重构淀粉链的键接方式，制备得到了高产丁酸淀粉基膳食纤维。所合成的淀粉基膳食纤维不仅具有高溶解度低粘度，而且易于被肠道微生物发酵高产丁酸，从而具有较高的营养品质。所得产品应用范围可涉及到功能食品、医药等领域。

Description

一种高产丁酸的淀粉基膳食纤维及其加工方法 技术领域
本发明涉及一种高产丁酸的淀粉基膳食纤维及其加工方法，属于现代食品营养食品加工技术领域。
背景技术
随着我国经济的飞速发展，中国居民的生活水平得到了大幅度提高。由于粗粮口感粗糙，使得我国居民的饮食习惯开始逐渐偏离以植物性食物为主的传统饮食模式，饮食结构日趋西方化。因此，我国居民的疾病谱也逐渐西方化，肥胖症、糖尿病和心血管疾病越来越普遍，高血压和II型糖尿病的患病率甚至已经达到并赶超美国。因此，研发新型淀粉基膳食纤维对于提高人民身体健康水平、加强慢性病防治工作、降低疾病负担、提高居民健康具有重要作用。
淀粉作为我国最丰富的碳水化合物资源，来源广泛、价格低廉。以淀粉为原料，合成具备一定抗消化性能的淀粉基膳食纤维对食品工业的发展具有重要意义。与淀粉相比，淀粉基膳食纤维在胃肠道多种消化酶的作用下仍保留大量的未消化部分，具有低血糖应达水平，有利于预防多种慢性疾病的患病率。此外，未消化的部分在进入大肠后在肠道微生物的作用下发酵产生大量短链脂肪酸，其中丁酸可以调节肠道有益菌的生长、维持结肠粘膜的完整性、降低诸如癌症和溃疡性结肠炎等疾病的患病率，可以有效地维持机体肠道健康。又因为淀粉基膳食纤维更容易添加到食品中，因而受到广泛关注。然而对于现有的淀粉基膳食纤维的生产主要是通过机械法、化学法、酶法以及混合法从豆渣、小麦麸皮、菊芋等农作物及其加工副产物中进行提取。例如，现有的酶法主要是采用蛋白酶、淀粉酶、纤维素酶等从农作物及加工副产物中进行提取，上述方法提取的淀粉基膳食纤维不仅产量低、纯度低、安全性差，而且制备工艺繁琐、成本高，无法应对日益增大的社会需求。基于上述原因，提供一种便捷、高效、安全的高产丁酸淀粉基膳食纤维的制备方法对食品工业的发展具有重要意义。
发明内容
为解决上述问题，本发明提供了一种便捷、高效、安全的高产丁酸淀粉基膳食纤维的制备方法。本发明提供的方法解决了目前淀粉基膳食纤维提取工艺繁琐、产量低、安全性差、成本高等难题，其应用范围可涉及到功能性食品、医药等领域，如流行病学研究表明淀粉基膳食纤作为益生元的一种，即可单独使用，也可以同益生菌联合使用，起到多种保健功能，可以降低肠道腹泻、便秘、身体肥胖、糖尿病；同时，淀粉基膳食纤维能够预防结肠癌，在 一定程度上治疗慢性疾病，具有“肠道清道夫”的美誉。
具体的，本发明通过如下技术方案实现：一种高产丁酸淀粉基膳食纤维的加工方法，以淀粉为底物，所述方法具体包括如下步骤：
(1)称取一定质量淀粉配成浓度10-30wt％的淀粉乳，置于70℃以上加热至完全糊化；
(2)待温度降至30-55℃，将淀粉糊溶液转移至酶-膜藕联反应器，按1-2.5U/g底物的加酶量添加α-淀粉酶和糖基转移酶组成的复合酶液并保温反应12-36h；
(3)收集膜滤液并灭酶活、离心处理，将所获得上清液进行干燥即得高产丁酸淀粉基膳食纤维。
在本发明的一种实施方式中，步骤(1)的加热优选为沸水浴加热。
在本发明的一种实施方式中，所述α-淀粉酶为中温型的商业化α-淀粉酶；所述糖基转移酶为来源于GH70家族微生物的转苷酶，所述α-淀粉酶和糖基转移酶的酶活比例为1:10-5:1。
在本发明的一种实施方式中，所述GH70家族微生物包括GH70家族真菌和细菌。
在本发明的一种实施方式中，所述糖基转移酶为来源于罗伊氏乳杆菌、发酵乳杆菌、嗜热链球菌、褐球固氮菌等GH70家族的多功能转苷酶。
在本发明的一种实施方式中，所述糖基转移酶能够酶解α-淀粉酶酶解淀粉后的产物生成α-1,4/6以及α-1,6-葡糖-α-1,4结构的膳食纤维。
在本发明的一种实施方式中，所述淀粉为玉米淀粉、马铃薯淀粉、木薯淀粉、稻米淀粉、小麦淀粉等中的任意一种，或是上述淀粉的普通淀粉、蜡质淀粉、高直链淀粉、淀粉糊精中的任意一种。
在本发明的一种实施方式中，步骤(1)中，称取一定质量淀粉配成浓度10-30wt％的淀粉乳，是指将淀粉分散于缓冲盐液(pH 6.5-7.0)后得到淀粉悬浮液。
在本发明的一种实施方式中，所述缓冲盐液包括醋酸-醋酸钠缓冲液、磷酸氢二钠-柠檬酸缓冲液、柠檬酸-柠檬酸钠缓冲液、磷酸二氢钾-氢氧化钠缓冲液、Tris-盐酸缓冲液等。
在本发明的一种实施方式中，所述酶-膜偶联反应器的膜截留分子量为10-60kDa。
在本发明的一种实施方式中，所述1-2.5U/g底物中“底物”指的是淀粉质量。
本发明的第二个目的是提供上述加工方法制备得到的高产丁酸淀粉基膳食纤维。
在本发明的一种实施方式中，所述高产丁酸淀粉基膳食纤维的分子量为0.5×10 7-9.5×10 7Da。
在本发明的一种实施方式中，所述高产丁酸淀粉基膳食纤维中的α-1,6糖苷键含量高于20％。
本发明的第三个目的是提供包含上述高产丁酸淀粉基膳食纤维的食品以及食品添加剂。
本发明的第四个目的是提供上述高产丁酸淀粉基膳食纤维的加工方法以及高产丁酸淀粉基膳食纤维在食品、医药领域的应用。
本发明具有以下优点：
(1)本发明利用多功能糖酶协同催化-膜滤分级藕联技术获得高产丁酸的淀粉基膳食纤维，方法步骤简便，反应条件温和、安全性高，实现了连续化和低成本绿色生产，且充分利用我国丰富的淀粉类资源，提升了现代食品的品质。
(2)本发明产物肠道发酵高产丁酸性能突出，保健功能更显著，真正满足广大消费者对现代功能食品的诉求。本发明所得的淀粉基膳食纤维在人体的胃部和小肠中不易被消化，在大肠中易于被微生物发酵产生短链脂肪酸，尤其是丁酸，肠道发酵产丁酸相比原底物提高了2倍以上，能够调节人体肠道健康。
(3)本发明制备得到的淀粉基膳食纤维可作为食品添加剂用于多种食品的加工，大大提升产品的价值，同时对提高人民健康水平也具有重要意义，因而具有较高的社会效益和经济效应。
附图说明
图1实施例1制备得到的淀粉基膳食纤维的 1H NMR图。
具体实施方式
测试方法：
分子量测定：将淀粉基膳食纤维配置成5mg/mL的水溶液，用HPSEC-MALL-RI系统测定其分子量分布。
1H NMR测试：称取25mg的淀粉基膳食纤维于洁净的1.5mL离心管中，加0.5mL的重水溶解，再冻干24h。如此重复2次，进行重水交换。再用0.5mL的重水将样品溶解，利用核磁共振谱仪进行 1H NMR分析。
淀粉基膳食纤维结构测定：准确称取淀粉基膳食纤维溶解于25mM的醋酸-醋酸钠缓冲液(pH 4.0)，配置成2.5mg/mL的反应液，分别向其中加入0.5U的酶组合(Ⅰ：异淀粉酶，异普鲁兰酶和β-淀粉酶；Ⅱ：异淀粉酶；Ⅲ：异淀粉酶和β-淀粉酶)，40℃水浴24h。随后用HPAEC-PAD测定上述反应液中水解产物的聚合度。
体外厌氧发酵产丁酸测定方法：以淀粉基膳食纤维或淀粉为碳源配制厌氧发酵培养基，按10％的添加量加入粪便稀释液，随后置于37℃的培养箱中进行厌氧发酵，在不同发酵时间取样用气相色谱GC测发酵液中丁酸含量。
玉米淀粉、玉米淀粉糊精、高直链玉米淀粉、马铃薯淀粉、小麦淀粉购自上海国民淀粉公司。
α-淀粉酶购自Sigma公司；糖基转移酶见文献Microbial Starch-Converting Enzymes:Recent Insights and Perspectives，Comprehensive Reviews in Food Science and Food Safety，2018,17(5)：1238-1260，包括酶基因挖掘、工程菌构建、发酵产酶等步骤。
下面结合实施例对本发明作进一步的描述，但本发明的实施方式不限于此。
实施例1
将200g玉米淀粉分散于醋酸-醋酸钠缓冲液(25mM,pH 6.5)得到20wt％的淀粉悬浮液，沸水浴至完全糊化。待温度降至50℃，转入酶膜反应器(膜截留尺寸10kDa)，同时加入400U的α-淀粉酶和糖基转移酶(二者酶活比例为5:1)复合酶液并保温反应36h，收集膜滤液并加热灭酶活、离心处理，将所获得上清液进行干燥即得淀粉基膳食纤维。
本实施例淀粉基膳食纤维纯度达98.3％，经测定分子量为1.1×10 7Da，α-1,6糖苷键含量为33％；在体外模拟厌氧发酵产丁酸含量是原底物玉米淀粉的2.7倍。
实施例2
将300g玉米淀粉糊精分散于醋酸-醋酸钠缓冲液(25mM,pH 6.5)得到30wt％的淀粉悬浮液，沸水浴糊化。待温度降至35℃，转入酶膜反应器(膜截留尺寸10kDa)，同时加入300U的α-淀粉酶和糖基转移酶(二者酶活比例1:1)复合酶液并保温反应24h，收集膜滤液并加热灭酶活、离心处理，将所获得上清液进行干燥即得淀粉基膳食纤维。
本实施例淀粉基膳食纤维纯度达96.5％，经测定分子量为0.6×10 7Da；α-1,6糖苷键含量为24％；在体外模拟厌氧发酵产丁酸含量是原底物玉米淀粉糊精的3.1倍。
实施例3
将200g高直链玉米淀粉分散于醋酸-醋酸钠缓冲液(25mM,pH 6.5)得到30wt％的淀粉悬浮液，沸水浴糊化。待温度降至40℃，转入酶膜反应器(膜截留尺寸10kDa)，同时加入500U的α-淀粉酶和糖基转移酶(二者酶活比例5:1)复合酶液并保温反应12h，收集膜滤液并加热灭酶活、离心处理，将所获得上清液进行干燥即得淀粉基膳食纤维。
本实施例淀粉基膳食纤维纯度为99.1％，经测定分子量为2.7×10 7Da；α-1,6糖苷键含量为30％；在体外模拟厌氧发酵产丁酸含量是原底物高直链玉米淀粉的2.7倍。
实施例4
将200g小麦淀粉分散于醋酸-醋酸钠缓冲液(25mM,pH 6.5)得到20wt％的淀粉悬浮液，沸水浴至完全糊化。待温度降至50℃，转入酶膜反应器(膜截留尺寸50kDa)，同时加入400U 的α-淀粉酶和糖基转移酶(二者酶活比例为5:1)复合酶液并保温反应36h，收集膜滤液并加热灭酶活、离心处理，将所获得上清液进行干燥即得淀粉基膳食纤维。
本实施例淀粉基膳食纤维纯度达97.2％，经测定分子量为4.6×10 7Da；α-1,6糖苷键含量为25％；在体外模拟厌氧发酵产丁酸含量是原底物小麦淀粉的2.5倍。
实施例5
将200g马铃薯淀粉分散于醋酸-醋酸钠缓冲液(25mM,pH 6.5)得到20wt％的淀粉悬浮液，沸水浴至完全糊化。待温度降至50℃，转入酶膜反应器(膜截留尺寸50kDa)，同时加入400U的α-淀粉酶和糖基转移酶(二者酶活比例为5:1)复合酶液并保温反应36h，收集膜滤液并加热灭酶活、离心处理，将所获得上清液进行干燥即得淀粉基膳食纤维。
本实施例淀粉基膳食纤维纯度为97.8％，经测定分子量为4.0×10 7Da；α-1,6糖苷键含量为29％；在体外模拟厌氧发酵产丁酸含量是原底物马铃薯淀粉的3.0倍。
实施例6
将200g稻米淀粉分散于醋酸-醋酸钠缓冲液(25mM,pH 6.5)得到20wt％的淀粉悬浮液，沸水浴至完全糊化。待温度降至50℃，转入酶膜反应器(膜截留尺寸50kDa)，同时加入400U的α-淀粉酶和糖基转移酶(二者酶活比例为5:1)复合酶液并保温反应36h，收集膜滤液并加热灭酶活、离心处理，将所获得上清液进行干燥即得淀粉基膳食纤维。
本实施例淀粉基膳食纤维纯度为99.1％，经测定分子量为2.7×10 7Da；α-1,6糖苷键含量为26％；在体外模拟厌氧发酵产丁酸含量是原底物马铃薯淀粉的2.8倍。
实施例7
将200g木薯淀粉分散于醋酸-醋酸钠缓冲液(25mM,pH 6.5)得到20wt％的淀粉悬浮液，沸水浴至完全糊化。待温度降至50℃，转入酶膜反应器(膜截留尺寸50kDa)，同时加入400U的α-淀粉酶和糖基转移酶(二者酶活比例为5:1)复合酶液并保温反应36h，收集膜滤液并加热灭酶活、离心处理，将所获得上清液进行干燥即得淀粉基膳食纤维。
本实施例淀粉基膳食纤维纯度为98.3％，经测定分子量为1.6×10 7Da；α-1,6糖苷键含量为21％；在体外模拟厌氧发酵产丁酸含量是原底物马铃薯淀粉的2.2倍。
本发明的方法对禾谷、块根、豆类淀粉以及各类淀粉的普通淀粉、高直链淀粉、淀粉糊精进行处理，均能获得分子量为0.5×10 7-9.5×10 7Da、α-1,6糖苷键含量高于20％、肠道发酵产丁酸相比原底物提高2.0倍以上的淀粉基膳食纤维。
对比例1
将200g玉米淀粉分散于醋酸-醋酸钠缓冲液(25mM,pH 6.5)得到20wt％的淀粉悬浮液， 沸水浴至完全糊化。待温度降至50℃，转入酶膜反应器(膜截留尺寸10kDa)，同时加入400U的糖基转移酶酶液并保温反应36h，收集膜滤液并加热灭酶活、离心处理，将所获得上清液进行干燥即得淀粉基膳食纤维。
经测定，制备得到淀粉基膳食纤维纯度92.2％，分子量为9.0×10 6Da；α-1,6糖苷键含量为18％；在体外模拟厌氧发酵产丁酸含量是原底物玉米淀粉的1.6倍。
对比例2
将200g小麦淀粉分散于醋酸-醋酸钠缓冲液(25mM,pH 6.5)得到20wt％的淀粉悬浮液，沸水浴至完全糊化。待温度降至50℃，转入酶膜反应器(膜截留尺寸10kDa)，同时加入400U的糖基转移酶酶液并保温反应36h，收集膜滤液并加热灭酶活、离心处理，将所获得上清液进行干燥即得淀粉基膳食纤维。
经测定，制备得到淀粉基膳食纤维纯度90.5％，分子量为1.5×10 8Da；α-1,6糖苷键含量为14％；在体外模拟厌氧发酵产丁酸含量是原底物小麦淀粉的1.1倍。
对比例3
将200g马铃薯淀粉分散于醋酸-醋酸钠缓冲液(25mM,pH 6.5)得到20wt％的淀粉悬浮液，沸水浴至完全糊化。待温度降至50℃，转入酶膜反应器(膜截留尺寸50kDa)，同时加入400U的糖基转移酶酶液并保温反应36h，收集膜滤液并加热灭酶活、离心处理，将所获得上清液进行干燥即得淀粉基膳食纤维。
经测定，制备得到淀粉基膳食纤维纯度85.7％，分子量为2.9×10 8Da；α-1,6糖苷键含量为11％；在体外模拟厌氧发酵产丁酸含量是原底物马铃薯淀粉的0.9倍。
对比例4
参照实施例1，将α-淀粉酶和糖基转移酶的用量由400U分别替换为80U、900U，制得相应的功能糖产品。所得功能糖产品的性能结果见表1。
表1 不同多功能酶系用量所得膳食纤维功效结果
用量(U/g) 发酵产丁酸增加倍数
80 0.7
900 1.1
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。

Claims (11)

  1. 一种高产丁酸淀粉基膳食纤维的加工方法，其特征在于，所述方法具体包括如下步骤：
    (1)称取一定质量淀粉配成浓度10-30wt％的淀粉乳，置于70℃以上加热至完全糊化；
    (2)待温度降至30-55℃，将淀粉糊溶液转移至酶-膜偶联反应器，按1-2.5U/g底物的加酶量添加α-淀粉酶和糖基转移酶组成的复合酶液并保温反应12-36h；
    (3)收集膜滤液并灭酶活、离心处理，将所获得上清液进行干燥即得高产丁酸淀粉基膳食纤维。
  2. 根据权利要求1所述的一种高产丁酸淀粉基膳食纤维的加工方法，其特征在于，所述α-淀粉酶为中温型的α-淀粉酶；所述糖基转移酶为来源于GH70家族微生物的转苷酶，其中，所述α-淀粉酶和糖基转移酶的酶活比例为1:10-5:1。
  3. 根据权利要求2所述的一种高产丁酸淀粉基膳食纤维的加工方法，其特征在于，所述GH70家族微生物包括GH70家族真菌和细菌。
  4. 根据权利要求2所述的一种高产丁酸淀粉基膳食纤维的加工方法，其特征在于，所述糖基转移酶能够酶解α-淀粉酶酶解淀粉后的产物生成α-1,4/6以及α-1,6-葡糖-α-1,4结构的膳食纤维。
  5. 根据权利要求1所述的一种高产丁酸淀粉基膳食纤维的加工方法，其特征在于，所述淀粉包括玉米淀粉、马铃薯淀粉、木薯淀粉、稻米淀粉、小麦淀粉中的任意一种.
  6. 根据权利要求5所述的一种高产丁酸淀粉基膳食纤维的加工方法，其特征在于，所述淀粉包括普通淀粉、蜡质淀粉、高直链淀粉、淀粉糊精中的任意一种。
  7. 根据权利要求1所述的一种高产丁酸淀粉基膳食纤维的加工方法，其特征在于，所述酶-膜偶联反应器的膜截留分子量为10-60kDa。
  8. 权利要求1～7任一所述的一种高产丁酸淀粉基膳食纤维的加工方法制备得到的高产丁酸淀粉基膳食纤维。
  9. 根据权利要求8所述的高产丁酸淀粉基膳食纤维，其特征在于，所述高产丁酸淀粉基膳食纤维的分子量为0.5×10 7-9.5×10 7Da。
  10. 包含权利要求8或9所述的高产丁酸淀粉基膳食纤维的食品以及食品添加剂。
  11. 权利要求1～7任一所述的一种高产丁酸淀粉基膳食纤维的加工方法或8或9所述的高产丁酸淀粉基膳食纤维在食品、医药领域的应用。
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