CN109134676B - Oligosaccharide and preparation method and application thereof - Google Patents
Oligosaccharide and preparation method and application thereof Download PDFInfo
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- CN109134676B CN109134676B CN201810824628.1A CN201810824628A CN109134676B CN 109134676 B CN109134676 B CN 109134676B CN 201810824628 A CN201810824628 A CN 201810824628A CN 109134676 B CN109134676 B CN 109134676B
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- 229920001542 oligosaccharide Polymers 0.000 title claims abstract description 53
- 150000002482 oligosaccharides Chemical class 0.000 title claims abstract description 53
- 238000002360 preparation method Methods 0.000 title abstract description 14
- 239000012528 membrane Substances 0.000 claims abstract description 62
- 239000011347 resin Substances 0.000 claims abstract description 58
- 229920005989 resin Polymers 0.000 claims abstract description 58
- 239000007788 liquid Substances 0.000 claims abstract description 44
- 238000000926 separation method Methods 0.000 claims abstract description 40
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 35
- 150000004676 glycans Chemical class 0.000 claims abstract description 31
- 229920001282 polysaccharide Polymers 0.000 claims abstract description 31
- 239000005017 polysaccharide Substances 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 29
- 238000001914 filtration Methods 0.000 claims abstract description 25
- 239000000706 filtrate Substances 0.000 claims abstract description 21
- 238000003809 water extraction Methods 0.000 claims abstract description 19
- 238000001556 precipitation Methods 0.000 claims abstract description 17
- 239000006041 probiotic Substances 0.000 claims abstract description 17
- 235000018291 probiotics Nutrition 0.000 claims abstract description 17
- 239000002699 waste material Substances 0.000 claims abstract description 17
- 108010059892 Cellulase Proteins 0.000 claims abstract description 15
- 108010059820 Polygalacturonase Proteins 0.000 claims abstract description 15
- 229940106157 cellulase Drugs 0.000 claims abstract description 15
- 108010093305 exopolygalacturonase Proteins 0.000 claims abstract description 15
- 239000012530 fluid Substances 0.000 claims abstract description 13
- 108090000790 Enzymes Proteins 0.000 claims abstract description 10
- 102000004190 Enzymes Human genes 0.000 claims abstract description 10
- 229940088598 enzyme Drugs 0.000 claims abstract description 10
- 239000000463 material Substances 0.000 claims abstract description 9
- 230000008569 process Effects 0.000 claims abstract description 8
- 238000001704 evaporation Methods 0.000 claims abstract description 3
- 230000000415 inactivating effect Effects 0.000 claims abstract description 3
- 238000000108 ultra-filtration Methods 0.000 claims description 24
- 238000004108 freeze drying Methods 0.000 claims description 16
- 239000000047 product Substances 0.000 claims description 10
- 238000004519 manufacturing process Methods 0.000 claims description 9
- 230000000968 intestinal effect Effects 0.000 claims description 8
- 235000013305 food Nutrition 0.000 claims description 5
- 230000000529 probiotic effect Effects 0.000 claims description 5
- 241000894006 Bacteria Species 0.000 claims description 4
- 230000036541 health Effects 0.000 claims description 4
- 239000000825 pharmaceutical preparation Substances 0.000 claims description 3
- 239000012465 retentate Substances 0.000 claims description 3
- 239000004480 active ingredient Substances 0.000 claims description 2
- 229940127557 pharmaceutical product Drugs 0.000 claims description 2
- 238000011282 treatment Methods 0.000 abstract description 35
- 230000035755 proliferation Effects 0.000 abstract description 11
- 239000002994 raw material Substances 0.000 abstract description 5
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- 230000000052 comparative effect Effects 0.000 description 12
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- 239000003814 drug Substances 0.000 description 7
- 230000002779 inactivation Effects 0.000 description 7
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- OQUKIQWCVTZJAF-UHFFFAOYSA-N phenol;sulfuric acid Chemical compound OS(O)(=O)=O.OC1=CC=CC=C1 OQUKIQWCVTZJAF-UHFFFAOYSA-N 0.000 description 6
- 230000001737 promoting effect Effects 0.000 description 6
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- 238000005259 measurement Methods 0.000 description 4
- 239000008213 purified water Substances 0.000 description 4
- 238000002390 rotary evaporation Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 240000006024 Lactobacillus plantarum Species 0.000 description 3
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000007865 diluting Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 229910017053 inorganic salt Inorganic materials 0.000 description 3
- 229940072205 lactobacillus plantarum Drugs 0.000 description 3
- 150000002772 monosaccharides Chemical class 0.000 description 3
- 239000008188 pellet Substances 0.000 description 3
- 239000000049 pigment Substances 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
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- 241000218588 Lactobacillus rhamnosus Species 0.000 description 2
- 235000019764 Soybean Meal Nutrition 0.000 description 2
- 239000002577 cryoprotective agent Substances 0.000 description 2
- 238000004042 decolorization Methods 0.000 description 2
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- 238000000605 extraction Methods 0.000 description 2
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- 235000021255 galacto-oligosaccharides Nutrition 0.000 description 2
- 150000003271 galactooligosaccharides Chemical class 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 210000000987 immune system Anatomy 0.000 description 2
- 210000004347 intestinal mucosa Anatomy 0.000 description 2
- DLRVVLDZNNYCBX-RTPHMHGBSA-N isomaltose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)C(O)O1 DLRVVLDZNNYCBX-RTPHMHGBSA-N 0.000 description 2
- 239000011812 mixed powder Substances 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000004455 soybean meal Substances 0.000 description 2
- 244000139693 Arctostaphylos uva ursi Species 0.000 description 1
- 235000012871 Arctostaphylos uva ursi Nutrition 0.000 description 1
- 244000170916 Paeonia officinalis Species 0.000 description 1
- 235000006484 Paeonia officinalis Nutrition 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- -1 extractum Substances 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 230000007661 gastrointestinal function Effects 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000006872 mrs medium Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229940023492 oral liquid product Drugs 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 235000019722 synbiotics Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940126680 traditional chinese medicines Drugs 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- 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
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
-
- 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
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- 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
- A61K31/7016—Disaccharides, e.g. lactose, lactulose
-
- 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
-
- 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
-
- 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
-
- 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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/175—Rhamnosus
Abstract
The invention relates to oligosaccharide and a preparation method and application thereof. The preparation method of the oligosaccharide comprises the following steps: collecting waste liquid generated in water extraction and alcohol precipitation process of Chinese medicinal materials, evaporating to remove ethanol, and filtering to obtain filtrate; performing membrane separation on the filtrate to obtain an effluent liquid, and performing membrane separation on the effluent liquid to obtain a trapped fluid; adding macroporous resin into the trapped fluid for decoloring, oscillating, and filtering to remove the macroporous resin to obtain polysaccharide solution; and adding cellulase into the polysaccharide solution for enzymolysis, adding pectinase for enzymolysis, and inactivating enzyme to obtain the polysaccharide. The method takes the waste liquid generated in the water extraction and alcohol precipitation treatment of the traditional Chinese medicinal materials as the raw material, firstly carries out membrane separation twice on the waste liquid, then carries out macroporous resin decoloration, and finally adopts cellulase and pectinase to carry out enzymolysis in sequence, so that the oligosaccharide with the molecular weight below 2000 can be obtained, the oligosaccharide is easier to be utilized by probiotics, and the proliferation of the probiotics can be well promoted.
Description
Technical Field
The invention relates to the technical field of oligosaccharide extraction, in particular to oligosaccharide and a preparation method and application thereof.
Background
The oligosaccharide is a novel functional carbohydrate source and is widely applied to the fields of food, health care products, medicines, feed additives and the like. The oligosaccharide can improve the micro-ecological environment in human body, regulate the gastrointestinal function, and is beneficial to the proliferation of probiotics and the growth inhibition of putrefying bacteria; meanwhile, the composition has the function of promoting the development of the intestinal mucosa immune system and also has the function of promoting the whole body immune system outside the intestinal tract; the oligosaccharide can also improve the activity of intestinal mucosa cells and increase the mineral absorption capacity of the intestinal tract by adjusting the microecological balance of intestinal flora.
The traditional oligosaccharide preparation processes, such as the process for preparing oligosaccharide by using peony seed meal and mulberry juice as raw materials, have weak functionality of oligosaccharide-containing products obtained by the traditional processes, and have very limited proliferation promoting effect on intestinal probiotics.
When traditional Chinese medicine enterprises produce products such as extractum, granules, oral liquid and the like, water extraction and alcohol precipitation treatment is often needed to be carried out on the traditional Chinese medicines, and waste liquid after treatment is often directly thrown away, so that the environmental damage is great.
At present, no method for producing oligosaccharide with obvious intestinal probiotic proliferation promoting effect by using waste liquid generated by water extraction and alcohol precipitation of traditional Chinese medicinal materials as a raw material exists.
Disclosure of Invention
Based on the above, the main purpose of the invention is to provide a method for preparing oligosaccharide with obvious effect of promoting intestinal probiotic proliferation by using the waste liquid of water extraction and alcohol precipitation of traditional Chinese medicinal materials as a raw material.
The purpose of the invention is realized by the following technical scheme:
a method for producing an oligosaccharide, said method comprising:
step one, taking waste liquid generated in the water extraction and alcohol precipitation process of the traditional Chinese medicinal materials, evaporating to remove ethanol, and filtering to obtain filtrate;
step two, performing membrane separation on the filtrate to obtain an effluent liquid, and performing membrane separation on the effluent liquid to obtain a trapped fluid;
step three, adding macroporous resin into the trapped fluid for decoloring, oscillating, and filtering to remove the macroporous resin to obtain polysaccharide solution;
and step four, adding cellulase into the polysaccharide solution for enzymolysis, adding pectinase for enzymolysis, and inactivating enzyme to obtain the polysaccharide.
In some embodiments, the filtrate is subjected to membrane separation by using an ultrafiltration membrane with the molecular weight cut-off of 250kDa to 300 kDa; when the effluent is subjected to membrane separation, an ultrafiltration membrane with the molecular weight cutoff of 3-5 kDa is adopted; the macroporous resin is selected from at least one of AB-8 resin, D101 resin, HPD-300 resin, X-5 resin, NK-2 resin, NKA-2 resin and NK-9 resin, and the mass of the macroporous resin added into each milliliter of the trapped fluid is 0.18g to 0.22 g; the conditions for enzymolysis by adding cellulase are as follows: the temperature is 30-50 ℃ and the time is 2-5 h; the conditions for adding pectinase for enzymolysis are as follows: 30-50 ℃ for 2-5 h.
In some embodiments, the filtrate is subjected to membrane separation by using an ultrafiltration membrane with the molecular weight cutoff of 280kDa to 300 kDa; when the effluent is subjected to membrane separation, an ultrafiltration membrane with the molecular weight cutoff of 3-5 kDa is adopted; the macroporous resin is selected from AB-8 resin, and the mass of the macroporous resin added into each milliliter of the trapped fluid is 0.20g to 0.22 g; the conditions for enzymolysis by adding cellulase are as follows: the temperature is 30-50 ℃ and the time is 2-5 h; the conditions for adding pectinase for enzymolysis are as follows: 30-50 ℃ for 2-5 h.
In some of the examples, the filtrate is subjected to membrane separation using an ultrafiltration membrane having a molecular weight cut-off of 300 kDa; and an ultrafiltration membrane with the molecular weight cutoff of 5kDa is adopted when the effluent is subjected to membrane separation.
In some of these embodiments, the macroporous resin is an AB-8 resin, and the mass of macroporous resin added per milliliter of the retentate is 0.2 g.
In some of these embodiments, step four comprises the step of freeze-drying the enzyme-inactivated product.
In some of these embodiments, the conditions of the freeze-drying are: -0.05MPa to-0.08 MPa, -45 ℃ to-35 ℃ and 2.5d to 3 d.
Another object of the present invention is to provide an oligosaccharide obtained by the above-mentioned preparation method.
The invention also aims to provide application of the oligosaccharide in preparing food, health-care products or medicines capable of improving intestinal probiotics.
Still another object of the present invention is to provide a food, health product or pharmaceutical product containing the oligosaccharide as an active ingredient, which can improve intestinal probiotics.
Compared with the prior art, the invention has the following beneficial effects:
the invention takes the waste liquid generated in the water extraction and alcohol precipitation treatment of the traditional Chinese medicinal materials as the raw material for the first time, firstly carries out membrane separation twice on the waste liquid, then carries out macroporous resin decoloration, and finally carries out enzymolysis sequentially by adopting cellulase and pectinase, particularly, the invention is based on the waste liquid generated in the water extraction and alcohol precipitation treatment of the traditional Chinese medicinal materials, and adopts a proper ultrafiltration membrane, macroporous resin and enzymolysis conditions correspondingly to form a specific oligosaccharide preparation process. Meanwhile, the preparation process successfully realizes the reutilization of the waste liquid of the traditional Chinese medicine enterprise, increases the economic benefit and is beneficial to protecting the environment.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The invention relates to a water extraction and alcohol precipitation method, namely a water extraction and alcohol precipitation method which is well known in the field of traditional Chinese medicine extraction, and the water extraction and alcohol precipitation method is a method for refining a water extraction solution after solid-liquid separation, wherein ethanol is added into a traditional Chinese medicine water extraction concentrated solution to achieve different alcohol contents, the solubility of certain medicine components in an alcohol solution is reduced, and precipitates are separated out. The waste liquid produced by water extraction and alcohol precipitation of the traditional Chinese medicinal materials is specifically the liquid part obtained after the solid-liquid separation.
Example 1
This example provides an oligosaccharide and a method for producing the same. The whole preparation process comprises the following steps:
taking waste liquid obtained after water extraction and alcohol precipitation of mulberry branches → rotary evaporation and volatilization of ethanol → filtration → membrane separation with molecular weight cutoff of 300kDa → effluent liquid → membrane separation with molecular weight cutoff of 5kDa → trapped liquid → macroporous resin decoloration → drying → polysaccharide concentration measurement → dilution → enzymolysis → enzyme inactivation → freeze drying; wherein:
and (3) filtering: filtering the extractive solution with 300 mesh filter cloth, filtering with 600 mesh filter cloth, and collecting filtrate and residue; adding 10 times of purified water into the filter residue, and extracting again according to the method; filtering, and mixing the filtrates.
Membrane separation: separating the filtrate by an ultrafiltration membrane with the molecular weight cutoff of 300kDa to remove most of proteins until the effluent liquid is determined to contain no polysaccharide by a phenol-sulfuric acid method, and taking the effluent liquid; then separating by an ultrafiltration membrane with the molecular weight cutoff of 5kDa until the effluent is colorless to remove small molecular substances such as monosaccharide, pigment and inorganic salt, and taking the cutoff solution; the membrane separation operation adopts a pressure of 0.3MPa, a temperature of room temperature and a membrane area of 0.2m2。
And (3) macroporous resin decoloring: adding pretreated macroporous resin AB-8 into the trapped fluid according to the proportion of resin/polysaccharide solution (0.2g/mL), placing in a constant temperature oscillator, oscillating at 37 deg.C (120r/min) for adsorption for 5h, filtering to remove macroporous resin to obtain decolorized polysaccharide solution.
Drying: drying at 60 ℃ for 5h to obtain blocks.
And (3) measuring the concentration of the polysaccharide: the concentration of the dried block-shaped polysaccharide was measured by a sulfuric acid-phenol method.
Diluting: the polysaccharide concentration was diluted to 50 mg/mL.
Enzymolysis: firstly, cellulase is used for enzymolysis for 4 hours at 40 ℃, and then pectinase is used for enzymolysis for 4 hours at 40 ℃.
Enzyme inactivation: heating the enzymolysis solution to boil for 4 min.
And (3) freeze drying: freeze drying the enzyme-inactivated liquid at-40 deg.C under-0.08 MPa for 3 days to obtain functional oligosaccharide powder.
Example 2
This example provides an oligosaccharide and a method for producing the same. The whole preparation process comprises the following steps:
taking waste liquid obtained after water extraction and alcohol precipitation of mulberry branches → rotary evaporation and volatilization of ethanol → filtration → membrane separation with molecular weight cutoff of 250kDa → effluent liquid → membrane separation with molecular weight cutoff of 3kDa → trapped liquid → macroporous resin decoloration → drying → polysaccharide concentration measurement → dilution → enzymolysis → enzyme inactivation → freeze drying; wherein:
and (3) filtering: filtering the extractive solution with 300 mesh filter cloth, filtering with 600 mesh filter cloth, and collecting filtrate and residue; adding 10 times of purified water into the filter residue, and extracting again according to the method; filtering, and mixing the filtrates.
Membrane separation: separating the filtrate with ultrafiltration membrane with molecular weight cutoff of 250kDa to remove most proteins until the effluent liquid is determined to contain no polysaccharide by phenol-sulfuric acid method, and taking the effluent liquid; then separating by an ultrafiltration membrane with the molecular weight cutoff of 3kDa until the effluent is colorless to remove small molecular substances such as monosaccharide, pigment and inorganic salt, and taking the cutoff solution; the membrane separation operation adopts a pressure of 0.3MPa, a temperature of room temperature and a membrane area of 0.2m2。
And (3) macroporous resin decoloring: adding pretreated macroporous resin AB-8 into the trapped fluid according to the proportion of resin/polysaccharide solution (0.18g/mL), placing in a constant temperature oscillator, oscillating at 37 deg.C (120r/min) for adsorption for 5h, filtering to remove macroporous resin to obtain decolorized polysaccharide solution.
Drying: drying at 60 ℃ for 4h to obtain blocks.
And (3) measuring the concentration of the polysaccharide: the concentration of the dried block-shaped polysaccharide was measured by a sulfuric acid-phenol method.
Diluting: the polysaccharide concentration was diluted to 1 mg/mL.
Enzymolysis: firstly, cellulase is used for enzymolysis for 2 hours at 30 ℃, and then pectinase is used for enzymolysis for 2 hours at 30 ℃.
Enzyme inactivation: and heating and boiling the enzymolysis liquid for 3-5 min.
And (3) freeze drying: freeze drying the enzyme-inactivated liquid at-40 deg.C under-0.08 MPa for 3 days to obtain functional oligosaccharide powder.
Example 3
This example provides an oligosaccharide and a method for producing the same. The whole preparation process comprises the following steps:
taking waste liquid obtained after water extraction and alcohol precipitation of mulberry branches → rotary evaporation and volatilization of ethanol → filtration → membrane separation with molecular weight cutoff of 280kDa → effluent liquid → membrane separation with molecular weight cutoff of 4kDa → trapped liquid → macroporous resin decoloration → drying → polysaccharide concentration measurement → dilution → enzymolysis → enzyme inactivation → freeze drying; wherein:
and (3) filtering: filtering the extractive solution with 300 mesh filter cloth, filtering with 600 mesh filter cloth, and collecting filtrate and residue; adding 10 times of purified water into the filter residue, and extracting again according to the method; filtering, and mixing the filtrates.
Membrane separation: separating the filtrate by an ultrafiltration membrane with the molecular weight cutoff of 280kDa to remove most of proteins until the effluent liquid is determined to contain no polysaccharide by a phenol-sulfuric acid method, and taking the effluent liquid; then separating by an ultrafiltration membrane with the molecular weight cutoff of 4kDa until the effluent is colorless to remove small molecular substances such as monosaccharide, pigment and inorganic salt, and taking the cutoff solution; the membrane separation operation adopts a pressure of 0.3MPa, a temperature of room temperature and a membrane area of 0.2m2。
And (3) macroporous resin decoloring: adding pretreated macroporous resin AB-8 into the trapped fluid according to the proportion of resin/polysaccharide solution (0.22g/mL), placing in a constant temperature oscillator, oscillating at 37 deg.C (120r/min) for adsorption for 5h, filtering to remove macroporous resin to obtain decolorized polysaccharide solution.
Drying: drying for 6h at 60 ℃ to block.
And (3) measuring the concentration of the polysaccharide: the concentration of the dried block-shaped polysaccharide was measured by a sulfuric acid-phenol method.
Diluting: the polysaccharide concentration was diluted to 100 mg/mL.
Enzymolysis: firstly, the enzymolysis is carried out for 5 hours at 50 ℃ by using cellulase, and then the enzymolysis is carried out for 5 hours at 50 ℃ by using pectinase.
Enzyme inactivation: heating the enzymolysis solution to boil for 5 min.
And (3) freeze drying: freeze drying the enzyme-inactivated liquid at-40 deg.C under-0.08 MPa for 3 days to obtain functional oligosaccharide powder.
Comparative example 1
The present example is a comparative example of example 1, and the main difference from example 1 is that the process is not ultrafiltration membrane separation, macroporous resin decolorization and final enzymolysis, but is enzymolysis, ultrafiltration membrane separation and macroporous resin decolorization, and the process is as follows:
taking waste liquid generated after water extraction and alcohol precipitation of mulberry branches → rotary evaporation and volatilization of ethanol → filtration → enzymolysis → enzyme inactivation → membrane separation with molecular weight cutoff of 300kDa → effluent liquid → membrane separation with molecular weight cutoff of 5kDa → trapped fluid → macroporous resin decoloration → drying → measurement of polysaccharide concentration → dilution → freeze drying.
Comparative example 2
The present example is a comparative example of example 1, and the main difference from example 1 is the step of enzymatic hydrolysis: in this case, the cellulase and the pectinase are not sequentially used for enzymolysis, but the cellulase and the pectinase are simultaneously added for enzymolysis.
Comparative example 3
This example is a comparative example to example 1, the main differences from example 1 being:
(1) when the membrane is separated, firstly adopting a 350kDa ultrafiltration membrane for separation, and then adopting a 10kDa ultrafiltration membrane for separation;
(2) during enzymolysis, cellulase is firstly adopted for enzymolysis for 6 hours at the temperature of 28 ℃, and then pectinase is adopted for enzymolysis for 1.5 hours at the temperature of 55 ℃.
Performance testing
The oligosaccharides obtained in example 1, example 2, example 3, comparative example 1, comparative example 2 and comparative example 3, and the commercial oligosaccharides (galacto-oligosaccharide, isomalto-oligosaccharide, as positive control) were added to MRS medium at an addition rate of 15% (w/v), and Lactobacillus plantarum was inoculated until the OD reached 0.5. And culturing and counting colonies.
Among them, the treatment with the oligosaccharide obtained in example 1 was referred to as treatment 1, the treatment with the oligosaccharide obtained in example 2 was referred to as treatment 2, the treatment with the oligosaccharide obtained in example 3 was referred to as treatment 3, the treatment with the oligosaccharide obtained in comparative example 1 was referred to as treatment 4, the treatment with the oligosaccharide obtained in comparative example 2 was referred to as treatment 5, the treatment with the oligosaccharide obtained in comparative example 3 was referred to as treatment 6, the treatment with commercial galactooligosaccharide was referred to as treatment 7, the treatment with commercial isomaltooligosaccharide was referred to as treatment 8 (positive control), and the treatment without oligosaccharide was referred to as treatment 9 (negative control).
From table 1 above, it can be seen that:
the results of treatments 1, 2 and 3 showed that the oligosaccharides obtained in examples 1, 2 and 3 exhibited a significant effect of promoting the proliferation of probiotics. However, the number of colonies obtained by treatment 1 was higher than that obtained by treatment, indicating that the oligosaccharide preparation process of the present invention has a better solution. The number of colonies is significantly increased in treatment 1 compared to treatment 6, which indicates that the selection of appropriate ultrafiltration membrane, macroporous resin, and enzymatic conditions in the preparation process is important.
The number of colonies increased significantly in treatment 1 compared to treatment 4 and treatment 5, indicating that the oligosaccharides produced by the process of the present invention are critical to the proliferation of probiotics, and if the process is adjusted, the resulting product, although it is also able to promote the proliferation of probiotics, is very limited.
Compared with treatment 1 and treatment 9, the proliferation rate of probiotics reaches 619%, and compared with treatment 7 and treatment 8, the proliferation rate of probiotics reaches 361% and 382%. This demonstrates that the oligosaccharides produced in example 1 are more readily available to the probiotic bacteria than commercially available oligosaccharides, thereby facilitating the proliferation of the probiotic bacteria.
Application example 1
Adding the oligosaccharide powder prepared in the embodiment 1 into an MRS culture medium according to the addition of 10-20% (w/v), inoculating lactobacillus plantarum, inoculating lactobacillus rhamnosus when the OD value reaches 0.5, culturing for 18h, then primarily concentrating thalli, then quickly freezing into small pellets, adding cryoprotectants such as soybean meal and the like, freeze-drying, grinding and mixing the small pellets, thus realizing uniformity, and finally subpackaging and making into independent small packages.
Application example 2
Adding the oligosaccharide powder prepared in the example 2 into an MRS culture medium according to the addition amount of 10-20% (w/v), inoculating lactobacillus plantarum, inoculating lactobacillus rhamnosus when the OD value reaches 0.5, culturing for 18h, then primarily concentrating the thallus, then quickly freezing into pellets, adding freeze-drying cryoprotectants such as soybean meal and the like, grinding and mixing the powder to obtain mixed powder, and finally adding the oligosaccharide powder of the example 2 again (the mass ratio of the mixed powder to the oligosaccharide powder is 4:1) to prepare synbiotics.
Application example 3
Dissolving the oligosaccharide powder prepared in the embodiment 3 in purified water according to 40-60% (w/v), adding 5% (w/v) xylitol, and subpackaging to prepare an independent oral liquid product.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A method for preparing oligosaccharides, comprising:
step one, taking a waste liquid generated by water extraction and alcohol precipitation of Chinese medicinal materials, evaporating to remove ethanol, and filtering to obtain a filtrate;
step two, performing membrane separation on the filtrate to obtain an effluent liquid, and performing membrane separation on the effluent liquid to obtain a trapped fluid;
step three, adding macroporous resin into the trapped fluid for decoloring, oscillating, and filtering to remove the macroporous resin to obtain polysaccharide solution;
adding cellulase into the polysaccharide solution for enzymolysis, adding pectinase for enzymolysis, and inactivating enzyme to obtain the polysaccharide;
adopting an ultrafiltration membrane with the molecular weight cutoff of 250-300 kDa when performing membrane separation on the filtrate; when the effluent is subjected to membrane separation, an ultrafiltration membrane with the molecular weight cutoff of 3-5 kDa is adopted;
the conditions for enzymolysis by adding cellulase are as follows: the temperature is 30-50 ℃ and the time is 2-5 h; the conditions for adding pectinase for enzymolysis are as follows: 30-50 ℃ for 2-5 h.
2. The method of claim 1, wherein the macroporous resin is at least one selected from the group consisting of AB-8 resin, D101 resin, HPD-300 resin, X-5 resin, NK-2 resin, NKA-2 resin, and NK-9 resin, and the mass of the macroporous resin per ml of the retentate is 0.18g to 0.22 g.
3. The method for preparing oligosaccharide according to claim 2, wherein the filtrate is subjected to membrane separation by using an ultrafiltration membrane with a molecular weight cut-off of 280kDa to 300 kDa; when the effluent is subjected to membrane separation, an ultrafiltration membrane with the molecular weight cutoff of 3-5 kDa is adopted; the macroporous resin is selected from AB-8 resin, and the mass of the macroporous resin added into each milliliter of the trapped fluid is 0.20g to 0.22 g; the conditions for enzymolysis by adding cellulase are as follows: the temperature is 30-50 ℃ and the time is 2-5 h; the conditions for adding pectinase for enzymolysis are as follows: 30-50 ℃ for 2-5 h.
4. The method for producing oligosaccharides according to claim 3, wherein an ultrafiltration membrane having a molecular weight cut-off of 300kDa is used for membrane separation of the filtrate; and an ultrafiltration membrane with the molecular weight cutoff of 5kDa is adopted when the effluent is subjected to membrane separation.
5. The method of claim 3, wherein the macroporous resin is AB-8 resin, and the mass of macroporous resin added to the retentate is 0.2 g/ml.
6. The method of any one of claims 1 to 5, wherein step four comprises the step of freeze-drying the enzyme-inactivated product.
7. The method for producing oligosaccharides according to claim 6, wherein the conditions for freeze-drying are as follows: -0.05MPa to-0.08 MPa, -45 ℃ to-35 ℃ and 2.5d to 3 d.
8. An oligosaccharide obtained by the process of any one of claims 1 to 7.
9. Use of an oligosaccharide according to claim 8 in the manufacture of a food, health product or pharmaceutical product for improving intestinal probiotic bacteria.
10. A food, health product or pharmaceutical preparation for improving intestinal probiotics, comprising the oligosaccharide of claim 8 as an active ingredient.
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| CN110128561B (en) * | 2019-05-09 | 2021-07-20 | 华南理工大学 | Preparation method of asparagus functional oligosaccharide |
| CN112480283B (en) * | 2020-12-28 | 2023-02-24 | 湖南中医药大学 | Method for preparing neutral oligosaccharide from rhizoma polygonati |
| CN113101250B (en) * | 2021-05-10 | 2022-09-27 | 广东省农业科学院蚕业与农产品加工研究所 | Method for preparing traditional Chinese medicine prebiotics toothpaste by using water extraction and alcohol precipitation waste and application thereof |
| CN113143825B (en) * | 2021-05-10 | 2022-09-27 | 广东省农业科学院蚕业与农产品加工研究所 | A collutory containing Chinese medicinal prebiotics and its preparation method |
| CN114916620A (en) * | 2022-05-23 | 2022-08-19 | 广西五和博澳药业有限公司 | Ramulus mori oligosaccharide prebiotics cattle feed and preparation method and application thereof |
| CN116694704A (en) * | 2023-06-26 | 2023-09-05 | 北京工商大学 | Preparation method and application of yellow water oligosaccharide |
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