CN105063128A - Preparation method of low molecular weight konjac oligosaccharide for regulating intestinal flora - Google Patents
Preparation method of low molecular weight konjac oligosaccharide for regulating intestinal flora Download PDFInfo
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Abstract
The invention relates to konjac oligosaccharide for regulating intestinal flora, in particular to a novel method, namely a method to acquire konjac oligosaccharide mannose mixture with low molecular weight, narrow distribution, low monosaccharide content and higher purity, by means of primary alcohol precipitation and secondary acetone treatment after enzymatic hydrolysis. The method includes the steps of hydrolyzing konjac mannan by beta- mannanase to obtain konjac mannose oligomer hydrolysate, adding ethanol precipitation of certain volume into the konjac mannose oligomer hydrolysate concentrated, concentrating supernate, adding acetone of certain volume, performing centrifuging after precipitating, removing supernate, and collecting precipitation to obtain the oligosaccharide mixture mainly containing konjac mannobiose, mannotriose, mannotetraose, mannopentaose, mannohexaose and mannoheptaose. The konjac oligosaccharide prepared by the method has a certain effect in regulating the intestinal flora.
Description
Technical field
The present invention relates to the preparation method of the konjac oligosaccharides with regulating intestinal canal flora, specifically, the present invention relates to a kind of have regulating intestinal canal biological community structure function low-molecular-weight, molecular weight is narrow, technique environmental protection, konjac oligosaccharides with low cost.
Background technology
The gi tract of people are the microecosystems of and complexity, its live away from home substantial amounts, miscellaneous microorganism.These microorganisms participate in a series of vital movement such as metabolism, immunity of human body thus maintain body health.Once this individual system balance is broken, alteration of intestinal flora will be caused, and then initiation even increases the weight of various diseases.In recent years, along with socioeconomic development, China resident especially developed area diet formula there occurs great variety, and the meat food etc. of high fat height hot drink food, refiningization is tended in these areas on diet.In addition larger life stress, the reason such as do not get enough athletic exercise, and makes enteron aisle sub-health state crowd increasing.In addition, many-sided factor such as edible additive long-term accumulated in vivo (as food dye, food flavour etc.), abuse of antibiotics, environmental pollution also affects micro-ecological environment in modern human body, brings the health problems such as some such as infect, constipation.
Konjac oligosaccharides is a kind of Non Digestible Oligosaccharides, and large quantity research found that it had numerous physiological activities in recent years, and at home and abroad feed, food, medicine and other fields are widely used.Its physiologically active mainly contains the following aspects: 1: regulate host intestine micro-ecological environment.The risk of the pathogenic agent such as Salmonellas, intestinal bacteria field planting in enteron aisle is reduced after it enters enteron aisle on the one hand, thus the diseases such as the diarrhoea that inhibit the corruption of harmful bacteria in enteron aisle to cause, reduce toxic metabolites (as indoles, nitroso-group ammonia etc.); On the other hand, it is optionally utilized by probioticss such as enteron aisle Lactobacterium acidophilum, bifidus bacilluss, produce some can bring powerful vitality organic acid to human body, intestinal cell for symbiosis provides DIRECT ENERGY source, and then stimulate the growth of knot mucomembranous cell, thus facilitating the balance of microorganism species, the effect of its short probiotic bacterium is up to 50 ~ 100 times of xylo-oligosaccharide; 2: improve lipid, metabolism of blood glucose, reduce blood fat and cholesterol.After Oligomeric manna sugar enters human body, probiotics is bred in a large number, Lactobacterium acidophilum absorbs cholesterol and suppresses intestines wall to the absorption of cholesterol; Bifidus bacillus, by affecting the activity of beta-hydroxy Beta-methyl glutaryl CoA-reductase, controls the synthesis of cholesterol; 3: promote trace element and B vitamin absorption.Oligomeric manna sugar has the ability retaining mineral element Ca, Fe, Zn, mineral substance is taken to caecum, colon.The important place of caecum, colon mineral absorption just.Bifidus bacillus amount reproduction, bifidus bacillus can produce endogenous V
b1, V
b2, V
b6, folic acid etc.4: anti-oxidant.Oligomeric manna sugar plays anti-oxidation efficacy through bifidus bacillus and lactobacillus 48h tunning by increase free radical scavenging and lipid peroxy thing.Therefore, it is acknowledged as the health factor of a new generation.
At present, konjac oligosaccharides adopts acid-hydrolysis method, enzymolysis process to obtain, and the former specificity is not high, and process relates to soda acid, easily causes environmental pollution.And for the molecular weight of oligosaccharide shortcoming distributed more widely that enzymolysis process obtains, a lot of report adopts ultrafiltration, a step alcohol deposition method carries out fractional separation to the oligose obtained, but the former is faced with cost intensive (industrialization ultrafiltration apparatus purchase commodity at least needs more than 100,000) in actual industrial production, the drawback that productive rate is lower, although the latter one step alcohol deposition method is prepared simply, is applicable to scale operation, but monose is often enriched in product, remove monose and need again new Production Flow Chart, these problems make the research and apply of konjaku functional component be restricted.Therefore the present invention devises a kind of new method, namely the method for secondary acetone treatment is added by enzymolysis rear stage alcohol precipitation, production unit demand is simple on the one hand, do not need to purchase specific installation, organic solvent on the other hand in this kind of procedure can recycle and reuse, avoid producing environment polluting, there is the advantage of green production.In addition, the konjaku Oligomeric manna sugar mixture that molecular weight is lower, narrow distribution, contents of monosaccharides are low, purity is higher can be obtained by this method, adopt this more at an easy rate, the more Oligomeric manna sugar prepared of environment protection method, therefore method of the present invention has more commercial competitiveness.
Summary of the invention
A preparation method for the konjac oligosaccharides of regulating intestinal canal flora, the step of preparation method is as follows:
(1) being dissolved in the water by konjak mannan, is 0.5%-3% by mass volume ratio, with the enzymic hydrolysis of β mannosans, obtains konjaku Oligomeric manna sugar hydrolyzed solution, and wherein enzyme concentration is lived with substrate mass ratio by enzyme is that 500-1500U/g calculates.
(2) konjaku Oligomeric manna sugar hydrolyzed solution goes out after enzyme, Rotary Evaporators is concentrated into the 1/5-1/10 of original volume, then in hydrolyzed solution, straight alcohol is added, to make in solution ethanol final volume concentration between 60%-85%, after precipitation, centrifugal, get supernatant, obtain the first step parting liquid, obtain containing the more Oligomeric manna sugar of monose.
(3) the first step parting liquid rotary evaporation is concentrated into the 1/5-1/10 of original volume, then adds 2-5 times of vol acetone, after precipitation, centrifugal, supernatant discarded, taking precipitate.Obtain mainly containing the oligosaccharide mixture of kanjak mannan disaccharides, trisaccharide, tetrose, pentasaccharides, six sugar.
Advantage of the present invention and positively effect are: the present invention prepares simply, and obtained konjac oligosaccharides molecular weight is little, narrow distribution, technique environmental protection, with low cost, also have the physiological function of regulating intestinal canal flora simultaneously.
Embodiment
Embodiment 1:
1. konjak mannan produces the processing method of specified molecular weight konjac oligosaccharides, and concrete steps are as follows:
(1) by konjak mannan through the enzymic hydrolysis of β mannosans, obtain konjaku Oligomeric manna sugar hydrolyzed solution, specific as follows, the concentration of konjak mannan is 2%, and adjusted to ph is 7.0, temperature 35 DEG C, and the concentration ratio of enzyme-to-substrate is 500U/g, enzymolysis aquation 6h..
(2), after konjaku Oligomeric manna sugar hydrolyzed solution 100 DEG C of enzymes that go out, Rotary Evaporators is concentrated into 1/10th (about 100mL) of original volume, and in hydrolyzed solution, add 900mL straight alcohol, stirring is spent the night, after precipitation, centrifugal, gets supernatant, obtains the first step parting liquid.
(3) the first step parting liquid rotary evaporation is concentrated into 1/10th (about 100mL) of original volume, then 200mL times of vol acetone is added, after precipitation, centrifugal, supernatant discarded, taking precipitate, obtain after vacuum-drying mainly containing the oligosaccharide mixture of kanjak mannan disaccharides, trisaccharide, tetrose, pentasaccharides, six sugar, seven sugar, product purity is greater than 95%.
Embodiment 2 the present invention studies the mouse experiment of the activity of the regulating intestinal canal flora of the Oligomeric manna sugar prepared
The impact of Oligomeric manna sugar on normal mouse intestinal form, intestinal microflora of this effects.Concrete grammar is: get mouse 50, be divided into four groups of experimental group and one group of control group at random, use various dose (1g/kgd respectively, 2g/kgd, 4g/kgd, Oligomeric manna sugar solution 8g/kgd) and isopyknic physiological saline gavage, after continuous 30d, observation and comparison Different adding amount is on the impact of mouse intestinal form, cecal content short chain fatty acid (SCFA) and microorganism species.Found that, when dosage reach addition be increased to 4g/kgd time, total acid content is increased to 103.35 ± 10.08 μm of ol/g, has had significance increase (p<0.05); When addition is up to 8g/kgd, total acid content (133.12 ± 15.4 μm of ol/g) improves very significantly (p<0.01).Mouse faecal samples bacterium colony analysis in the 30th day shows the change that feeding konjac oligosaccharides can cause enteric microorganism structure and forms.Mouse intestinal flora digitizing and cluster analysis result find that control group and experimental group define boundary to a certain degree, dendrogram obviously divide into two large bunches, wherein maximum dose level group LMOS8 group is all gathered in cluster, similarity is greater than 60%, and other samples are polymerized to other cluster, after showing feeding 30d, the intestinal microflora composition of high dose group has had obvious change.In order to understand Oligomeric manna sugar further, biological community structure in mouse caecum is analyzed, sequencing analysis has been carried out to band relevant in structure of community, sequencing result produces sour bacterioide (Bacteroidesacidifaciensstrain) intestines Bacterium lacticum (Lactobacillusintestinalisstrain) after showing feeding Oligomeric manna sugar, Lactobacillus animalis (Lactobacillusanimalisstrain), the probioticss such as lactobacillus reuteri (Lactobacillusreuteristrain) and clostridium (Clostridiumsp.) are all increased.
Embodiment 3
The difference of the product adopting different methods to prepare:
| method | one step alcohol precipitation | one step acetone treatment | one step alcohol precipitation two step acetone treatment |
| contents of monosaccharides | >10% | >10% | <2% |
| 2-7 sugar content | <50% | <50% | >90% |
| >7 sugar content | >35% | >35% | <5% |
| product molecule weight range | oligose composition containing a large amount of monose and >2 sugar, molecular weight distribution is wider | oligose composition containing a large amount of monose and >2 sugar, molecular weight distribution is wider | monose is considerably less, and 2-7 sugar accounts for 90%, >6 sugar content lower than 5%. |
Embodiment 4
Establish constipation type mouse model, the konjac oligosaccharides 1(2-7 sugar that feeding the inventive method obtains) with konjac oligosaccharides 2(>7 sugar) after 24h, find compared with konjac oligosaccharides 2 groups, the stool in mice first grain defecation time of feeding konjac oligosaccharides 1 is shorter, ight soil humidity improves, defecation grain number, defecation weight, enteron aisle carbon powder propelling rate increase, and the flatulence situation of konjac oligosaccharides 1 group of mouse is obviously less than konjac oligosaccharides 2 groups.
Claims (3)
1. a preparation method for the konjac oligosaccharides of regulating intestinal canal flora, is characterized in that: the step of preparation method is as follows:
Konjak mannan is dissolved in distilled water, and mass volume ratio is 0.5%-3%, through the enzymic hydrolysis of β mannosans, obtains konjaku Oligomeric manna sugar hydrolyzed solution, and wherein enzyme concentration is lived with substrate mass ratio as 500-1500U/g calculates with enzyme; Konjaku Oligomeric manna sugar hydrolyzed solution goes out after enzyme, and Rotary Evaporators is concentrated into the 1/5-1/10 of original volume, then in hydrolyzed solution, adds straight alcohol, make in solution that ethanol final volume concentration is between 60%-85%, after precipitation, centrifugal, get supernatant, namely containing the Oligomeric manna sugar that monose is more; Supernatant liquor rotary evaporation is concentrated into the 1/5-1/10 of original volume, then adds 2-5 times of vol acetone, after precipitation, centrifugal, get precipitation; To vacuum-drying be precipitated, obtain konjac oligosaccharides.
2. preparation method as claimed in claim 1, is characterized in that, described β mannase is the mixture of one or more in neutral beta mannonase acidic beta mannonase alkalescence β mannase.
3. preparation method as claimed in claim 1, is characterized in that, described konjac oligosaccharides is the mixture of mannobiose, trisaccharide, tetrose, pentasaccharides, six sugar, seven sugar.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048508A (en) * | 2018-01-31 | 2018-05-18 | 西安源森生物科技有限公司 | A kind of method that Oligomeric manna sugar is prepared using konjaku |
| CN108359701A (en) * | 2018-02-28 | 2018-08-03 | 浙江天草生物科技股份有限公司 | A method of mannan-oligosaccharides being prepared from coffee bean with enzymatic isolation method |
| CN112679563A (en) * | 2021-01-06 | 2021-04-20 | 澳门大学 | Glucosidotetraose and preparation method and application thereof |
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| CN1406944A (en) * | 2001-08-28 | 2003-04-02 | 中国科学院微生物研究所 | Sulphonated modified mannose and preparation thereof |
| CN101418326A (en) * | 2008-12-03 | 2009-04-29 | 中国海洋大学 | Method for producing galacto-mannan-oligosaccharides by enzymatic degradation of sophora bean gum |
| CN103060399A (en) * | 2013-01-04 | 2013-04-24 | 西南大学 | Method for producing konjac glucomannan |
| CN103382493A (en) * | 2012-05-03 | 2013-11-06 | 桂林微邦生物技术有限公司 | Method for fast producing high-purity mannan-oligosaccharide |
| CN103468766A (en) * | 2013-09-18 | 2013-12-25 | 恩施天天佳生物科技有限公司 | Preparation method of high-purity mannan oligosaccharide |
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2015
- 2015-07-16 CN CN201510417997.5A patent/CN105063128B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1406944A (en) * | 2001-08-28 | 2003-04-02 | 中国科学院微生物研究所 | Sulphonated modified mannose and preparation thereof |
| CN101418326A (en) * | 2008-12-03 | 2009-04-29 | 中国海洋大学 | Method for producing galacto-mannan-oligosaccharides by enzymatic degradation of sophora bean gum |
| CN103382493A (en) * | 2012-05-03 | 2013-11-06 | 桂林微邦生物技术有限公司 | Method for fast producing high-purity mannan-oligosaccharide |
| CN103060399A (en) * | 2013-01-04 | 2013-04-24 | 西南大学 | Method for producing konjac glucomannan |
| CN103468766A (en) * | 2013-09-18 | 2013-12-25 | 恩施天天佳生物科技有限公司 | Preparation method of high-purity mannan oligosaccharide |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048508A (en) * | 2018-01-31 | 2018-05-18 | 西安源森生物科技有限公司 | A kind of method that Oligomeric manna sugar is prepared using konjaku |
| CN108048508B (en) * | 2018-01-31 | 2021-08-13 | 西安源森生物科技有限公司 | Method for preparing mannose oligomer by utilizing konjak |
| CN108359701A (en) * | 2018-02-28 | 2018-08-03 | 浙江天草生物科技股份有限公司 | A method of mannan-oligosaccharides being prepared from coffee bean with enzymatic isolation method |
| CN112679563A (en) * | 2021-01-06 | 2021-04-20 | 澳门大学 | Glucosidotetraose and preparation method and application thereof |
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