CN109497558A - Kuh-seng dregs of a decoction polysaccharide synbiotic and the preparation method and application thereof - Google Patents
Kuh-seng dregs of a decoction polysaccharide synbiotic and the preparation method and application thereof Download PDFInfo
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- CN109497558A CN109497558A CN201811345915.0A CN201811345915A CN109497558A CN 109497558 A CN109497558 A CN 109497558A CN 201811345915 A CN201811345915 A CN 201811345915A CN 109497558 A CN109497558 A CN 109497558A
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- kuh
- polysaccharide
- decoction
- seng
- dregs
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- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 101
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 101
- 235000019722 synbiotics Nutrition 0.000 title claims abstract description 63
- 238000002360 preparation method Methods 0.000 title claims abstract description 27
- 229940072205 lactobacillus plantarum Drugs 0.000 claims abstract description 25
- 240000006024 Lactobacillus plantarum Species 0.000 claims abstract description 24
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims abstract description 24
- 230000000968 intestinal effect Effects 0.000 claims abstract description 24
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 40
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- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 claims 1
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Classifications
-
- 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
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- 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
- A23V2400/169—Plantarum
Abstract
The invention discloses a kind of kuh-seng dregs of a decoction polysaccharide synbiotics and the preparation method and application thereof, the present invention is fermented as unique raw material through lactobacillus plantarum using kuh-seng dregs of a decoction polysaccharide obtains kuh-seng dregs of a decoction polysaccharide synbiotic, the experimental results showed that, the synbiotic being prepared is remarkably improved mice organs index and mouse intestinal digestive enzyme activity, keep the integrality and permeability of intestinal mucosa, safeguard gut barrier function, reduce body endotoxin, diamine oxidase, the level of D-ALPHA-Hydroxypropionic acid and inflammatory factor, and it can be by improving mouse intestinal flora diversity and flora composed structure, promote the growth of beneficial bacterium, the quantity for reducing harmful bacteria adjusts animal intestinal micro-ecology, and the digestion power and immunocompetence of animal can be enhanced, with important application value.
Description
Technical field
The present invention relates to kuh-seng dregs of a decoction polysaccharide synbiotics and preparation method thereof, belong to Chinese medicine slag resource technology and Tiny ecosystem
Formulation art.
Background technique
Natural resources of Chinese medicinal materials product is the Chinese medicine strategic materials of country, is to ensure national health, development of national medicinal industry
Material base, the sound development of traditional Chinese medicine depend on the efficient utilization and sustainable development of natural resources of Chinese medicinal materials.As China is big
Blueness of the fast-developing continuous propulsion and international market with health service industry of Chinese Medicine Industry for natural resources product
It looks at, Chinese medicine and the production scale of natural medicinal living resources raw material is promoted constantly to expand.At the same time, in natural resources of Chinese medicinal materials industrialization
The traditional Chinese medicine waste of flood tide is also produced in the process, is mainly derived from discarded histoorgan during production of crude drugs, leftover bits and pieces
Waste residue, waste water, the exhaust gas etc. generated in material, Chinese medical extract and resource products manufacturing process.At present to the place of Chinese medicine dreg
Reason method mainly has: 1. concentrating as waste and stacks or bury, do not only take up a large amount of soils in this way, generates a large amount of harmful fermentations
Gas, and underground water easy to pollute；2. by burning after Chinese medicine dreg drying, but generating volume of smoke, carbon dioxide etc. after burning
Emission causes the significant wastage of environmental pollution and resource.How to efficiently use Chinese medicine slag is to realize Chinese medicine sustainability and show
One urgent problem to be solved of generationization.
It is more for the trans-utilization technology of Chinese medicine dreg at present, such as the extracting again of resource chemical component, microorganism hair
Ferment digests, prepares active carbon, as organic fertilizer, feed addictive and fungal culture matrix etc..Wherein using living in Chinese medicine dreg
Property component and probiotic combination application (synbiotic) research it is more prominent.Synbiotic is one kind by probiotics (such as lactic acid bacteria, bud
Spore bacillus, photosynthetic bacteria etc.) and prebiotics (such as oligosaccharide, saponins, Anthraquinones) be formulated according to certain technique it is micro-
Ecologic active substance.Its main effect is to adjust intestinal microflora, promotes beneficial microbe colonizing in enteron aisle, is reduced
The quantity of pathogenic microorganism improves immunity of organisms, improves function of intestinal mucosa barrier in patient, improves enteron aisle digestion power, promotes nutrition
The absorption of substance.At present there are mainly two types of the preparation methods of synbiotic: compatibility type synbiotic and fermented type synbiotic.Compatibility type closes
Raw member is that beneficial bacterium is directly used cooperatively with Chinese medicine.It the advantage is that the stability for guaranteeing the respective performance of beneficial bacterium and Chinese medicine,
But this method does not give full play to the effect that mutual facilitation between the two, especially probiotics promote prebiotics metabolism
Using extremely limited.Fermented type synbiotic is then with the obvious advantage in this respect, in probiotics while fermentation, own growth generation
It thanks and is often also promoted.Therefore last tunning had not only contained higher beneficial bacteria concentration, but also contained excellent prebiotics
Ingredient can be used as standard and ideal synbiotic.The production of synbiotic mostly uses conventional compatibility type, fermented type synbiotic at present
Theoretical research it is more mature, but its application and development is less.
Kuh-seng is the dry root of leguminous plant kuh-seng SophoraflavescensAit., and bitter is cold in nature, has heat-clearing
Eliminating dampness, desinsection diuresis and other effects.As commonly using large traditional Chinese medicinal materials assortment, market demand increases kuh-seng year by year, at the same time its
The solid waste and by-product enormous amount that production process generates, and discarded without Processes For Effective Conversion, it results in waste of resources,
If things go on like this it is unfavorable for green, the sustainable development of kuh-seng resource industries.Studies have reported that polysaccharide rich in radix sophorae
Constituents, polysaccharide are mainly made of galactolipin, mannose, have good antioxidant activity.Research shows that in the kuh-seng dregs of a decoction
Containing about 10% or so polysaccharide component, the resource utilization to polysaccharide component in the kuh-seng dregs of a decoction rarely seen at present is reported, benefit
Research with polysaccharide component preparation synbiotic in Chinese medicine slag is also less, also has no Related product.
It therefore, not only can effective land productivity by kuh-seng dregs of a decoction polysaccharide and lactobacillus plantarum co-fermentation production preparation synbiotic
With kuh-seng dregs of a decoction resource, excellent synbiotic preparation also can be obtained, which utilizes the comprehensive high-efficiency of Chinese medicine waste material
With positive impetus.
Summary of the invention
Goal of the invention: the technical problem to be solved by the present invention is in view of the deficiencies of the prior art, provide a kind of kuh-seng medicine
Slag polysaccharide synbiotic and preparation method thereof.Another object of the present invention is to provide kuh-seng dregs of a decoction polysaccharide synbiotic to prepare enteron aisle micro-
Ecological regulation, the health care product or the application in animal feed additive for improving gut barrier and digestion power and strengthen immunity.
Technical solution；In order to achieve the goal above, the technical scheme adopted by the invention is as follows:
A kind of kuh-seng dregs of a decoction polysaccharide synbiotic, it is prepared by lactobacillus plantarum fermentation kuh-seng dregs of a decoction polysaccharide.
Preferably, a kind of above-described kuh-seng dregs of a decoction polysaccharide synbiotic, kuh-seng dregs of a decoction polysaccharide are by molar ratio
Galactolipin, mannose, glucose, xylose, rhamnose and the arabinose group of 0.20:0.44:2.14:1.03:1.00:0.31
At.
A kind of preparation method of kuh-seng dregs of a decoction polysaccharide synbiotic, comprising the following steps:
(1) control of MRS culture medium (lactic acid bacteria culture medium) initial pH value is gone out between 6.4~6.6, and through high pressure
Bacterium, cooling, standing；
(2) kuh-seng dregs of a decoction polysaccharide is added in the MRS culture medium of step (1) with the concentration of 1~5g/L；Then through micropore
Filtrate is collected in membrane filtration, separation；
(3) under aseptic operating platform, by the lactobacillus plantarum after activation by volume 1%~5% inoculum concentration be seeded in
It in the filtrate of MRS culture medium of the above-mentioned steps (2) containing kuh-seng dregs of a decoction polysaccharide, dispenses, sealing；
(4) above-mentioned filtrate is placed in 37 DEG C of anaerobic culture box with static condition and is cultivated, incubation time be 12~
30h；After bacterium solution after culture is freeze-dried, both kuh-seng dregs of a decoction polysaccharide synbiotic freeze-dried powder.
Preferably, the preparation method of above-described kuh-seng dregs of a decoction polysaccharide synbiotic, lactic acid bacteria culture medium
The initial pH value of (MRS culture medium) is 6.5.
Preferably, the preparation method of above-described kuh-seng dregs of a decoction polysaccharide synbiotic, miillpore filter aperture is
0.22μm。
Preferably, the preparation method of above-described kuh-seng dregs of a decoction polysaccharide synbiotic, kuh-seng dregs of a decoction polysaccharide be by
What following methods were prepared:
Kuh-seng dregs of a decoction drying sample is weighed, petroleum ether degreasing is added, sample is dried after degreasing, addition ethyl alcohol, refluxing extraction,
It filters, the ethyl alcohol of filter residue heat, washs while hot, after filter residue and drying, water is added, heating and refluxing extraction obtains extracting solution, is concentrated under reduced pressure
Aqueous extracts are slowly added to dehydrated alcohol, alcohol content are made to reach 80~85%, stand overnight, be centrifuged, obtain supernatant filter to proper volume
Liquid and precipitating, precipitating are kuh-seng dregs of a decoction Thick many candies；
Kuh-seng dregs of a decoction Thick many candies are weighed, is dissolved in pure water, obtains polysaccharide solution, weigh trichloroacetic acid, be dissolved in pure water
In, solution of trichloroacetic acid is made；Thick many candies and solution of trichloroacetic acid are mixed, stood overnight, centrifuging and taking supernatant, add and on
The solution of trichloroacetic acid of clear liquid same volume carries out secondary removing protein；By except it is finished it is white after polysaccharide solution be concentrated under reduced pressure into centainly
Volume is slowly added to dehydrated alcohol, makes its alcohol content up to 80~85%, after standing overnight, is filtered with Buchner funnel, and what is obtained is heavy
Forming sediment is the kuh-seng polysaccharide removed after deproteinized；
Kuh-seng polysaccharide precipitation is dissolved in deionization ultrapure water, is adsorbed through AB-8 type macroporous absorbent resin, is decolourized, removed
Reason is lived together, up to kuh-seng dregs of a decoction polysaccharide after freeze-drying.
As more preferred scheme, the preparation method of above-described kuh-seng dregs of a decoction polysaccharide synbiotic, kuh-seng dregs of a decoction polysaccharide
It is prepared as follows to obtain:
Kuh-seng dregs of a decoction drying sample 1.0kg is weighed, petroleum ether degreasing is added twice in 1:10 (m/V) ratio, sample after degreasing
Product drying, is added 80% ethyl alcohol of 10 times of amounts, and 80 DEG C of water-bath refluxing extraction 2h are filtered while hot, and 80% ethyl alcohol of filter residue heat is washed
It washs 2~3 times, after filter residue and drying, the pure water that 10 times of amounts are added is heated to reflux in 150 DEG C, is extracted twice, and each 2h merges and extracts
Liquid.Aqueous extracts are concentrated under reduced pressure to proper volume, is slowly added to dehydrated alcohol, alcohol content is made to reach 80%, 4 DEG C stand overnight, from
The heart, obtains supernatant fluid filtrate and precipitating, and precipitating is kuh-seng dregs of a decoction Thick many candies.
Kuh-seng dregs of a decoction Thick many candies 100g is weighed, is dissolved in 1.0L pure water, obtains polysaccharide solution.Weigh trichloroacetic acid
100g is dissolved in pure water, and 5% solution of trichloroacetic acid is made.By Thick many candies and solution of trichloroacetic acid, 1:1 is mixed by volume, and 4
It DEG C stands overnight, centrifuging and taking supernatant adds the solution of trichloroacetic acid with supernatant same volume, carries out secondary removing protein.It will remove
It is finished it is white after polysaccharide solution be concentrated under reduced pressure into certain volume, be slowly added to dehydrated alcohol, make its alcohol content up to 80%, in 4 DEG C
It after standing overnight, is filtered with Buchner funnel, obtained precipitating is except the polysaccharide after deproteinized.The precipitating is dissolved in deionization to surpass
It in pure water, adsorbs, is decolourized, removal of impurities processing, up to kuh-seng dregs of a decoction polysaccharide after freeze-drying through AB-8 type macroporous absorbent resin.
Preferably, the preparation method of above-described kuh-seng dregs of a decoction polysaccharide synbiotic, step (2) kuh-seng dregs of a decoction are more
The final concentration of 2.0g/L of fermentation of sugar.
Kuh-seng dregs of a decoction polysaccharide synbiotic provided by the invention can be formed by improving mouse intestinal flora diversity and flora
Structure promotes the growth of beneficial bacterium, reduces the quantity of harmful bacteria, and enteron aisle digestive enzyme activity can be improved, and keeps the complete of intestinal mucosa
Whole property and permeability safeguard gut barrier function, reduce the water of body endotoxin, diamine oxidase, D-ALPHA-Hydroxypropionic acid and inflammatory factor
It is flat.
The utility model has the advantages that
1, present invention extraction creative first obtains kuh-seng dregs of a decoction polysaccharide, and is added into preferred lactobacillus plantarum, bitter
Join dregs of a decoction polysaccharide and lactobacillus plantarum co-fermentation production preparation synbiotic, can not only effectively utilize kuh-seng dregs of a decoction resource, also
Excellent synbiotic preparation can be obtained, which utilizes the comprehensive high-efficiency of Chinese medicine waste material makees with positive promotion
With.
2, the experimental results showed that, and kuh-seng dregs of a decoction polysaccharide provided by the invention is as fermentable sources, as shown in Figure 1, can be very
The growth of good promotion lactobacillus plantarum.Show that kuh-seng dregs of a decoction polysaccharide provided by the invention is the excellent battalion of lactobacillus plantarum breeding
The source of supporting.
3, enteron aisle digestive enzyme activity can be improved in the kuh-seng dregs of a decoction polysaccharide synbiotic that the present invention is prepared, and keeps intestinal mucosa
Integrality and permeability, safeguard gut barrier function, reduce body endotoxin, diamine oxidase, D-ALPHA-Hydroxypropionic acid and inflammatory factor
Level, can effectively adjust the state that body enteron aisle is kept fit, promote absorption of the body to nutriment, body can be improved
Immunity has important application value.
Detailed description of the invention
Fig. 1 is the growth curve that single plant lactobacillus and kuh-seng dregs of a decoction polysaccharide-lactobacillus plantarum co-culture.
Specific embodiment
The technical characteristic of the invention sent out and advantage are described in detail with test data with reference to embodiments.
The domestication of 1 lactobacillus plantarum of embodiment
Lactobacillus plantarum is inoculated in the MRS culture medium of the kuh-seng dregs of a decoction polysaccharide containing 5g/L, 30 DEG C are cultivated for 24 hours, continuously
It passed for 5 generations, is then seeded into the MRS culture solution of the dregs of a decoction polysaccharide of kuh-seng containing 2g/L and cultivates for 24 hours, it is continuous to pass for 3 generations, it obtains and adapts to kuh-seng
The lactobacillus plantarum of dregs of a decoction polysaccharide is placed in 2~8 DEG C of refrigerators, saves backup.
The preparation of 2 kuh-seng dregs of a decoction polysaccharide synbiotic of embodiment
(1) MRS Medium's PH Value is adjusted with the NaOH of 1mol/L, makes its pH value 6.5, then autoclave sterilization 20 divides
Clock.100mLMRS fluid nutrient medium is fitted into 250mL conical flask, addition 0.2g kuh-seng dregs of a decoction polysaccharide be (the kuh-seng dregs of a decoction polysaccharide
The preparation method comprises the following steps: weighing kuh-seng dregs of a decoction drying sample 1.0kg, petroleum ether degreasing is added twice in 1:10 (m/V) ratio, after degreasing
Sample drying, is added 80% ethyl alcohol of 10 times of amounts, and 80 DEG C of water-bath refluxing extraction 2h are filtered while hot, 80% ethyl alcohol of filter residue heat,
Washing 2~3 times, after filter residue and drying, the pure water that 10 times of amounts are added is heated to reflux in 150 DEG C, is extracted twice, each 2h, merging mentions
Take liquid.Aqueous extracts are concentrated under reduced pressure to proper volume, is slowly added to dehydrated alcohol, alcohol content is made to reach 80%, 4 DEG C stand overnight,
Centrifugation, obtains supernatant fluid filtrate and precipitating, and precipitating is kuh-seng dregs of a decoction Thick many candies.Kuh-seng dregs of a decoction Thick many candies 100g is weighed, is dissolved in
In 1.0L pure water, polysaccharide solution is obtained.Trichloroacetic acid 100g is weighed, is dissolved in pure water, 5% solution of trichloroacetic acid is made.It will
1:1 is mixed by volume for Thick many candies and solution of trichloroacetic acid, and 4 DEG C stand overnight, centrifuging and taking supernatant, is added and supernatant consubstantiality
Long-pending solution of trichloroacetic acid carries out secondary removing protein.By except it is finished it is white after polysaccharide solution be concentrated under reduced pressure into certain volume, slowly
Dehydrated alcohol is added, makes its alcohol content up to 80%, after 4 DEG C stand overnight, is filtered with Buchner funnel, obtained precipitating is to remove
Polysaccharide after deproteinized.The precipitating is dissolved in deionization ultrapure water, is adsorbed through AB-8 type macroporous absorbent resin, decolourized,
Removal of impurities processing, up to kuh-seng dregs of a decoction polysaccharide after freeze-drying), it is then 0.22 μm of filtering with microporous membrane, separating filtrate with aperture.
(2) under aseptic operating platform, by embodiment 1 activate after lactobacillus plantarum by volume 2% inoculum concentration be inoculated with
In the filtrate of MRS culture medium of the above-mentioned steps (1) containing kuh-seng dregs of a decoction polysaccharide, sealed after packing.
(3) filtrate by above-mentioned containing lactobacillus plantarum stands to be placed in 37 DEG C of anaerobic culture boxes and cultivate for 24 hours.After culture
Bacterium solution it is freeze-dried after both kuh-seng dregs of a decoction polysaccharide synbiotic freeze-dried powder, be placed in 2~8 DEG C of refrigerators, save backup.
Comparative examples
(1) MRS Medium's PH Value is adjusted with the NaOH of 1mol/L, makes its pH value 6.5, then autoclave sterilization 20 divides
Clock.100mLMRS fluid nutrient medium is fitted into 250mL conical flask.
(2) under aseptic operating platform, by embodiment 1 activate after lactobacillus plantarum by volume 2% inoculum concentration be inoculated with
In the filtrate of above-mentioned steps (1) MRS culture medium, sealed after packing.
(3) filtrate by above-mentioned containing lactobacillus plantarum stands to be placed in 37 DEG C of anaerobic culture boxes and cultivate for 24 hours.After culture
Bacterium solution it is freeze-dried after, both lactobacillus plantarum ferment reference material, be placed in 2~8 DEG C of refrigerators, save backup.
Embodiment 3 is made the present invention further below with reference to feeding experiment, biochemistry detection test and microbiological Test
Description:
One, Animals fed experimental
(1) selection, grouping and experimental design of experimental animal
ICR mouse (4 week old, male, 18-22g)；Purchased from Nanjing Qinglongshan animal-breeding field, all animal feeding conditions
It is SPF grades, 22 DEG C, raises under the conditions of day-night cycle 12h, adapt to after feeding 1 week.Random grouping, fills daily by every group 8
Stomach is primary, continues stomach-filling 28d.Test medicine: lactobacillus plantarum, kuh-seng dregs of a decoction polysaccharide (being prepared by 1 method of embodiment), kuh-seng
Dregs of a decoction polysaccharide synbiotic (embodiment 2 is prepared, hereinafter referred to as synbiotic).Comparative examples.Specific experimental animal grouping
And dosage is shown in Table 1.
The grouping of 1 experimental animal of table and dosage
| Group | Dosage | Stomach-filling amount/mL |
| Blank group | Physiological saline | 0.3 |
| Low dosage lactobacillus plantarum | 100mg/kg | 0.3 |
| High dose lactobacillus plantarum | 200mg/kg | 0.3 |
| Low dosage kuh-seng dregs of a decoction polysaccharide | 108CFU/kg | 0.3 |
| High dose kuh-seng dregs of a decoction polysaccharide | 109CFU/kg | 0.3 |
| 2 low dosage synbiotic of embodiment | 100mg/kg | 0.3 |
| 2 high dose synbiotic of embodiment | 200mg/kg | 0.3 |
| Comparative examples fermentation material low concentration | 100mg/kg | 0.3 |
| Comparative examples fermentation material high concentration | 200mg/kg | 0.3 |
(2) test index measures
It is dissected for 24 hours after intragastric administration on mice, it is fasting for solids and liquids before dissection, it after weighing, plucks eyeball and takes blood, the neck that breaks is put to death.
Blood is centrifuged (3,000rpm, 10min, 4 DEG C) after 4 DEG C of standing 1h, takes upper serum.Serum diamine oxidase (DAO), D- cream
Sour (D-LA), endotoxin (ET), anti-tumor necrosis factor (TNF-α) are according to the operating procedure for corresponding to ELISA kit specification
Measurement.When dissected takes out complete heart, liver, spleen, thymus gland, lung and renal tissue and uses filter paper after cleaning in physiological saline
It blots, its quality of accurate weighing.Clip duodenum, ileum position intestinal segment about 2cm are distinguished with scissors, use normal saline flushing
Afterwards, ileum intestinal segment is placed in neutral formalin solution fixed, through paraffin embedding, is sliced, is used hematoxylin-eosin after dewaxing
(HaematoxylinEosin, H&E) dyeing, observes intestinal mucosa structure under the microscope.Duodenum intestinal segment is immediately placed in liquid nitrogen
Middle preservation.Intestinal segment is taken out when measurement, with 10 times of ice physiological saline dilutions, homogenate, collects homogenate, be centrifuged (6,000rpm,
10min, 4 DEG C), Aspirate supernatant, according to amylase (Amylase) Activity Assay Kit, trypsase (trypsin) activity
Assay kit, lipase (Lipase) Activity Assay Kit, alkaline phosphatase (ALP) Activity Assay Kit specification
In operating procedure measure the activity of each digestive ferment.
Every group of mouse fresh excreta about 0.2g is collected in stomach-filling 28d.With 2mL deionized water dissolving, it is vortexed and mixes.Centrifugation
(6,000rpm, 10min) Aspirate supernatant with pH meter measures its pH value afterwards.Fresh excreta about 0.1g is taken, is measured with auto moisture
Instrument measures the water content in excrement.
(3) test result
Lactobacillus plantarum group, kuh-seng dregs of a decoction polysaccharide group and kuh-seng dregs of a decoction polysaccharide synbiotic it can be seen from data in table 2
Group can significantly improve the Lung Exponent of mouse.High dose symphysis tuple can significantly improve the cardiac index of mouse, thymus index,
Its administration group is to the organ index of mouse without significant impact.This result prompt kuh-seng dregs of a decoction polysaccharide synbiotic can improve small
Mouse constitution, the differentiation of enhancing internal organs and immunity, increase its organ index.
The result of 2 mice organs index of table
Note: compared to the blank group,*Indicate that p < 0.05 indicates significant difference, * * indicates that p < 0.01 indicates extremely significant difference
It is different.
The stool in mice water content of kuh-seng dregs of a decoction polysaccharide symphysis tuple is lower it can be seen from data in table 3, Binding experiment
In observation, blank group excrement is slightly dilute, is not easy to shape, and the excrement quality of symphysis tuple is harder, is readily formed, and is conducive to arrange
Just.The pH value of intestinal environment is extremely important for maintaining intestinal health, and the growth of the various digestive ferments of enteron aisle and beneficial bacterium needs
Stablize suitable pH value, gut pH is excessively high to will lead to some saprophytic bacterias mass propagation in enteron aisle, and metabolism generates some carcinogenic
Substance.Single lactobacillus, kuh-seng dregs of a decoction polysaccharide and kuh-seng dregs of a decoction polysaccharide synbiotic different degrees of downward mouse intestinal of energy
PH value, wherein kuh-seng dregs of a decoction polysaccharide symphysis tuple effect is optimal.Illustrate that kuh-seng dregs of a decoction polysaccharide synbiotic can reduce in mouse intestinal
Free ammonia substance, lower gut pH, improve intestinal microenvironment.
3 stool in mice water content of table, pH value and ammonia content result
Note: compared to the blank group,*Indicate that p < 0.05 indicates significant difference,**Indicate that p < 0.01 indicates extremely significant difference
It is different.
Mice serum index is as shown in table 4.As can be seen from Table 4, synbiotic can be significantly reduced in mice serum
The content of TNF-α, DAO and D-Lac.Illustrate that synbiotic can prevent DAO and D-Lac from entering blood, the enteron aisle for improving mouse is glutinous
Envelope barrier function maintains the integrality of intestinal mucosa.The decline of TNF-α and level of endotoxin illustrates that synbiotic can reduce in serum
The generation of internal inflammatory reaction, enhances the immunocompetence of mouse.
The content results of ET, TNF-α, DAO and D-Lac in 4 mice serum of table
Note: compared to the blank group,*Indicate that p < 0.05 indicates significant difference,**Indicate that p < 0.01 indicates extremely significant difference
It is different.
The expression of mouse ileum tissue pathological slice result, height of naps, Crypt depth, mucous membrane thickness and the suede of ileal tissue
Hair height/Crypt depth (V/C) is shown in Table 5.As can be seen from the results in the table that synbiotic can be obviously improved the villus of mouse ileum
Highly, mucous membrane thickness and V/C value.Illustrate that synbiotic can promote mouse ileum tissue surface product, accelerates cell growth, to nutrition
The digestion of substance and absorption function enhancing.
5 mouse ileum height of naps of table, Crypt depth, mucous membrane thickness and height of naps/Crypt depth (V/C)
Note: compared to the blank group,*Indicate that p < 0.05 indicates significant difference,**Indicate that p < 0.01 indicates extremely significant difference
It is different.
The digested enzymatic activity of mouse intestinal is as shown in table 6.The experimental results showed that the synbiotic that the present invention is prepared can be shown
The activity for promoting various digestive ferments in mouse intestinal is write, is conducive to starch, protein and lipid in degradation food, improves small intestine
Digestion power, enhance absorption of the small intestine to nutriment.
The digested enzymatic activity of 6 mouse intestinal of table
Note: compared to the blank group,*Indicate that p < 0.05 indicates significant difference,**Indicate that p < 0.01 indicates extremely significant difference
It is different.
Dominant microflora is followed successively by Firmicutes (Firmicutes), Bacteroidetes in each group mouse intestinal content
(Bacteroidetes), Proteobacteria (Proteobacteria), actinomyces door are (Actinobacteria) and some other
Flora such as Tenericutes (Tenericutes), deferrization bacillus door (Deferribacteres) etc..Each group Bacterial community is similar, but
Composition ratio is different.Firmicutes (Firmicutes), Bacteroidetes (Bacteroidetes) between different groups and
Proteobacteria (Proteobacteria) composition ratio and F/B/P value are shown in Table 7, compared to blank group, the F/B/P value of symphysis tuple
There is apparent rising, illustrates that synbiotic can change the flora composed structure of mouse intestinal.
Firmicutes (F), Bacteroidetes (B) under 7 different dosing dosage of table between each group
And Proteobacteria (P) composition ratio and F/B/P value
Note: compared to the blank group,*Indicate that p < 0.05 indicates significant difference,**Indicate that p < 0.01 indicates extremely significant difference
It is different.
The results are shown in Table 8 for characteristic differences Pseudomonas in mouse intestinal, and symphysis tuple can significantly improve in mouse intestinal
The quantity of some bacterium.For example, abundance of the Lactobacillus (lactobacillus) in symphysis tuple is significantly higher than blank group, list
One lactobacillus plantarum, single kuh-seng polysaccharide group and reference examples culture.Lactobacillus in enteron aisle can promote digestive ferment
Secretion, the metabolite generated can inhibit the growth for producing the pernicious bacteria of amine in enteron aisle, safeguard the integrality of gut barrier,
The immunity of body is improved by activation enteron aisle self immune system function.On the other hand, synbiotic can reduce in enteron aisle
The quantity of some harmful bacterias inhibits its growth.Such as Desulfovibrio (desulfovibrio) is substantially less than just in symphysis tuple
Normal group.Desulfovibrio is considered closely related with the generation of intestinal polyp and ulcerative colitis, reduces
Quantity of the Desulfovibrio in enteron aisle has important meaning to maintenance intestinal health.
Comparing result shows to give mouse exogenous plant lactobacillus, kuh-seng dregs of a decoction polysaccharide or reference examples culture merely
The quantity for increasing lactobacillus in enteron aisle can not be changed, and synbiotic can be promoted in mouse intestinal significantly
The quantity of Lactobacillus.
Characteristic differences Pseudomonas in 8 mouse intestinal of table
Note: compared to the blank group,*Indicate that p < 0.05 indicates significant difference,**Indicate that p < 0.01 indicates extremely significant difference
It is different.
Show in present invention screening, kuh-seng dregs of a decoction polysaccharide can promote the growth of lactobacillus, and by kuh-seng dregs of a decoction polysaccharide with
Lactobacillus plantarum fermentation preparation synbiotic, lactobacillus can utilize and decompose kuh-seng dregs of a decoction polysaccharide and colonize in enteron aisle, while it is bitter
Ginseng dregs of a decoction polysaccharide can induce and stimulate the growth of some healthy intestinal bacterium in enteron aisle, inhibit the breeding of harmful bacteria.Lactobacillus and hardship
Join the collaboration mutually of both dregs of a decoction polysaccharide, the common micro-ecological environment for improving mouse intestinal.
From above-described embodiment it is found that the adjustable animal intestinal micro-ecology of kuh-seng dregs of a decoction polysaccharide synbiotic of the present invention, improves
Animal intestinal tract barrier and digestion power enhance the immunity of animal.It should be understood that coming to those of ordinary skill in the art
It says, it can be modified or changed according to the above description, and all these modifications and variations all should belong to right appended by the present invention
It is required that protection scope.
Claims (8)
1. a kind of kuh-seng dregs of a decoction polysaccharide synbiotic, which is characterized in that it is by lactobacillus plantarum fermentation kuh-seng dregs of a decoction polysaccharide preparation
It obtains.
2. a kind of kuh-seng dregs of a decoction polysaccharide synbiotic according to claim 1, which is characterized in that kuh-seng dregs of a decoction polysaccharide by mole
Than for the galactolipin of 0.20:0.44:2.14:1.03:1.00:0.31, mannose, glucose, xylose, rhamnose and arabinose
Composition.
3. a kind of preparation method of kuh-seng dregs of a decoction polysaccharide synbiotic, which comprises the following steps:
(1) by the control of MRS initial pH value of medium between 6.4~6.6, and through high pressure sterilization, cooling, standing；
(2) kuh-seng dregs of a decoction polysaccharide is added in the MRS culture medium of step (1) with the concentration of 1~5g/L；Then through miillpore filter
Filtrate is collected in filtering, separation；
(3) under aseptic operating platform, by the lactobacillus plantarum after activation by volume 1%~5% inoculum concentration be seeded in it is above-mentioned
It in the filtrate of MRS culture medium of the step (2) containing kuh-seng dregs of a decoction polysaccharide, dispenses, sealing；
(4) above-mentioned filtrate is placed in 37 DEG C of anaerobic culture box with static condition and is cultivated, incubation time is 12~30h；Training
Support after bacterium solution it is freeze-dried after, both kuh-seng dregs of a decoction polysaccharide synbiotic freeze-dried powder.
4. the preparation method of kuh-seng dregs of a decoction polysaccharide synbiotic according to claim 2, it is characterised in that: kuh-seng dregs of a decoction polysaccharide
By molar ratio be the galactolipin of 0.20:0.44:2.14:1.03:1.00:0.31, mannose, glucose, xylose, rhamnose and Ah
Draw uncle's sugar composition.
5. the preparation method of kuh-seng dregs of a decoction polysaccharide synbiotic according to claim 3, which is characterized in that kuh-seng dregs of a decoction polysaccharide
It is prepared as follows to obtain: weighing kuh-seng dregs of a decoction drying sample, petroleum ether degreasing is added, sample is dried after degreasing, is added
Entering ethyl alcohol, refluxing extraction filters while hot, the ethyl alcohol of filter residue heat, washing, after filter residue and drying, addition water, heating and refluxing extraction,
Extracting solution is obtained, Aqueous extracts are concentrated under reduced pressure to proper volume, is slowly added to dehydrated alcohol, alcohol content is made to reach 80~85%, stand
Overnight, centrifugation, obtains supernatant fluid filtrate and precipitating, and precipitating is kuh-seng dregs of a decoction Thick many candies；
Kuh-seng dregs of a decoction Thick many candies are weighed, is dissolved in pure water, obtains polysaccharide solution, weigh trichloroacetic acid, be dissolved in pure water, are made
At solution of trichloroacetic acid；Thick many candies and solution of trichloroacetic acid are mixed, stood overnight, centrifuging and taking supernatant, is added and supernatant
The solution of trichloroacetic acid of same volume carries out secondary removing protein；By except it is finished it is white after polysaccharide solution be concentrated under reduced pressure into certain volume,
It is slowly added to dehydrated alcohol, makes its alcohol content up to 80~85%, after standing overnight, is filtered with Buchner funnel, obtained precipitating is i.e.
For except the kuh-seng polysaccharide after deproteinized；
Kuh-seng polysaccharide precipitation is dissolved in deionization ultrapure water, is adsorbed through AB-8 type macroporous absorbent resin, is decolourized, except living together
Reason, up to kuh-seng dregs of a decoction polysaccharide after freeze-drying.
6. the preparation method of kuh-seng dregs of a decoction polysaccharide synbiotic according to claim 5, it is characterised in that: kuh-seng dregs of a decoction polysaccharide
It is prepared as follows to obtain:
Kuh-seng dregs of a decoction drying sample 1.0kg is weighed, petroleum ether degreasing is added twice in volume ratio 1:10 ratio by weight, sample after degreasing
Product drying, is added 80% ethyl alcohol of volumetric concentration of 10 times of amounts, and 80 DEG C of water-bath refluxing extraction 2h are filtered while hot, filter residue heat
80% ethyl alcohol washs 2~3 times, and after filter residue and drying, the pure water that 10 times of amounts are added is heated to reflux in 150 DEG C, extracts twice, every time
2h, combined extract；Aqueous extracts are concentrated under reduced pressure to proper volume, are slowly added to dehydrated alcohol, alcohol content is made to reach 80%, 4 DEG C
It stands overnight, is centrifuged, obtain supernatant fluid filtrate and precipitating, precipitating is kuh-seng dregs of a decoction Thick many candies.
Kuh-seng dregs of a decoction Thick many candies 100g is weighed, is dissolved in 1.0L pure water, obtains polysaccharide solution, weighs trichloroacetic acid 100g, it is molten
5% solution of trichloroacetic acid is made in pure water in solution；By Thick many candies and solution of trichloroacetic acid, 1:1 is mixed by volume, 4 DEG C of standings
Overnight, centrifuging and taking supernatant adds the solution of trichloroacetic acid with supernatant same volume, carries out secondary removing protein, will remove finished white
Polysaccharide solution afterwards is concentrated under reduced pressure into certain volume, is slowly added to dehydrated alcohol, makes its alcohol content up to 80%, stood in 4 DEG C
It after night, is filtered with Buchner funnel, obtained precipitating is except the kuh-seng polysaccharide after deproteinized；
It will be dissolved in deionization ultrapure water except the kuh-seng polysaccharide after deproteinized, adsorb, taken off through AB-8 type macroporous absorbent resin
Color, removal of impurities processing, up to kuh-seng dregs of a decoction polysaccharide after freeze-drying.
7. the preparation method of kuh-seng dregs of a decoction polysaccharide synbiotic according to claim 3, which is characterized in that step (2) kuh-seng
The final concentration of 2.0g/L of the fermentation of dregs of a decoction polysaccharide.
8. kuh-seng dregs of a decoction polysaccharide synbiotic of any of claims 1 or 2 preparation adjust intestinal microecology, improve gut barrier and
The drug or the application in health care product of digestion power and strengthen immunity.
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| CN114397221A (en) * | 2021-12-16 | 2022-04-26 | 福建傲农生物科技集团股份有限公司 | Method for determining true protein content in whey powder |
| CN116425901A (en) * | 2023-06-13 | 2023-07-14 | 西南民族大学 | Bitter bamboo shoot polysaccharide and preparation method and application thereof |
| CN116425901B (en) * | 2023-06-13 | 2023-08-18 | 西南民族大学 | Bitter bamboo shoot polysaccharide and preparation method and application thereof |
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