CN109988725A - The preparation technique and application of reducing weight and blood fat microbial bacterial agent and its derivative - Google Patents
The preparation technique and application of reducing weight and blood fat microbial bacterial agent and its derivative Download PDFInfo
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- CN109988725A CN109988725A CN201810665158.9A CN201810665158A CN109988725A CN 109988725 A CN109988725 A CN 109988725A CN 201810665158 A CN201810665158 A CN 201810665158A CN 109988725 A CN109988725 A CN 109988725A
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Abstract
The invention discloses the preparation methods of a kind of active complex micro organism fungicide and its derivative Tiny ecosystem product, and by health food, health care product or drug with reducing blood lipid and auxiliary weight losing function, applies in reducing blood lipid, prevents and treats in the related diseases such as fat and cardiovascular and cerebrovascular.Natural a variety of strains of the activity complex microorganism bacterial classification both from healthy population.According to analysis of biological information and management platform, optimize active complex microorganism bacterial classification formula, obtains good active complex micro organism fungicide and its derivative；Remove the harm such as addition any chemical reagents from；Retain the multifarious symbiosis flora function of enteron aisle, adjusts colony balance, fat metabolism and other effects；It solves the single production technology of tradition, provides new way for the high-valued production of Tiny ecosystem product, fill up industry blank, have a good application prospect；Meanwhile using and perfect " micro ecology of gastrointestinal tract imbalance and fat and closely related cardio-cerebrovascular diseases scientific judgment.
Description
Technical field
The invention belongs to the technical fields of natural microecological health foods, health care product or drug personalized customization, more specifically
Ground is said, is related to the formulating method of the reducing weight and blood fat Tiny ecosystem personalization formula in medical and health field and is precisely applied.
Background technique
Mankind's in the 21st century, living standard has significant raising, disease relevant to life style such as obesity, heart and brain
Vascular diseases etc. seriously threaten the health of the mankind.A large amount of epidemiological survey and studies have shown that hyperlipidemia is these diseases
The important risk factor of disease.
But the drug of current market sales of reducing blood lipid disease is mostly chemical synthetic drug, curative effect is general, and side effect is big.?
There are some (for Different Individual, flora ratio is unalterable) composite bacterias using single culture or blindness to ferment, at
It is point relatively fixed, it is not able to satisfy the Tiny ecosystem products of different races, Different Individual.For example, Chinese Patent Application No. is CN
03128995, data of publication of application is that on 2 8th, 2004 patent application documents disclose a kind of changeless strain group of application
At technical solution.Chinese Patent Application No. is 200610126889.3, and data of publication of application is the patent Shen on May 12nd, 2010
Please file be also to disclose similar Tiny ecosystem product.For example a certain patent of invention (103598594 B of CN) data of publication of application again
For the patent application document on October 28th, 2013, appliable plant lactobacillus, which individually ferments, is made dairy products and drinks etc.；
The present invention is using the dynamic formula of natural activity complex microorganism bacterial classification and its Tiny ecosystem product preparation process of derivative
In, significantly improve the quality and effect of Tiny ecosystem product, remove from chemical substance and additive etc. endangered caused by human body and
Improve the value precisely applied.Can promote the metabolic response of substance in fermentation preparation process, it is a large amount of generate and accumulation it is primary and
The multiple beneficials ingredients such as cometabolism (derivative) product；Meanwhile retaining the function of itself multifarious symbiosis flora.These are beneficial
Ingredient can promote the proliferation of profitable strain, and the breeding of pernicious bacteria and corrupt substance is inhibited to be formed, and play adjusting colony balance
Metabolism with fat reduces taken in excess and accumulation to fat, prevents and treats fat disease related to cardiovascular and cerebrovascular etc.
Disease；Enhancing is immunized, delays the effects of failure.This method combines the biology information technology of current forefront, solves traditional single
One production technology；And include medicine, health food and/or additive, material intermediate as fine Tiny ecosystem product, it is micro-
The high-valued production of ecological product provides new way.Resource is set further to be optimized and be circulated benignly, in fat and heart and brain
It obtains fully precisely applying in the prevention and treatment application of vascular diseases, fills up industry blank, have a good application prospect.
Summary of the invention
It solves the problems, such as
Product for the reducing weight and blood fat sold on existing market is mostly chemical synthetic drug, and curative effect is general, and side effect is big.Though having
Some Tiny ecosystem products unalterable using the single culture or flora ratio of blindness, but it is not able to satisfy different races, difference
Individual eventually leads to unsatisfactory curative effect.The present invention provides a kind of formulating method of reducing weight and blood fat Tiny ecosystem personalization microbial inoculum formula
With accurate application, unique microbial bacterial agent is determined according to the case where not agnate, Different Individual intestinal flora microbial profile
Formula, ferments to natural plants, obtains personalized Tiny ecosystem product, has significant health giving quality, specific aim and uniqueness
Property.
2. technical solution
The present invention is inoculated with access in the mixture of natural plants (fruit, rhizome, edible mushroom, fructification and herbal class etc.), excellent
Change and the Viability complex micro organism fungicide of personalized set carry out harmless treatment and continuously ferment.Utilize the activity naturally mixed
A kind of active complex micro organism fungicide is made in substance, and the metabolite generated by the fermentation of active complex micro organism fungicide
And its Tiny ecosystem product of derivative.The product has the derivative of more nutritional ingredients and microorganism, and can fully protect
The original function for protecting beneficial natural a variety of strains, integrates curative effect and natural ecosystem activity.
Specifically, the present invention provides a kind of natural, optimization and personalization Tiny ecosystem product, which can be by including
The method of following steps is prepared;
(1) human excrement and urine's sample process: extracting and purifies the microbial DNA in sample, using 515F/ 806R PCR (515F-
GTGCCAGCMGCCGCGGTAA, 806R-GGACTACHVGGGTWTCTAAT) primer pair microbial DNA 16S rRNA gene
The hypervariable region V4 expanded；16S rRNA gene is that the corresponding DNA sequence dna of rRNA is encoded on bacterium, is present in all bacteriums
Genome in, conservative of the 16S rRNA with height and specificity and the gene order have more complete reference library
It is analyzed to typing of bacteria.
(2) it measures human body intestinal canal flora content: extracting the total DNA of microorganism in human excrement and urine, then with 16S rRNA work
For target sequence, absolute quantitation is carried out to total microbial flora by quantitatively instant PCR using 515F/ 806R PCR primer,
Using Escherichia coli as standard items；
(3) identify different microorganisms flora ratio in human excrement and urine: by high-throughput gene sequencing technology to the product of amplification into
(sequencing length can reach 500 ~ 600 base of both-end, can completely include 16S genetic fragment for row sequencing；It can be simultaneously to being more than
The pcr amplification product of 10000 samples is measured, and greatly reduces the sequencing price of single sample, removes Price Bottleneck),
Analysis assessment is carried out to sequencing result, the microbial gene sequences database of sequencing result and 16S rRNA is compared, really
Determine the relative amount and ratio of different activities microbial flora in human excrement and urine's sample.
(4) microbial inoculum formula is determined: will be in the microbial flora content of measurement and race's human excrement and urine's microbiological data library
The content distribution of the microbial flora is compared, if relative amount≤25%, the microbial flora in the microbial inoculum that provides formula
Active quantities be not less than 5 × 109cfu/g (mL)；If relative amount > 25%, the microbial bacteria in the microbial inoculum that provides formula
The active quantities of group are not less than 1 × 109cfu/g (mL)；
(5) reducing weight and blood fat microorganism fungus kind is fermented: carrying out the microbial inoculum formula determined in step (4) to mix hair with fermentation substrate
Ferment, when the pH value of fermentation substrate reaches or when close to 5.0, then fermenting-ripening.Fermentation liquid is cooled down, by mixing, filtering
Separation, pasteurization and cooling detect, allotment, are housed in without sunlight direct projection, at shady and cool ventilation.
Further, it is smashed using microballon in the step (1) and/or the method for ultrasonic (KHz of 130 W × 20)
The microbial DNA in sample is extracted, uses fluorescence spectrometry DNA concentration.
Further, the 515F/ 806R PCR primer used in the step (1) is 515F:
GTGCCAGCMGCCGCGGTAA806R；806R:GGACTACHVGGGTWTCTAAT.Bacterial 16 S rRNA gene includes from V1, V2
To totally nine hypervariable regions V9.The 515F/ 806R PCR primer encoded in the present invention by using both-end expands 16S rRNA base
The hypervariable region V4 of cause.The product of amplification carries out high-flux sequence by Illumina or other microarray datasets.
Further, the system of a kind of reducing weight and blood fat Tiny ecosystem personalization microbial inoculum formula according to claim 1
Determine method, it is characterised in that: the microbial flora in human excrement and urine includes lactobacillus acidophilus, lactobacillus plantarum
(Lactobacillus plantarum), Lactobacillus casei, Shi Shi methane brevibacterium, Bifidobacterium, streptococcus thermophilus intend bar
Pseudomonas (Bacteroides) and Prey irrigate Pseudomonas (Prevotella), Ruminococcus gnavus (Ruminococcus gnavus) and
Collins bacterium (Coriobacteriaceae) etc..
Further, the system of a kind of reducing weight and blood fat Tiny ecosystem personalization microbial inoculum formula according to claim 1
Determine method, it is characterised in that: the construction method of the microbial gene sequences database of 16S rRNA is to select by public database
The DNA sequence dna of the 16SrRNA gene of the title and Qi Yue 1.8kb size of common several hundred kinds of bacterial strains in human excrement and urine's sample is selected, number
According in library include bacterial strain detailed system specific name and DNA sequence dna information, public database include RDP, SILVA,
GreenGene、UNITE。
Further, the system of a kind of reducing weight and blood fat Tiny ecosystem personalization microbial inoculum formula according to claim 4
Determine method, it is characterised in that: the method for building up in human excrement and urine's microbiological data library is to pass through high-throughput 16S rRNA gene sequencing
Obtain in a large amount of fecal samples of a certain race microorganism group at distribution percent information, as shown in Figure 2.
Further, a kind of reducing weight and blood fat Tiny ecosystem personalization microbial inoculum formula according to claim 1 or 4
Formulating method, it is characterised in that: the fermentation substrate, including edible vegetable, fruit, rhizome, fungi plant, fructification and sheet
Careless class.Fermentation substrate will through cleaning, stripping and slicing/crushing (length < 1.5cm) and remove impurity (iron wire, stone, plastics, clod,
Meat, oils etc.).
It further, include 70% water and 5 8% sucrose, mass fraction in fermentation substrate.
Further, the sucrose be it is a kind of without by highly refined, band it is sweet it is molding, color is deep
Sugarcane commerieal sugar, is primarily referred to as brown sugar and brown sugar.
Further, fermentation process in step (5) are as follows: 25 ~ 30 DEG C at a temperature of ferment, active complex microorganism into
Field headquarters is supported and breeding, and after the 7d that ferments, when generating without bulk gas, sealed fermenter is to kill pathogen, worm's ovum etc.
Raising harmless treatment degree, this stage at least keep 15-20d.Fermentation process continues, and it is more stable for gradating
Tiny ecosystem product.After temperature is stablized, and pH is reached or close to 5.0 when shows fermenting-ripening, and cooling；Fermentation ends, will
Fermentation liquid mixes, and is concentrated, and is centrifuged, filtering, and pasteurization and cooling, detection obtain the micro- life of reducing weight and blood fat of liquid after allotment
State product；It is housed in without sunlight direct projection, at shady and cool ventilation.Through detecting, above-mentioned liquid-type Tiny ecosystem product is in brown color；pH
5.0-7.5, various formula bacterium total contents in 12 months109Cfu/g(mL), comply with standard.
Beneficial effect
Compared with the prior art, the invention has the benefit that
(1) our zoopery is shown, active complex micro organism fungicide and its derivative can not only efficiently reduce serum
The level of total cholesterol, triglycerides and low density lipoprotein cholesterol, and can be reduced rat body caused by high fat diet
The increase of weight and the accumulation of fat, the purpose for playing control and losing weight have effects that weight-reducing.Therefore, mesh of the invention
Be to provide for one kind be effectively reduced serum total cholesterol, triglyceride levels and weight-reducing Tiny ecosystem biological agent and health care food
Product, the accurate personalized application of especially original active complex micro organism fungicide and its derivative.It is multiple that the present invention relates to activity
Close the application of microbial bacterial agent and its derivative in preparation reducing blood lipid and in terms of assisting diet products.The composite bacteria agent and its derivative
Object can prepare drinks, health food or drug etc..
(2) aforementioned present invention carries out analysis of biological information, optimization and formulation tool by the microbial flora to fecal sample
Targetedly, dynamic active complex micro organism fungicide formula, sends out Nantural non-toxic plant using the microbial inoculum formula
Ferment, it is decomposed after decomposing plant component sufficiently under the action of the active complex micro organism fungicide of personalization building, it obtains high-quality
The active complex micro organism fungicide of personalization and its derivative；The method have the characteristics that by compound microorganism ferments, it can
The stability for improving product removes the harm such as addition any chemical reagents from；It can promote the metabolism of substance in fermentation preparation process
Reaction, it is a large amount of to generate and accumulate primary and cometabolism (derivative) product, it is a variety of to generate oligosaccharide, sugar alcohol, enzyme, oligopeptide etc.
Beneficiating ingredient；Meanwhile retaining the function of itself multifarious symbiosis flora；These beneficiating ingredients can promote the increasing of profitable strain
It grows, the breeding of pernicious bacteria and corrupt substance is inhibited to be formed, play the effects of adjusting colony balance, there are many beneficial to function to individual
Effect.
(3) present invention is using the natural various active complex microorganism bacterial classification and diversification composition from large natural environment
Multiple types natural plants are carried out fermentation steady in a long-term by (according to analysis of biological information result), can make fermentation process more
Sufficiently, make the metabolism for the rich in nutrients for including in natural plants more complete, generate more nutritional ingredients and microorganism
Derivative, and can fully protect the original function of beneficial natural a variety of strains.
(4) present invention solves the single production technology of tradition, mistake by the biology information technology of the current forefront of combination
The reducing weight and blood fat Tiny ecosystem product of liquid is obtained after filter separation, so that resource is further optimized and is circulated benignly, in the modern times
It obtains fully precisely applying in agriculture field, fills up industry blank, greatly tally with the national condition, have a extensive future.
(5) present invention can promote the metabolic response of substance in fermentation preparation process, it is a large amount of generate and accumulation it is primary and
Cometabolism (derivative) product generates the multiple beneficials ingredients such as oligosaccharide, sugar alcohol, enzyme, oligopeptide；Meanwhile it is more to retain itself
The function of the symbiosis flora of sample；These beneficiating ingredients can promote the proliferation of profitable strain, inhibit pernicious bacteria breeding and
Corrupt substance is formed, and is played and is adjusted human body intestinal canal flora balance, enhance individual immunity, delay the effects of failure, has to individual more
Kind beneficial functional.
Detailed description of the invention
Fig. 1 is reducing weight and blood fat microbial bacterial agent preparation method synoptic diagram in the present invention；
Fig. 2 is the flow diagram that enteric microorganism database is constructed in the present invention；
Fig. 3 is the flow diagram that reducing weight and blood fat Tiny ecosystem personalization microbial inoculum formula is formulated in the present invention.
Fig. 4 is the production simple process flow of personalized active complex micro organism fungicide and its derivative in the present invention
Specific embodiment
In order to which the invention will be further elaborated, following individual case study on implementation are enumerated.It solemnly declares simultaneously: these institutes
It states case not limit the scope of the invention in any way, the present invention is not limited solely to these described examples, in claim
In the range of certain changes for being made and adjustment also should belong to the scope of the present invention.
Case study on implementation (zoopery)
In the world, a large amount of experiment has verified that, the health food and/or action of drug evaluation experimental of regulating plasma lipid are carried out with rat
Method it is very mature and reliable.China's ministry standard method " function of health food assessment process and the method for inspection " clear stipulaties
Rat can be used as experimental material to carry out blood fat reducing healthcare food and/or the screening of drug effect, accurate detection and evaluation experimental.
Therefore, reducing blood lipid and weight loss that the present invention designs.In order to customize the microbial inoculum for meeting its individual demand, firstly, building is normal
Rat microbiological data library obtains 30 Wistar male rat fecal samples by high-throughput 16S rRNA gene sequencing
Middle microorganism group then determines dynamic microbial inoculum formula at distribution percent information (process is as illustrated in fig. 1 and 2) by the following method
(as shown in Figure 3.Rat animal test is as follows:
(1) fecal sample is handled: being smashed method using microballon and is extracted and purify the microbial DNA in sample, using 515F/ 806R
The hypervariable region V4 of the 16S rRNA gene of PCR primer pair microbial DNA is expanded；515F/ 806R PCR primer is
515F:GTGCCAGCMGCCGCGGTAA, 806R: GGACTACHVGGGTWTCTAAT.
((2) measure microorganism total amount in excrement: the total DNA of microorganism in excrement are extracted, then using 16S rRNA as mesh
Sequence is marked, carries out absolute quantitation, using Escherichia coli as standard items, gradient 10 using 515F/ 806R PCR primer9, 5 ×
109, 1010, 5 × 1010, 1011, 5 × 1011, 1012Copy number/g；Total content of microorganisms is 2 × 10 in the fecal sample11Copy
Number/g.
(3) identify different microorganisms flora ratio in excrement: by high-throughput gene sequencing technology to the product of amplification into
Row sequencing, carries out analysis assessment to sequencing result, and the microbial gene sequences database of sequencing result and 16S rRNA are carried out
Comparison (construction method of the microbial gene sequences database of 16S rRNA are as follows: by public database (RDP, SILVA,
GreenGene, UNITE etc.) in bacterial genetic information sifting selection fecal sample in common several hundred kinds of bacterial strains about 1.8kb size
16S rRNA gene DNA sequence dna, include the detailed system specific name and DNA sequence dna information of bacterial strain in database), determine
The relative amount and ratio of different activities microbial flora in sample；It is sequenced, is determined in the sample containing lactobacillus plantarum: 5.0
x 108Copy number/ml (g)；Lactobacillus casei: 2.0 x 108Copy number/ml (g)；Bifidobacterium: 3.0 x 109Copy number/
ml(g)；Streptococcus thermophilus: 1.0 x 108Copy number/ml (g)；And bacteroides thetaiotaomicron: 1.0 x 1010Copy number/ml (g)
Deng.
(4) microbial inoculum formula is determined: by the content point of the microbial flora in the microbial flora content of measurement and database
Cloth is compared that (method for building up of fecal microorganism database is by high-throughput 16S rRNA gene sequencing to obtain the race's
Microorganism group is at distribution percent information in rat fecal sample, as shown in Figure 2), relative amount≤25%, the then microbial inoculum provided is matched
The active quantities of the microbial flora are not less than 5 × 10 in side10cfu/g (mL)；If relative amount > 25%, the microbial inoculum provided
The active quantities of the microbial flora are not less than 1 × 10 in formula10cfu/g (mL)；
Lactobacillus plantarum content≤25% is 2.2 × 10 in race difference fecal sample8Copy number/g(25% critical value, under
Together), lactobacillus casei content≤25% is 1.6 × 108Copy number/g, Bifidobacterium content≤25% are 8 × 109Copy number/g, it is thermophilic
Hot streptococcus≤25% is 5 × 108Copy number/g；Bacteroides thetaiotaomicron content≤25% is 5.6 × 109Copy number/g.
Case one
(5) experimental animal is grouped: hyperlipemia rat animal model-present invention selects health 8 week old of cleaning, 200-225g
After Wistar male rat 30, adaptive feeding 3 days, be randomly divided into 2 groups (normal group: 10, hyperlipidemia model group: 20
Only), specific grouping is as shown in table 1.
1 experimental animal of table is grouped situation
| Group | It feeds | Duration is extremely |
| Normal group (10) | Basal feed+water | 30th day |
| Hyperlipidemia model group (20) | High lipid food+water | 30th day |
Method is fed using high lipid food to build hyperlipidemia rats animal model.The formula of high lipid food are as follows: 78.8% basis
Feed (crude protein >=18.0%, crude fat >=4.0%, crude fibre≤5.0%, lysine >=0.82%, coarse ash≤
8.0%, moisture≤10.0%, calcium 1.0-1.8%, phosphorus 0.6-1.2%, salt 0.3-0.8%), 1% cholesterol, 10% yolk
Powder, 10% lard, 0.2% cholate.Normal group feeds arm's length basis feed, hyperlipidemia model group and hyperlipidemia model treatment
Group feed high lipid food, three groups of equal drinking public water supplies of animal (through disinfection by ultraviolet light).
(6) the 30th days, blood is taken from normal rat and hyperlipidemia model group rat eye socket respectively, it is solid to detect total gallbladder in rat blood serum
The content of alcohol (TC), triglycerides (TG) and low density lipoprotein cholesterol (LDL-C).The measurement of rat blood serum: according to
The mode of table 1 feeds rat, and fasting 12h, the method for taking eye socket to take blood collect the blood of each group rat after the test within 30 days
And add a small amount of heparin to prevent blood clotting, after standing 10min, 3000g is centrifuged 15min, collects serum.Select lipids detection reagent
Box measures the content of TC, TG and LDL-C of rat blood serum, and the results are shown in Table 2.
The analysis of 2 rat fat of table: influence of the high fat diet in 30 days to rat fat
| Group | TC(mmol/L) | TG(mmol/L) | LDL-C(mmol/L) |
| Normal group | 1.41±0.29 | 0.76±0.10 | 1.74±0.09 |
| Hyperlipidemia model group | 2.37±0.27* | 0.81±0.11 | 1.7±0.11 |
As shown in Table 2, the TC of hyperlipidemia model group is compared with Normal group, significant difference, illustrates that hyperlipidemia rat is built
It stands successfully.
(7) measurement of rat body weight: every rat surveys weight before starting experiment, then tests the weight of survey in every 30 days,
Fasting 12h before survey weight, the results are shown in Table 3.
The variation of 3 rat body weight of table: influence of the high fat diet in 30 days to rat body weight
| Group | 0 day (Weight averages g) | 30th day (Weight averages g) |
| Normal group (10) | 214.53±3.87 | 243.61±3.96 |
| Hyperlipidemia model group (20) | 213.71±4.04 | 282.16±5.28* |
As shown in Table 3, the rat body weight of hyperlipidemia model group is compared with Normal group, significant difference, further proof obesity,
Hyperlipidemia rat is successfully established.
After (8) 30 days, due to Establishment of Rat Model success, then at random by hyperlipidemia model group be divided into 2 groups (hyperlipidemia model group and
Hyperlipidemia model treatment group), every group 10, specific grouping is as shown in table 4.It detects different in hyperlipidemia model treatment group rat excrement
Microbial flora ratio, and the content of the normal rat microbial flora in the microbial flora content of measurement and database is divided
Cloth is compared, to formulate the flora formula of hyperlipidemia model treatment group rat personalization respectively.At this point, continue Normal group into
Arm's length basis feed is eaten, high lipid food, the three groups of equal drinking public water supply of rat (warps are fed by hyperlipidemia model group and hyperlipidemia model treatment group
Disinfection by ultraviolet light), it is specific as shown in table 4.
4 experimental animal of table is grouped situation
| Group | It feeds | Stomach-filling: 2 times/day, 300 μ l/ times/only | Duration is extremely |
| Normal group (10) | Basal feed+water | Physiological saline | 90th day |
| Hyperlipidemia model group (10) | High lipid food+water | Physiological saline | 90th day |
| Hyperlipidemia model treatment group (10) | High lipid food+water | Personalized activity complex micro organism fungicide and its derivative | 90th day |
Hyperlipidemia model treatment group: it preferably and by personalized complex micro organism fungicide and its derivative (is set in the allotment of following ratio
Meter), stomach-filling is carried out to rat, it is secondary daily (morning and afternoon is each primary), 300 μ l/ times/only；The complex microorganism of personalization formula
Microbial inoculum formula are as follows: lactobacillus plantarum: 5.0 x 1010(cfu)/ml(g)；Lactobacillus casei: 2.0 x 1010 (cfu)/ ml
(g)；Bifidobacterium: 3.0 x 1010 (cfu)/ ml(g)；Streptococcus thermophilus: 1.0 x 1010(cfu)/ ml(g)；With it is many types of
Bacteroid: 1.0 x 1010 (cfu)/ ml(g)。
(9) the 90th days, blood is taken from normal rat, hyperlipidemia model and hyperlipidemia model treatment group rat eye socket respectively, detection is big
The content of total cholesterol (TC), triglycerides (TG) and low density lipoprotein cholesterol (LDL-C) in mouse serum.
The measurement of rat blood serum
Rat is fed in the way of table 4, fasting 12h, the method for taking eye socket to take blood collect each group after the test within the 90th day
The blood of rat simultaneously adds a small amount of heparin to prevent blood clotting, and after standing 10min, 3000g is centrifuged 15min, collects serum.Select blood
Rouge detection kit measures the content of TC, TG and LDL-C of rat blood serum, and the results are shown in Table 5.
The analysis of 5 rat fat of table: after 90 days, influence of the personalized activity complex micro organism fungicide to rat fat
| Group | TC(mmol/L) | TG(mmol/L) | LDL-C(mmol/L) |
| Normal group (10) | 1.64±0.59 | 0.82±0.14 | 1.81±0.07 |
| Hyperlipidemia model group (10) | 3.01±0.57* | 1.51±0.31* | 3.49±0.28* |
| Hyperlipidemia model treatment group (10) | 2.04±0.51** | 0.91±0.19** | 2.01±0.32** |
As shown in Table 5, for TC, TG, LDL-C of hyperlipidemia model group compared with Normal group, difference is extremely significant, illustrates hyperlipidemia
The success of disease Establishment of Rat Model.
Known again by table 5, compared with hyperlipidemia model group, the height of personalized activity complex micro organism fungicide and its derivatives for treatment
Rat TC, TG and LDL-C conspicuousness of rouge model treatment group reduces；Show active complex micro organism fungicide to the big of high fat diet
Mouse has significant effect for reducing blood fat.It can be seen that active complex micro organism fungicide, which has, adjusts blood lipid metabolism, in addition prevention and
Treat cardiovascular and cerebrovascular disease relevant to Dyslipidemia etc..
(10) measurement of rat body weight: every rat surveys weight before starting experiment, tests the weight of survey in every 30 days, surveys body
Fasting 12h before weight, the results are shown in Table 6.
The variation of 6 rat body weight of table: after 90 days, the shadow of personalized complex micro organism fungicide and high fat diet to rat body weight
It rings
| Group | 30th day (g) | 60th day (g) | 90th day (g) |
| Normal group (10) | 243.61±3.96 | 265.42±3.07 | 283.46±2.34 |
| Hyperlipidemia model group (10) | 280.33±4.21* | 337.81±4.23* | 379.63±3.79* |
| Hyperlipidemia model treatment group (10) | 283.99±3.25* | 309.17±6.54# | 331.72±5.27# |
As shown in Table 6, the rat body weight of hyperlipidemia model group is compared with Normal group, significant difference, illustrates fat, hyperlipidemia
The success of disease Establishment of Rat Model.
Can significantly it be found out according to the variation tendency of rat body weight between the different groups of table 6, after 60 days, personalized activity is multiple
The hyperlipidemia model treatment group rat body weight for closing microbial bacterial agent and its derivatives for treatment is obviously lower than hyperlipidemia model group；In particular,
In the 90th day weight substantially close to rats in normal control group, display effect was significant.The above result shows that personalized activity is compound
Microbial bacterial agent and its derivative have apparent effect in terms of controlling and mitigating rat body weight, be suitble to it is original develop with it is auxiliary
Help the relevant Tiny ecosystem product of antiobesity action.
Case two
(11) experimental animal is grouped: hyperlipemia rat animal model-present invention selects health 16 week old of cleaning, 230-250g
After Wistar male rat 30, adaptive feeding 3 days, be randomly divided into 2 groups (normal group: 10, hyperlipidemia model group: 20
Only), specific grouping is as shown in table 1.Method is fed using same high lipid food to build hyperlipidemia rats animal model.
(12) the 30th days, blood is taken from normal rat and hyperlipidemia model group rat eye socket respectively, detects total gallbladder in rat blood serum
The content of sterol (TC), triglycerides (TG) and low density lipoprotein cholesterol (LDL-C).The measurement of rat blood serum: it adopts
Serum is collected with identical method.The content of TC, TG and LDL-C of lipids detection kits rat blood serum are selected, as a result
As shown in table 7.
The analysis of 7 rat fat of table: influence of the high fat diet in 30 days to rat fat
| Group | TC(mmol/L) | TG(mmol/L) | LDL-C(mmol/L) |
| Normal group | 1.61±0.32 | 0.91±0.21 | 1.93±0.11 |
| Hyperlipidemia model group | 2.71±0.29* | 0.95±0.08 | 2.01±0.13 |
As shown in Table 7, the TC of hyperlipidemia model group is compared with Normal group, significant difference, illustrates that hyperlipidemia rat is built
It stands successfully.
(13) measurement of rat body weight: every rat surveys weight before starting experiment, then tests the weight of survey in every 30 days,
Fasting 12h before survey weight, the results are shown in Table 8.
The variation of 8 rat body weight of table: influence of the high fat diet in 30 days to rat body weight
| Group | 0 day (Weight averages g) | 30th day (Weight averages g) |
| Normal group (10) | 234.27±2.26 | 253.41±2.45 |
| Hyperlipidemia model group (20) | 231.31±2.07 | 307.43±4.17* |
As shown in Table 8, the rat body weight of hyperlipidemia model group is compared with Normal group, significant difference, further proof obesity,
Hyperlipidemia rat is successfully established.
After (14) 30 days, due to Establishment of Rat Model success, then hyperlipidemia model group is divided into 2 groups of (hyperlipidemia model groups at random
With hyperlipidemia model treatment group), every group 10, specific grouping is as shown in table 9.It detects in hyperlipidemia model treatment group rat excrement not
With microbial flora ratio, and by the content of the normal rat microbial flora in the microbial flora content of measurement and database
Distribution is compared, to formulate the flora formula of hyperlipidemia model treatment group rat personalization respectively.At this point, continuing Normal group
Arm's length basis feed is fed, high lipid food, three groups of equal drinking public water supplies of rat are fed by hyperlipidemia model group and hyperlipidemia model treatment group
(through disinfection by ultraviolet light), it is specific as shown in table 9.
9 experimental animal of table is grouped situation
| Group | It feeds | Stomach-filling: 2 times/day, 300 μ l/ times/only | Duration is extremely |
| Normal group (10) | Basal feed+water | Physiological saline | 90th day |
| Hyperlipidemia model group (10) | High lipid food+water | Physiological saline | 90th day |
| Hyperlipidemia model treatment group (10) | High lipid food+water | Personalized activity complex micro organism fungicide and its derivative | 90th day |
Hyperlipidemia model treatment group: it preferably and by personalized complex micro organism fungicide and its derivative (is set in the allotment of following ratio
Meter), stomach-filling is carried out to rat, it is secondary daily (morning and afternoon is each primary), 300 μ l/ times/only；The complex microorganism of personalization formula
Microbial inoculum formula are as follows: lactobacillus plantarum: 4.0 x 1010(cfu)/ml(g)；Lactobacillus casei: 3.0 x 1010 (cfu)/ ml
(g)；Bifidobacterium: 6.0 x 1010 (cfu)/ ml(g)；Streptococcus thermophilus: 2.0 x 1010(cfu)/ ml(g)；With it is many types of
Bacteroid: 1.0 x 1010 (cfu)/ ml(g)。
(15) the 90th days, blood is taken from normal rat, hyperlipidemia model and hyperlipidemia model treatment group rat eye socket respectively, detection is big
The content of total cholesterol (TC), triglycerides (TG) and low density lipoprotein cholesterol (LDL-C) in mouse serum.
The measurement of rat blood serum
Rat is fed in the way of table 9, fasting 12h, the method for taking eye socket to take blood collect each group after the test within the 90th day
The blood of rat simultaneously collects serum.The content of TC, TG and LDL-C of lipids detection kits rat blood serum are selected, as a result
As shown in table 10.
The analysis of 10 rat fat of table: after 90 days, influence of the personalized activity complex micro organism fungicide to rat fat
| Group | TC(mmol/L) | TG(mmol/L) | LDL-C(mmol/L) |
| Normal group (10) | 1.71±0.73 | 0.94±0.2 | 1.89±0.12 |
| Hyperlipidemia model group (10) | 3.34±0.68* | 1.83±0.52* | 3.78±0.29* |
| Hyperlipidemia model treatment group (10) | 1.99±0.63** | 1.01±0.36** | 2.03±0.28** |
As shown in Table 10, for TC, TG, LDL-C of hyperlipidemia model group compared with Normal group, significant difference illustrates hyperlipemia
Establishment of Rat Model success.
Known again by table 10, compared with hyperlipidemia model group, personalized activity complex micro organism fungicide and its derivatives for treatment
Rat TC, TG and LDL-C conspicuousness of hyperlipidemia model treatment group reduces；Show active complex micro organism fungicide to high fat diet
Rat has significant effect for reducing blood fat.It can be seen that personalized activity complex micro organism fungicide, which has, adjusts blood lipid metabolism, very
To preventing and treating relevant to Dyslipidemia cardiovascular and cerebrovascular disease etc..
(16) measurement of rat body weight: every rat surveys weight before starting experiment, tests the weight of survey in every 30 days, surveys body
Fasting 12h before weight, as a result as shown in table 11.
The variation of 11 rat body weight of table: after 90 days, personalized complex micro organism fungicide and high fat diet are to rat body weight
It influences
| Group | 30th day (g) | 60th day (g) | 90th day (g) |
| Normal group (10) | 253.41±2.45 | 297.23±4.19 | 311.23±3.25 |
| Hyperlipidemia model group (10) | 305.56±5.24* | 371.37±4.51* | 419.61±4.41* |
| Hyperlipidemia model treatment group (10) | 309.61±4.17* | 321.39±5.18# | 337.19±4.09# |
As shown in Table 11, the rat body weight of hyperlipidemia model group is compared with Normal group, significant difference, illustrates fat, hyperlipidemia
The success of disease Establishment of Rat Model.
Can significantly it be found out according to the variation tendency of rat body weight between the different groups of table 11, after 60 days, personalized activity
The hyperlipidemia model treatment group rat body weight of complex micro organism fungicide and its derivatives for treatment is obviously lower than hyperlipidemia model group；Especially
It is that, in the 90th day weight substantially close to rats in normal control group, display effect is significant.The above result shows that personalized activity is multiple
Close microbial bacterial agent and its derivative has apparent effect in terms of controlling and mitigating rat body weight, be suitble to original exploitation and
Assist the relevant Tiny ecosystem product of antiobesity action.
(17) the production simple process flow (Fig. 4) of personalized active complex micro organism fungicide and its derivative.
Claims (10)
1. the Tiny ecosystem product preparation method of reducing weight and blood fat natural activity complex micro organism fungicide and its derivative, including it is following
Step:
(1) human excrement and urine's sample process: extracting and purifies the microbial DNA in sample, using 515F/806R PCR primer to micro-
The hypervariable region V4 of the 16S rRNA gene of biological DNA is expanded；
(2) it measures human body intestinal canal flora content: the total DNA of microorganism in human excrement and urine is extracted, then using 16S rRNA as mesh
Sequence is marked, absolute quantitation is carried out using 515F/806R PCR primer, using Escherichia coli as standard items；
(3) identify different microorganisms flora ratio in human excrement and urine: by high-throughput gene sequencing technology to the product of amplification into
Row sequencing, carries out analysis assessment to sequencing result, and the microbial gene sequences database of sequencing result and 16S rRNA are carried out
Comparison, determines the relative amount and ratio of different activities microbial flora in human excrement and urine's sample；
(4) microbial inoculum formula is determined: the microbial flora content of measurement and this in race's human excrement and urine's microbiological data library is micro-
The content distribution of biological flora is compared, if relative amount≤25%, the microbial flora in the microbial inoculum that provides formula
Active quantities are not less than 5 × 109cfu/g(mL)；If relative amount > 25%, the microbial flora in the microbial inoculum that provides formula
Active quantities be not less than 1 × 109cfu/g(mL)；
(5) reducing weight and blood fat microorganism fungus kind is fermented: carrying out the microbial inoculum formula determined in step (4) to mix hair with fermentation substrate
Ferment, when the pH value of fermentation substrate reaches or when close to 5.0, then fermenting-ripening.
2. a kind of formulating method of reducing weight and blood fat Tiny ecosystem personalization microbial inoculum formula according to claim 1, feature
It is: is smashed in the step (1) using microballon and/or ultrasonic method extracts the microbial DNA in sample.
3. a kind of formulating method of reducing weight and blood fat Tiny ecosystem personalization microbial inoculum formula according to claim 1 or 2, special
Sign is: the 515F/806R PCR primer used in the step (1) is 515F-GTGCCAGCMGCCGCGGTAA, 806R-
GGACTACHVGGGTWTCTAAT。
4. a kind of formulating method of reducing weight and blood fat Tiny ecosystem personalization microbial inoculum formula according to claim 1, feature
It is: the microorganism fungus kind of reducing weight and blood fat Tiny ecosystem personalization microbial inoculum formula, including lactobacillus acidophilus, lactobacillus plantarum
(Lactobacillus plantarum), Lactobacillus casei, Shi Shi methane brevibacterium, Bifidobacterium, streptococcus thermophilus intend bar
Pseudomonas (Bacteroides) and Prey irrigate Pseudomonas (Prevotella), Ruminococcus gnavus (Ruminococcus gnavus),
With Collins bacterium (Coriobacteriaceae) etc..
5. a kind of formulating method of reducing weight and blood fat Tiny ecosystem personalization microbial inoculum formula according to claim 4, feature
Be: the construction method of the microbial gene sequences database of 16S rRNA is to select human excrement and urine's sample by public database
In common several hundred kinds of bacterial strains title and Qi Yue 1.8kb size 16SrRNA gene DNA sequence dna, include bacterial strain in database
Detailed system specific name and DNA sequence dna information, public database include RDP, SILVA, GreenGene, UNITE.
6. a kind of formulating method of reducing weight and blood fat Tiny ecosystem personalization microbial inoculum formula according to claim 4, feature
It is: the method for building up in human excrement and urine's microbiological data library are as follows: a large amount of human body excrement of a certain race are obtained by high-flux sequence
Just in sample microorganism group at distribution percent information.
7. a kind of formulating method of reducing weight and blood fat Tiny ecosystem personalization microbial inoculum formula according to claim 1 or 4, special
Sign is: the fermentation substrate, including veterinary antibiotics, rhizome, fungi plant, fructification and herbal class.
8. a kind of formulating method of reducing weight and blood fat Tiny ecosystem personalization microbial inoculum formula according to claim 7, feature
It is: includes 70% water and 5~8% sucrose, mass fraction in fermentation substrate.
9. a kind of formulating method of reducing weight and blood fat Tiny ecosystem personalization microbial inoculum formula according to claim 8, feature
Be: the sucrose is a kind of without leading by highly refined, band honey is molding, sugarcane commerieal sugar that color is deep
Refer to brown sugar and brown sugar.
10. a kind of formulating method of reducing weight and blood fat Tiny ecosystem personalization microbial inoculum formula according to claim 7 or 8,
Be characterized in that: fermentation process in step (5) are as follows: 25~30 DEG C at a temperature of fermentation 7d after, without bulk gas generate when, it is close
Seal fermentation tank at least keeps 15-20d；After temperature is stablized, and pH is reached or close to 5.0 when shows fermenting-ripening, and cooling；Hair
Ferment terminates, and fermentation liquid is mixed, and is concentrated, and is centrifuged, filtering, pasteurization and cooling, detection, and the weight-reducing drop of liquid is obtained after allotment
Blood lipid Tiny ecosystem product；It is housed in without sunlight direct projection, at shady and cool ventilation.
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