CN110522035A - A kind of humanized's probiotics and its application in terms of assisting in reducing blood sugar - Google Patents
A kind of humanized's probiotics and its application in terms of assisting in reducing blood sugar Download PDFInfo
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- CN110522035A CN110522035A CN201910939473.0A CN201910939473A CN110522035A CN 110522035 A CN110522035 A CN 110522035A CN 201910939473 A CN201910939473 A CN 201910939473A CN 110522035 A CN110522035 A CN 110522035A
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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
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/143—Fermentum
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Mycology (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention provides a kind of humanized's probiotics, it include: 11 freeze-dried vaccine powder of lactobacillus fermenti ZLT, 305 freeze-dried vaccine powder of lactobacillus fermenti ZLT, 22 freeze-dried vaccine powder of lactobacillus plantarum ZLT, 25 freeze-dried vaccine powder of lactobacillus plantarum ZLT are mixed according to mass ratio 1:1:1:1, further comprise polydextrose and oligofructose.Application the present invention also provides above-mentioned humanized's probiotics as the food and health care product of assisting in reducing blood sugar.
Description
Technical field
The invention belongs to microorganism fields, are related to a kind of probiotics preparation, and in particular to a kind of humanized's probiotics and
Its application on assisting in reducing blood sugar.
Background technique
Diabetes are a kind of metabolic diseases, and main feature is exactly that blood glucose level increases, and are divided into Type I diabetes and II type sugar
Urine disease.The wherein 85%-95% of the total number of patients of the most Zhan of patients with NIDDM.Type-1 diabetes mellitus be T- it is cell-mediated from
Body immunological diseases, the pancreatic beta cell for showing as generating insulin are destroyed, patient must insulin injection treat.II type sugar
Urine disease be beta Cell of islet caused by insulin resistance destruction and (or) insulin it is opposite lack it is caused characterized by hyperglycemia
Metabolic disease, number of the infected constantly increases.If cannot obtain it is effective must control, lasting hyperglycemia can damage heart and brain blood
Pipe, kidney etc., can trigger multiple complications.Some researches show that the changes of enteric microorganism and obesity, cardiovascular disease, glycosuria
The metabolic diseases such as disease are related.Type II diabetes is complicated gene- environment interaction as a result, not only related with human body gene
It is also related with enteric microorganism group.Existing research shows the plan by changing the metabolic diseases such as enteric microorganism group control diabetes
Slightly.
Since conventional medicament has very strong side effect, probiotics is highly-safe, and side effect is low, for probiotics in anti-glycosuria
The research in sick direction is more and more, plays the role of test and confirms probiotics in alleviating type II diabetes.Research also turns out benefit
Raw bacterium has improvement inflammation, reduces blood lipid, reduces blood glucose, alleviates the effect of metabolic syndrome.Lactic acid bacteria is fermentation carbon hydrate
A kind of general name without gemma, gram-positive bacterium of object, it can generate cream from the fermentation process of glucose or lactose
The bacterium of acid, is that one kind is present in the intracorporal probiotics of the mankind, it, which has, adjusts function of human body, is delayed senescence, prevention disease etc.
Effect, is widely used in food and health care product.Effect of the lactic acid bacteria in terms of resisting diabetes is also increasingly by research people
The attention of member.Currently, diabetes are other than using drug therapy, it is more and more it is demonstrated experimentally that lactic acid bacteria has anti-diabetic
Effect, mainly passing through reduces oxidative stress, inhibits alpha-glucosaccharase enzyme activity, balance intestinal microorganism and adjusts the side such as immune
Formula reduces blood glucose.Alpha-glucosidase is the key enzyme of the promotion carbohydrate inversion positioned at enteric cavity and brush edge film,
Function is that the carbohydrate inversions such as starch and polysaccharide Cheng Yi is promoted to be absorbed monosaccharide, inhibits its activity, it is possible to reduce glucose
Absorption, reduce level of postprandial blood sugar, effectively type II diabetes delayed to fall ill, be to reduce before type II diabetes controls risk and most have
One of the therapeutic strategy on way.In addition, alpha-amylase inhibitor can effectively inhibit ptyalin and Pancreatic arnylase activity, slow down
The decomposition of carbohydrate, to achieve the purpose that reduce blood glucose.Alpha-amylase inhibitory activity is normally used for dropping as evaluation
One of the Critical policies of one of index of blood glucose ability and type II diabetes control blood glucose.
But existing it is found to have alpha-amylase inhibition and the compound of alpha-glucosaccharase enzyme inhibition is mostly
It is studied as drug, medication period is long, at high cost.
Summary of the invention
To solve the above-mentioned problems, the purpose of the present invention is to provide a kind of humanized's probiotics, the humanized is prebiotic
Alpha-glucosidase and alpha-amylase can be effectively suppressed in microbial inoculum, so as to slow down decomposition and the oligosaccharide hydrolysis of carbohydrate
For the process of monosaccharide, slow down the rising of postprandial blood sugar, thus assisting in reducing blood sugar.
Another object of the present invention is to provide a kind of preparation methods of above-mentioned humanized's probiotics.
Third object of the present invention is to provide a kind of food of above-mentioned humanized's probiotics as assisting in reducing blood sugar
The application of product and health care product.
To achieve the goals above, the present invention provides a kind of humanized's probiotics, including following bacterial strain:
11 freeze-dried vaccine powder of lactobacillus fermenti ZLT, 305 freeze-dried vaccine powder of lactobacillus fermenti ZLT, lactobacillus plantarum ZLT 22
Freeze-dried vaccine powder, 25 freeze-dried vaccine powder of lactobacillus plantarum ZLT are mixed according to mass ratio 1:1:1:1；
Wherein, 11 freeze-dried vaccine powder of lactobacillus fermenti ZLT, 305 freeze-dried vaccine powder of lactobacillus fermenti ZLT, lactobacillus plantarum ZLT
22 freeze-dried vaccine powder, 25 freeze-dried vaccine powder of lactobacillus plantarum ZLT viable bacteria concentration be 1010cfu/g；
Wherein, the classification naming of lactobacillus plantarum ZLT 22 are as follows: lactobacillus plantarum (Lactobacillus plantarum)
ZLT 22；Depositary institution are as follows: China Committee for Culture Collection of Microorganisms's common micro-organisms center；Address are as follows: court, Beijing
No. 3 Institute of Microorganism, Academia Sinica, institute of positive area's North Star West Road 1；Deposit number are as follows: CGMCC No.18208；Preservation date
Are as follows: on July 11st, 2019；
The classification naming of lactobacillus plantarum ZLT 25 are as follows: lactobacillus plantarum (Lactobacillus plantarum) ZLT
25；Depositary institution are as follows: China Committee for Culture Collection of Microorganisms's common micro-organisms center；Address are as follows: Chaoyang District, Beijing City
No. 3 Institute of Microorganism, Academia Sinica, institute of North Star West Road 1；Deposit number are as follows: CGMCC No.18209；Preservation date are as follows:
On July 11st, 2019；
The classification naming of lactobacillus fermenti ZLT 11 are as follows: lactobacillus fermenti (Lactobacillus fermentum) ZLT
11；Depositary institution are as follows: China Committee for Culture Collection of Microorganisms's common micro-organisms center；Address are as follows: Chaoyang District, Beijing City
No. 3 Institute of Microorganism, Academia Sinica, institute of North Star West Road 1；Deposit number are as follows: CGMCC No.18206；Preservation date are as follows:
On July 11st, 2019；
The classification naming of lactobacillus fermenti ZLT 305 are as follows: lactobacillus fermenti (Lactobacillus fermentum) ZLT
305；Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center；Address are as follows: Chaoyang District, Beijing City north
No. 3 Institute of Microorganism, Academia Sinica, institute of occasion West Road 1；Deposit number are as follows: CGMCC No.18207；Preservation date are as follows:
On July 11st, 2019.
A more progressive ground further includes polydextrose and oligofructose, wherein polydextrose and oligofructose and acidified milk bar
11 freeze-dried vaccine powder of bacterium ZLT, 305 freeze-dried vaccine powder of lactobacillus fermenti ZLT, 22 freeze-dried vaccine powder of lactobacillus plantarum ZLT, plant cream bar
The ratio of 25 freeze-dried vaccine powder of bacterium ZLT are as follows: 11 freeze-dried vaccine powder of lactobacillus fermenti ZLT, 305 freeze-dried vaccine powder of lactobacillus fermenti ZLT,
22 freeze-dried vaccine powder of lactobacillus plantarum ZLT, 25 freeze-dried vaccine powder of lactobacillus plantarum ZLT, polydextrose, oligofructose are according to quality
Than 1:1:1:1:(1-2): (0.5-1.5) is mixed.
The preferred mass of above-mentioned bacterial strains and prebiotics ratio are as follows: 1:1:1:1:1.5:1.
The present invention also provides a kind of preparation methods of above-mentioned humanized's probiotics, include the following steps:
1) by lactobacillus fermenti ZLT 11, lactobacillus fermenti ZLT 305, lactobacillus plantarum ZLT 22, lactobacillus plantarum ZLT
25 bacterial strains are activated respectively, amplify culture, and above-mentioned four kinds of bacterium solutions are respectively obtained；
2) above-mentioned four kinds of bacterium solutions are centrifugated respectively and collect wet thallus, then be put into freeze-dryer and freeze after washing respectively
It is dry to collect, the 11 bacterium powder of lactobacillus fermenti ZLT after being lyophilized, 305 bacterium powder of lactobacillus fermenti ZLT, lactobacillus plantarum ZLT
25 bacterium powder of 22 bacterium powder and lactobacillus plantarum ZLT；
3) by 11 bacterium powder of lactobacillus fermenti ZLT, 305 bacterium powder of lactobacillus fermenti ZLT, 22 bacterium powder of lactobacillus plantarum ZLT and
25 bacterium powder of lactobacillus plantarum ZLT mixes in proportion.
Further, further includes: polydextrose and oligofructose are proportionally added into step 3).
Further, the activation method are as follows: aforementioned four bacterial strain is inoculated into respectively in MRS broth bouillon,
37 DEG C of stationary culture 12h；The method of the expansion culture are as follows: be inoculated into aforementioned four bacterial strain with 5% inoculum concentration respectively
In MRS broth bouillon, 37 DEG C of stationary culture 12h.
Application the present invention also provides above-mentioned humanized's probiotics as the food and health care product of assisting in reducing blood sugar.
The beneficial effects of the present invention are:
The present invention provides a kind of humanized's probiotics and preparation method thereof, and humanized's probiotics, said preparation can fit
It is colonized in human gastrointestinal tract, alpha-glucosidase and alpha-amylase can be effectively suppressed, so as to slow down point of carbohydrate
Solution and oligosaccharide hydrolysis are the process of monosaccharide, slow down the rising of postprandial blood sugar, thus assisting in reducing blood sugar, auxiliary alleviates II type sugar
Urinate the symptom of disease.Experiment shows that the optimum combination of four kinds of bacterium powder can achieve 93.18% to alpha-amylase activity inhibition, to α-Portugal
Polyglycoside inhibition of enzyme activity can achieve 75.27%；It presses down alpha-glucosidase activity after polydextrose, oligofructose is added
System reaches 82.33%.
Specific embodiment
The separation and screening of probiotics are mainly derived from traditional fermented food, natural environment, animal and human body etc. at present, by
It differs greatly in the growing environment of external source probio and the environment of human gastrointestinal tract, thus many scholars think ideal probiotics
Preferably from the gastrointestinal tract of human body itself.Research finds that there is the passes that specificity mutually selects between host and intestinal flora
System will enhance bacterial strain to the specificity and specific aim of host's beneficial function when the source of bacterial strain is consistent with using object.This
Outside, source of people probiotics compares external source probio, in resistance to bile, acidproof and resistant to gastric juice, gastrointestinal tract field planting and adhesive force, and bacteriostasis
Etc. functionality have more advantage.Accordingly, with respect to the probiotics in other sources, source of people probiotics with high security, no
Be easy repelled by the immune system of human body intestinal canal, have genetic stability, thus be more suitable for mankind probiotics come using.
Recently research have indicated that probiotics can be used as effective biologic medical means to achieve the purpose that intervene diabetes, and it is newborn
Sour bacterium has the function of potential prevention and treatment diabetes, and probiotics is acknowledged as edible safety.Lactobacillus plantarum and hair
Kefir milk bacillus belongs to lactobacillus in lactic acid bacteria, they are edible in the Ministry of Public Health " the strain list that can be used for food "
The probiotics of safety.Probiotic composition may include one or more probiotics, have to the effect of human body because of the difference of its type
Institute's difference.And the prebiotic effect of bacterial strain also has specificity, and the different strains of same strain may generate different effects,
It prevents diabetes and reduces definite molecular mechanism involved in blood glucose to still need to carry out deeply and systematic research.
Prebiotics are the functional food for having the multi-functional factor, have the defaecation factor, the increment factor of Bifidobacterium, poison
The functions such as plain clearing factor can pass through the active matter of the intracorporal one or several kinds of microorganism growths of selective stimulating host
Matter, so as to change enteric microorganism composition and then generate wholesome effect to body.Prebiotics are by promoting beneficial bacterium
Breeding inhibits harmful bacterial growth, to maintain the equilibrium state of intestinal flora, promotes body health.Oligofructose belongs to benefit
Raw member also known as fructooligosaccharide, bifidus factor, are that the probiotics such as Bifidobacterium are proliferated rapidly proliferant agent in enteron aisle, be it is a kind of not by
Human consumption absorbs, and directly reaches the oligosaccharides of large intestine.Correlative study, which shows that functional oligose has, reduces gallbladder in serum
Sterol content reduces blood glucose and decomposes the functions such as carcinogenic substance.With researcher deepening continuously to the research of intestinal flora,
Dietary fiber is considered as a kind of good prebiotics, it, which can partially be fermented by intestinal flora, generates small molecule bioactive object
Matter improves intestinal flora composition, and then adjusts body metabolism, has preferable control action to the state of an illness of diabetic.Poly- Portugal
Grape sugar is a kind of low in calories, low-glycemic special carbohydrate, has the characteristics that water-soluble dietary fiber and prebiotics,
It is rapidly developed in recent years.Intestines and stomach micro-ecological environment, the physiological properties such as defaecation, prevention intestines problem are adjusted possessed by it
Can be widely applied to it in various food.
The main metabolic of diabetes is characterized in that blood glucose persistently increases, and slows down that starch decomposes in small intestine and monosaccharide is in small intestine
In absorption be resistant to one of important method of diabetes.Alpha-amylase and alpha-glucosidase are that starch decomposes in absorption process
Important enzyme.The two enzymatic activitys are inhibited to can effectively reduce the raising of blood glucose.Now many hypoglycemic medicines using the two enzymes as
Target spot, especially α glucuroide.
The present invention carries out count plate by the screening lactobacillus in healthy human faecal mass, to the lactic acid bacteria being separated to, in vitro
Two plants of lactobacillus fermenti (lactobacillus fermentis are selected in anti-alpha-amylase and alpha-glucosidase experiment, in vitro digestion experiment
ZLT 11, lactobacillus fermenti ZLT 305) and two lactobacillus plantarums (lactobacillus plantarum ZLT 22, lactobacillus plantarum ZLT
25).Four plants of bacterium are freeze-dried respectively and are made into bacterium powder.By four factors, three horizontal quadrature, determination inhibits four kinds of bacterium powder in vitro in fact
Primary and secondary sequence and concentration level in alpha-amylase activity and alpha-glucosidase activity experiment.Again by polydextrose and oligomeric
The experiment of single factor of two kinds of prebiotics of fructose determines three concentration of polydextrose (10%, 15%, 20%), and oligofructose three dense
It spends (5%, 10%, 15%) and four kinds of bacterium powder carries out inhibiting alpha-amylase activity and alpha-glucosaccharase outside six factors, three level body
The orthogonal experiment of enzymatic activity.It determines optimum combination and influences primary and secondary sequence.Bacterium powder is compared again and bacterium powder adds two kinds of situations of prebiotics,
The two has significant difference (p < 0.05).
Since lactobacillus plantarum and lactobacillus fermenti belong to lactic acid bacteria major class, for the ease of expression, it be not related to specifically
When bacterial strain, with lactic acid bacteria refer to the present embodiments relate to whole lactobacillus plantarums and lactobacillus fermenti.
Material:
1.MRS broth bouillon (liquid) and MRS agar medium (solid) purchase are limited from Beijing overpass technical concern
Company.
It is configured to fluid nutrient medium to specifications using preceding MRS broth bouillon and sterilize spare.
It is prepared into MRS solid plate to specifications using preceding MRS agar medium and sterilize spare.
2. alpha-amylase, alpha-glucosidase are bought from sigma company.
The purchase of 3.4- nitrobenzophenone-α-D- glucopyranoside likes (Shanghai) chemical conversion industry Development Co., Ltd from ladder is uncommon.
4. oligofructose, polydextrose are purchased from hundred garden Long Chuan biotech inc of Shandong.
5. other reagents are that analysis is pure, buy from Beijing Chemical Plant.
Equipment
1. high-pressure steam sterilizing pan is bought from Autoclave company.
2. constant incubator is bought from the permanent Science and Technology Ltd. in Shanghai one.
3.Varioskan Flash multi-function microplate reader is bought from Thermo Fischer Scient Inc., the U.S..
4. freeze drier is bought from Beijing Bo Yikang laboratory apparatus Co., Ltd.
5. low-temperature and high-speed centrifuge is bought from Eppendorf company, Germany.
6. Chinese mugwort card KS 4000i temperature control shaking table purchase ends certainly card (Guangzhou) experimental instruments and equipment limited (IKA China).
Embodiment 1
1. strain isolation and identification
It takes healthy human faecal mass 10g to be put into cultivate in 100mL MRS broth bouillon and carry out gradient dilution afterwards for 24 hours, chooses and close
Suitable gradient (10-6、10-7、10-8) be applied on MRS solid plate.37 DEG C of stationary cultures for 24 hours after size, shape according to bacterium colony
Shape, color choose bacterium colony, draw inclined-plane and are saved and numbered, and the bacterial strain of all screenings is carried out 16S rDNA Molecular Identification, is obtained
To 5 lactobacillus plantarums, 18 plants of lactobacillus fermentis, the results are shown in Table 1.
The 16S rDNA Molecular Identification of 1 isolated strains of table
Embodiment 2
1. viable count measures
By above-mentioned 5 lactobacillus plantarum, 18 plants of lactobacillus fermentis are respectively from one ring bacterium of picking on the inclined-plane of respective preservation
At to 37 DEG C of MRS broth bouillon after stationary culture 12h, the strain cultured solution after activation is inoculated into MRS meat by 5% inoculum concentration
Soup culture medium, stationary culture 12h at 37 DEG C.0.9mL bacterium solution is taken to carry out 10 times of gradient dilutions.Choose suitable gradient (10-6、10-7、10-8) be coated.Each gradient takes 0.1mL bacterium solution to be placed on MRS solid plate, uniformly spreadable with spreader, stands 1h
After be inverted in 37 DEG C of constant incubator cultures for 24 hours, carry out bacterium colony counting.
Carrying out viable count measurement by bacteria concentration after calculating fermented and cultured is the growing state to understand bacterium, is subsequent experimental
Data are provided to support.
2. prepared by bacteria suspension
By above-mentioned 5 lactobacillus plantarum, 18 plants of lactobacillus fermentis are seeded in activation culture in MRS broth bouillon respectively,
Then 4 DEG C, 8000r/min is centrifuged 15min；Supernatant is outwelled, bacterium mud is resuspended with sterile water, adjustment bacteria concentration reaches
109Sample liquid of the cfu/mL as subsequent experimental.
3. lactic acid bacteria inhibits alpha-amylase activity
The 0.02M PBS buffer solution that 10 μ L include alpha-amylase solution (0.5mg/mL) is added in centrifuge tube
(pH6.9), 50 μ L sample liquids, 37 DEG C of water-bath 10min are added；Then the 0.02M PBS that 50 μ L include 1% (w/v) starch is added
Buffer, 37 DEG C of water-bath 10min；20 μ L DNS reagents are added, boiling water bath 10min is cooled to room temperature and adds 1mL distilled water, takes 200
μ L spreads 96 orifice plates, surveys absorbance at 540nm with microplate reader.It uses buffer to replace enzyme solutions as blank, is replaced with buffer
Sample liquid is as control.
4 lactic acid bacterias are to alpha-glucosaccharase enzyme inhibition
50 μ L PBS buffer solution (pH6.9), 20 μ L bacteria suspensions, 20 μ L 4- nitrobenzophenone-α-D- pyrroles are added in centrifuge tube
In 37 DEG C of reaction 10min 10 μ L alpha-glucosidases (0.5U/ml) are added, 37 DEG C are reacted again in glucopyranoside glycosides (5mM)
100 μ L 0.02M stopping reaction with sodium carbonate solution are added in 10min, add distilled water to 1mL, take 200 μ L to spread 96 orifice plates, with enzyme mark
Instrument surveys absorbance under 405nm again.Meanwhile PBS buffer solution (pH6.9) being used to replace enzyme solutions as blank, use PBS buffer solution
(pH6.9) replace sample liquid as control.
The counting of above-mentioned viable bacteria and its results are shown in Table 2 to the inhibition of alpha-amylase and alpha-glucosidase.
The counting of 2 viable bacteria of table and its to the inhibition of alpha-amylase and alpha-glucosidase
In the maximum inhibition of alpha-amylase and alpha-glucosidase, the big bacterium of the two inhibiting rates is chosen.It can from table 2
To find out, two lactobacillus plantarums and two plants of lactobacillus fermentis are selected, wherein No. 11 and No. 305 two in lactobacillus fermenti
The inhibiting rate of enzymatic activity is highest；No. 22 and No. 25 are highest two to alpha-glucosaccharase enzyme inhibition rate in lactobacillus plantarum
It is a.No. 22 are highest to the inhibiting rate of alpha amylase.No. 25 inhibiting rates to alpha amylase are more slightly lower than No. 10, but gap is not
Big and 25 number variances are smaller, comprehensively consider selection No. 22 and No. 25.This time all bacteria concentrations of experimental selection are same
The order of magnitude and gap is little.
Embodiment 3
The digestion of 1 simulated gastrointestinal tract
In order to verify the survival ability of lactic acid bacteria in the gastrointestinal tract, alpha-amylase and alpha-glucosidase in table 2 are selected
Higher 4 lactobacillus plantarum of maximum inhibition overall merit and 7 plants of lactobacillus fermentis carry out simulated gastrointestinal tract digestion experiment.
According to De-QuanZhu et al. (Zhu D Q, Liu F, Sun Y, et al.Genome-Wide
Identification of Small RNAs in Bifidobacterium animalis subsp.lactis KLDS
2.0603 and Their Regulation Role in the Adaption to Gastrointestinal
Environment [J] .Plos One, 2015,10 (2): e0117373.) method manufacturing artificial saliva, gastric juice and intestinal juice.In
It takes a ring bacterium to be seeded in the MRS broth bouillon of 10mL on inclined-plane, is connect after 37 DEG C of constant incubator stationary culture 12h with 5%
Kind amount is inoculated into 30mL fluid nutrient medium, then after 37 DEG C of constant incubator stationary culture 12h, and 4 DEG C, 8000r/min centrifugation
15min, it is centrifuged again after being cleaned with PBS solution, adjustment cell concentration is 1010cfu/mL.One milliliter is taken to be put into 50mL centrifuge tube,
5mL artificial saliva is added, is put into temperature control shaking table, 37 DEG C, 100r/min, 5min.After centrifugation plus 10mL gastric juice is resuspended, and is put into temperature control
Shaking table 2h.After centrifugation, adds 10mL intestinal juice to be resuspended, be put into temperature control shaking table 2h.Thalline were collected by centrifugation, is centrifuged weight after being cleaned with PBS again
It is outstanding, carry out gradient (10-4、10-5、10-6) dilution after be coated with, culture count afterwards for 24 hours, the results are shown in Table 3.
The digestion of 3 bacterial strain simulated gastrointestinal tract of table
From table 3 it can be seen that alpha-amylase inhibiting rate and alpha-glucosaccharase enzyme inhibition rate are higher in lactobacillus plantarum
No. 22 and No. 25 bacterial strains are also relatively high on digestion amount of survival, therefore as selected bacterial strain.And α-shallow lake in lactobacillus fermenti
Although powder enzyme inhibition rate and alpha-glucosaccharase enzyme inhibition rate higher No. 11 and No. 305 bacterial strains on digestion amount of survival slightly below
No. 26 bacterial strains, but the alpha-amylase inhibiting rate of No. 11 and No. 305 bacterial strains and alpha-glucosaccharase enzyme inhibition rate are much higher than No. 26 bacterial strains,
And the digestion amount of survival and No. 26 bacterial strains of No. 11 and No. 305 bacterial strains do not reach the difference of an order of magnitude yet, therefore, still
So select No. 11 and No. 305 bacterial strains to select bacterial strain as final.
To sum up, the count plate of 5 lactobacillus plantarum of integrated survey and 18 plants of lactobacillus fermentis, to alpha-amylase and α-Portugal
The inhibiting rate and simulated gastrointestinal tract of polyglycoside enzyme digest, and select No. 22 and No. 25 lactobacillus plantarum (Lactobacillus
Fermentum), No. 11 and No. 305 lactobacillus fermenti (Lactobacillus fermentum) this 4 plants of bacterial strains are as preferred bacterium
Strain, and microbial preservation is carried out, wherein No. 22 lactobacillus plantarums (Lactobacillus fermentum) are named as plant cream
Bacillus (Lactobacillus plantarum) ZLT 22 (deposit number are as follows: CGMCC No.18208), abbreviation plant cream bar
Bacterium ZLT 22；No. 25 lactobacillus plantarums (Lactobacillus fermentum) are named as lactobacillus plantarum
(Lactobacillus plantarum) ZLT 25 (deposit number are as follows: CGMCC No.18209), abbreviation lactobacillus plantarum ZLT
25；No. 11 lactobacillus fermentis (Lactobacillus fermentum) are named as lactobacillus fermenti (Lactobacillus
Fermentum) ZLT 11 (deposit number are as follows: CGMCC No.18206), abbreviation lactobacillus fermenti ZLT 11；No. 305 acidified milks
Bacillus (Lactobacillus fermentum) is named as lactobacillus fermenti (Lactobacillus fermentum) ZLT 305
(deposit number are as follows: CGMCC No.18207), abbreviation lactobacillus fermenti ZLT 305.
The preparation of 2 bacterium powder and count plate
The lactobacillus fermenti ZLT 11 that will be screened, lactobacillus fermenti ZLT 305, lactobacillus plantarum ZLT 22, plant cream
25 bacterial strain of bacillus ZLT is inoculated into respectively in the MRS broth bouillon of 250mL, and 37 DEG C of cultures stand 12h, is activated；Then
37 DEG C of standing 12h in 3L MRS broth bouillon are inoculated into the inoculum concentration of volume 5%, are expanded culture；4 DEG C, 8000r/
Min is centrifuged 15min；With sterile water wash, similarity condition is centrifuged again, is repeated twice；Bacterium mud pre-cooling (- 20 DEG C) is collected, is put into cold
Freeze freeze-drying in drier and collects bacterium powder (condition: 48h, temperature: subzero 40 DEG C -50 DEG C subzero, vacuum degree 20-30Pa).Take 0.1g
Bacterium powder is dissolved in 1mL sterile water, is then carried out gradient dilution according to the method for taking 0.1mL to be added in 0.9mL sterile water, is taken conjunction
Suitable gradient (10-7、10-8、10-9) be applied on MRS solid medium, 37 DEG C of stationary cultures count afterwards for 24 hours, as a result such as 4 institute of table
Show.
The wet when count plate of 4 bacterium dried bean noodles of table
By calculating psychrometric ratio and count plate, as shown in table 4, the yield of bacterium powder after fermented and cultured, every gram of bacterium powder are understood
In bacteria-containing quantity.
The orthogonal experiment that 3 bacterium powder inhibit alpha-glucosidase and alpha-amylase
4 kinds of bacterium powder prepared above are as 4 factors, and wherein A indicates that lactobacillus fermenti ZLT 11, B indicate acidified milk bar
Bacterium ZLT 305, C indicate that lactobacillus plantarum ZLT 22, D indicate lactobacillus plantarum ZLT 25, and each factor takes 3 levels according to L9
(34) design scheme and tested.Experimental method is in 2.3 lactic acid bacteria of embodiment to alpha-amylase activity inhibition and embodiment
It is modified slightly on the basis of the step of 2.4 lactic acid bacterias are to alpha-glucosaccharase enzyme inhibition, will be under the premise of total addition level be constant, bacterium
Powder is configured to equivalent (volume) after required bacteria concentration solution, is added, and 4 DEG C before spreading 96 orifice plates, 5000r/min is centrifuged 3min, real
It tests factor and level is shown in Table 5.
The factor and level of 5 kinds of bacterium powder orthogonal experiments of table
In table 5 in factor and horizontal expression, A1 indicates that viable bacteria concentration is 1010No. 11 bacterium of cfu/mL, A2 are indicated
Viable bacteria concentration is 109No. 11 bacterium of cfu/mL, A3 indicate that viable bacteria concentration is 108No. 11 bacterium of cfu/mL, BCD can similarly deduce.
Bacterium powder is as shown in table 6 to alpha-glucosaccharase enzyme inhibition rate Orthogonal experiment results:
6 bacterium powder of table analyzes alpha-glucosaccharase enzyme inhibition rate Orthogonal experiment results
1-9 indicates 9 groups of experiments in table 6.Three test indexes are (with other three bacterium corresponding to the 1st horizontal A1 of factor A
The different viable bacteria concentrations of strain assemble) average value is respectively k1=63.72% (average value of the inhibiting rate of group 1-3).Other values
Similarly obtain.
(A1, A2, A3 indicate three different viable bacterias of lactobacillus fermenti ZLT 11 in three groups of tests that investigation factor A is carried out
Concentration), all there is same number in each level of B, C, D, and therefore, for A1, A2 and A3, the experimental condition of three groups of tests is
It is duplicate.If factor A on test index without influence, then the corresponding K of A1、K2、K3Should be equal, but from above
And it is unequal.This is because caused by the horizontal variation of factor A, therefore the size of K value reflects A1, A2 and A3 to test index
The size of influence.Since inhibiting rate is the bigger the better, so can determine whether that the excellent water that A1 is factor A is flat.Similarly, can determine whether factor B, C,
The optimal level of D.
R indicates the corresponding K of A, B, C, D1、K2、K3Middle maximum value subtracts minimum value.
Bacterium powder is as shown in table 7 to alpha-glucosaccharase enzyme inhibition rate Orthogonal experiment results variance analysis:
7 bacterium powder of table is to alpha-glucosaccharase enzyme inhibition rate Orthogonal experiment results variance analysis
Note " * " expression has conspicuousness (p < 0.05) to result
RB > RA > RC > RD known to the R value of comparison sheet 6, so the primary and secondary that 4 kinds of bacterium powder influence alpha-glucosaccharase enzyme inhibition rate
Sequence is BACD, and optimum combination is A1B1C1D1.It can be seen that the preferred viable bacteria concentration of 4 plants of bacterium powder is 1010(cfu/mL).Wherein certainly
It is obtained by spending according to calculation formula, tables look-up and can obtain when calculating F value.As shown in Table 7,4 plants of bacterium powder inhibit alpha-glucosidase
Rate has a significant impact.As it can be seen that 4 plants of bacterium powder are 10 to the preferred viable bacteria concentration of alpha-glucosaccharase enzyme inhibition rate10(cfu/mL),
10 are prepared as when preparing freeze-dried vaccine powder10Cfu/g is used.
Bacterium powder analyzes alpha-amylase inhibiting rate Orthogonal experiment results as shown in table 8:
8 bacterium powder of table analyzes alpha-amylase inhibiting rate Orthogonal experiment results
Three test index average values corresponding to the 1st horizontal A1 of factor A are respectively k1=63.50%.Other values are same
Reason obtains.
In three groups of tests that investigation factor A is carried out (A1, A2, A3), all there is same number in each level of B, C, D, therefore,
For A1, A2 and A3, the experimental condition of three groups of tests is duplicate.If factor A on test index without influence, then
A corresponding K1, K2, K3 should be equal, but known to above and unequal.This is because caused by the horizontal variation of factor A, because
The size of this K value reflects the size that A1, A2 and A3 influence test index.Since inhibiting rate is the bigger the better, so can determine whether
The excellent water that A1 is factor A is flat.Similarly, the optimal level of factor B, C, D be can determine whether.
R indicates the corresponding K of A, B, C, D1、K2、K3Middle maximum value subtracts minimum value.
Four kinds of bacterium powder are as shown in table 9 to alpha-amylase enzyme inhibition rate Orthogonal experiment results variance analysis:
9 four kinds of bacterium powder of table are to alpha-amylase enzyme inhibition rate Orthogonal experiment results variance analysis
Note " * " expression has conspicuousness (p < 0.05) to result
RA > RD > RB > RC known to comparison sheet 8R value, so the primary and secondary that 4 kinds of bacterium powder influence alpha-glucosaccharase enzyme inhibition rate is suitable
Sequence is ADBC, and optimum combination is A1B1C1D1.As shown in Table 9, lactobacillus fermenti ZLT 11, lactobacillus fermenti ZLT 305 and plant
This 3 plants of bacterium powder of lactobacillus ZLT 25 have a significant impact alpha-amylase enzyme inhibition rate.It can be seen that 4 plants of bacterium powder press down alpha-amylase enzyme
The preferred viable bacteria concentration of rate processed is 1010(cfu/mL), 10 are prepared as when preparing freeze-dried vaccine powder10Cfu/g is used.
4. confirmatory experiment
After orthogonal experiment obtains optimum combination, confirmatory experiment is carried out with optimum combination, inhibiting rate the results are shown in Table 10.Knot
It is all highest that fruit, which shows that optimum combination obtains inhibiting rate to two kinds of enzymatic activitys, wherein to alpha-amylase obtain inhibiting rate be 93.18 ±
1.19%, obtaining inhibiting rate to α glucuroide is 75.33 ± 2.89%.
The confirmatory experiment that 10 bacterium powder of table inhibits alpha-glucosidase and alpha-amylase
Embodiment 4
1. the experiment of single factor that polydextrose and oligofructose inhibit alpha-glucosidase
Be 0% by polydextrose solid powder and oligofructose solid powder difference compound concentration (mass/volume),
5%, 10%, 15%, 20% polydextrose and the solution of oligofructose inhibit single factor test to carry out alpha-glucosidase respectively
The step of experiment, experimental procedure inhibits part to alpha-amylase activity referring to embodiment 2.3. lactic acid bacteria, operates, and as a result sees
Table 11.
The experiment of single factor that 11 polydextrose of table and oligofructose inhibit alpha-glucosidase
As can be seen from Table 11, when polydextrose is 15%, it is to alpha-glucosaccharase enzyme inhibition rate highest；When oligomeric fruit
When sugar is 10%, it is to alpha-glucosaccharase enzyme inhibition rate highest.Therefore, it is selected according to the principle that selection peak value and its two sides obtain concentration
The polydextrose solution that concentration is 10%, 15%, 20% and the oligofructose solution that concentration is 5%, 10%, 15% is taken to carry out
Subsequent orthogonal experiment.
2. the orthogonal experiment that bacterium powder collaboration polydextrose and oligofructose inhibit alpha-glucosidase
Polydextrose, oligofructose, four kinds of bacterium powder, 6 influence factors are selected according to experimental result, each factor takes 3 water
It puts down according to L18(36) design scheme and tested.3.33 bacterium powder of experimental procedure embodiment is to α-glucuroide and alphalise starch
The orthogonal experiment that enzyme inhibits is identical.Wherein, A indicates lactobacillus fermenti ZLT 11；B indicates lactobacillus fermenti ZLT 305；C is indicated
Lactobacillus plantarum ZLT 22；D indicates lactobacillus plantarum ZLT 25；E indicates polydextrose；F indicates oligofructose.Empirical factor with
Level is shown in Table 12:
12 polydextrose of table, oligofructose and four kinds of bacterium powder orthogonal experiment factors and level
In table 12 in factor and horizontal expression, A1 indicates that viable bacteria concentration is 1010The lactobacillus fermenti ZLT 11 of cfu/mL,
A2 indicates that viable bacteria concentration is 109The lactobacillus fermenti ZLT 11 of cfu/mL, A3 indicate that viable bacteria concentration is 108The hair of cfu/mL
Kefir milk bacillus ZLT 11, BCD can similarly deduce.E1 indicates that concentration is 10% polydextrose, E2 indicate concentration be 15% it is poly-
Glucose, E3 indicate that concentration is 20% polydextrose；F can similarly deduce.
Carry out alpha-glucosidase activity Inhibition test according to factor level table, experimental procedure is referring to embodiment 2.4, bacterium powder
The Orthogonal experiment results that collaboration polydextrose and oligofructose inhibit alpha-glucosidase are shown in Table 13.
13 bacterium powder of table collaboration polydextrose and oligofructose analyze the Orthogonal experiment results that alpha-glucosidase inhibits
As can be seen from Table 13, three test index average values corresponding to the 1st horizontal A1 of factor A are respectively K1=
67.69%.Other values similarly obtain.
In three groups of tests that investigation factor A is carried out (A1, A2, A3), all there is same number in each level of B, C, D, E, F,
Therefore, for A1, A2 and A3, the experimental condition of three groups of tests is duplicate.If factor A is to test index without shadow
It rings, then A corresponding K1, K2, K3 should be equal, but known to above and unequal.This is because the horizontal variation of factor A is drawn
It rises, therefore the size of K value reflects the size that A1, A2 and A3 influence test index.Since inhibiting rate is the bigger the better, so
It can determine whether that the excellent water that A1 is factor A is flat.Similarly, it can determine whether that the optimal level R of factor B, C, D, E, F indicate A, B, C, D, E, F couple
Maximum value subtracts minimum value in K1, K2, the K3 answered.
The Orthogonal experiment results variance analysis that bacterium powder collaboration polydextrose and oligofructose inhibit alpha-glucosidase, knot
Fruit is shown in Table 14:
The orthogonal experiment variance analysis that 14 bacterium powder of table collaboration polydextrose and oligofructose inhibit alpha-glucosidase
Note " * " expression has conspicuousness (p < 0.05) to result
R as shown in Table 13B>RA>RC>RE>RD>RFSo the primary and secondary sequence of influence of the experimental factor to test index is
BACEDF, optimum combination A1B1C1D1E2F2.As shown in Table 14, lactobacillus fermenti ZLT 11 and lactobacillus fermenti ZLT 305 are to α-
Glucosidase inhibitor rate has a significant impact.It can be seen that more excellent condition are as follows: the viable bacteria concentration of 4 plants of bacterium powder is 1010Cfu/mL gathers
Concentration of glucose is 15% (polydextrose containing 15g in every 100 milliliters of solution), and the concentration of oligofructose is 10% (every 100 milliliters
Oligofructose containing 10g in solution), 10 are prepared as when preparing freeze-dried vaccine powder10Cfu/g is used.
3 comparative experimentss
Optimal solution rule is selected according to two groups of orthogonal experiments and carries out alpha-glucosidase activity Inhibition test, the results are shown in Table
Shown in 15.It can be seen that bacterium powder collaboration polydextrose and oligofructose are up to 82.33 ± 1.53% to alpha-glucosaccharase enzyme inhibition rate.
15 two kinds of optimum combinations of table are to alpha-glucosaccharase enzyme inhibition rate
Two groups of result independent sample T check analysis result interpretations of result are shown in Table 16.
16 two groups of result independent sample T check analysis results of table
From table 15 and table 16 as can be seen that illustrating there is significant difference between two groups according to independent sample T inspection result, benefit
Raw bacterium lactic acid bacteria bacterium powder has significantly alpha-glucosidase inhibitory effect after two kinds of prebiotics polydextroses and oligofructose is added
It is promoted.
From above-described embodiment as can be seen that the present invention provides a kind of humanized's probiotics suitable for diabetes, the people
Property probiotics in source have the function of human body pipe intestinal digesting, suitable for being colonized in human body alimentary canal.α-glucuroide is
It is present in a kind of enzyme of small intestine, inhibits its activity that can delay the absorption of carbohydrate, reduces postprandial blood sugar.Pass through separation screening
And Molecular Identification, 5 lactobacillus plantarums and 18 plants of lactobacillus fermentis are obtained from Healthy People enteron aisle.Their viable bacteria of integrated survey
It counts, the inhibiting rate and simulated gastrointestinal tract of alpha-amylase and alpha-glucosidase is digested, obtain performance preferably plant cream
Bacillus ZLT 22, lactobacillus plantarum ZLT 25, lactobacillus fermenti ZLT 11 and lactobacillus fermenti ZLT 305, this 4 plants of bacterial strains are
Preferred strain.Freeze-drying is fabricated to bacterium powder and is counted after being fermented, being collected by centrifugation, the results showed that their work
Bacterium number is respectively 2.10 ± 0.21*1011cfu/g、1.48±0.68*1011cfu/g、2.27±0.56*1011cfu/g、 2.04
±0.81*1011cfu/g.Orthogonal test shows that the more excellent viable bacteria concentration of 4 plants of bacterium powder is 1010(cfu/g).Experiment of single factor table
Bright, when polydextrose is 15%, it is to alpha-glucosaccharase enzyme inhibition rate highest；When oligofructose is 10%, it is to α-Portugal
Polyglycoside enzyme inhibition rate highest.The orthogonal test of polydextrose, oligofructose and 4 kinds of bacterium powder obtains more excellent condition are as follows: 4 plants of bacterium powder
Viable bacteria concentration be 1010* cfu/mL, polydextrose concentration are 15%, and the concentration of oligofructose is 10%.Orthogonal test is tested
Confirmation, which is tested, shows that the optimum combination of four kinds of bacterium powder can achieve 93.18 ± 1.19% to alpha-amylase activity inhibition, to α-grape
Glycosidase activity inhibition can achieve 75.33 ± 2.89%；The orthogonal test of polydextrose, oligofructose and 4 kinds of bacterium powder obtains
Under optimal conditions, 82.33 ± 1.53% are reached to alpha-glucosidase activity inhibition, and with bacterium powder optimum combination when exists
Significant difference.As it can be seen that obtaining the humanized lactic acid bacteria probiotics suitable for diabetes from the separation of healthy enteron aisle, screening and optimization
Agent and lactic acid bacteria cooperate with prebiotics microbial inoculum.
Therefore, the present invention provides a kind of humanized's probiotics, separates, screens from health adult's excrement, identifying and is excellent
Dissolving reduces hypoglycemic effect preferably humanized's lactobacillus, and may also be combined with oligofructose and polydextrose, advanced optimizes
To with assisting in reducing blood sugar effect preferably humanized's probiotics, it can be used as the food and health care product of assisting in reducing blood sugar.
Claims (7)
1. a kind of humanized's probiotics, which is characterized in that including following bacterial strain:
11 freeze-dried vaccine powder of lactobacillus fermenti ZLT, 305 freeze-dried vaccine powder of lactobacillus fermenti ZLT, 22 freeze-dried vaccine of lactobacillus plantarum ZLT
Powder, 25 freeze-dried vaccine powder of lactobacillus plantarum ZLT are mixed according to mass ratio 1:1:1:1；
Wherein, 11 freeze-dried vaccine powder of lactobacillus fermenti ZLT, 305 freeze-dried vaccine powder of lactobacillus fermenti ZLT, lactobacillus plantarum ZLT 22
Freeze-dried vaccine powder, 25 freeze-dried vaccine powder of lactobacillus plantarum ZLT viable bacteria concentration be 1010cfu/g；
Wherein, the classification naming of lactobacillus plantarum ZLT 22 are as follows: lactobacillus plantarum (Lactobacillus plantarum) ZLT
22；Depositary institution are as follows: China Committee for Culture Collection of Microorganisms's common micro-organisms center；Address are as follows: Chaoyang District, Beijing City
No. 3 Institute of Microorganism, Academia Sinica, institute of North Star West Road 1；Deposit number are as follows: CGMCC No.18208；Preservation date are as follows:
On July 11st, 2019；
The classification naming of lactobacillus plantarum ZLT 25 are as follows: lactobacillus plantarum (Lactobacillus plantarum) ZLT 25；It protects
Hide unit are as follows: China Committee for Culture Collection of Microorganisms's common micro-organisms center；Address are as follows: Chaoyang District, Beijing City North Star west
No. 3 Institute of Microorganism, Academia Sinica, institute of road 1；Deposit number are as follows: CGMCC No.18209；Preservation date are as follows: 2019 7
The moon 11；
The classification naming of lactobacillus fermenti ZLT 11 are as follows: lactobacillus fermenti (Lactobacillus fermentum) ZLT 11；It protects
Hide unit are as follows: China Committee for Culture Collection of Microorganisms's common micro-organisms center；Address are as follows: Chaoyang District, Beijing City North Star west
No. 3 Institute of Microorganism, Academia Sinica, institute of road 1；Deposit number are as follows: CGMCC No.18206；Preservation date are as follows: 2019 7
The moon 11；
The classification naming of lactobacillus fermenti ZLT 305 are as follows: lactobacillus fermenti (Lactobacillus fermentum) ZLT 305；
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center；Address are as follows: Chaoyang District, Beijing City North Star west
No. 3 Institute of Microorganism, Academia Sinica, institute of road 1；Deposit number are as follows: CGMCC No.18207；Preservation date are as follows: 2019 7
The moon 11.
2. humanized's probiotics as described in claim 1, which is characterized in that it further include polydextrose and oligofructose,
Middle polydextrose and oligofructose and 11 freeze-dried vaccine powder of lactobacillus fermenti ZLT, 305 freeze-dried vaccine powder of lactobacillus fermenti ZLT, plant
The ratio of 22 freeze-dried vaccine powder of lactobacillus ZLT, 25 freeze-dried vaccine powder of lactobacillus plantarum ZLT are as follows: 11 freeze-dried vaccine of lactobacillus fermenti ZLT
Powder, 305 freeze-dried vaccine powder of lactobacillus fermenti ZLT, 22 freeze-dried vaccine powder of lactobacillus plantarum ZLT, 25 freeze-dried vaccine of lactobacillus plantarum ZLT
Powder, polydextrose, oligofructose are according to mass ratio 1:1:1:1:(1-2): (0.5-1.5) is mixed.
3. humanized's probiotics as claimed in claim 2, which is characterized in that 11 freeze-dried vaccine of lactobacillus fermenti ZLT
Powder, 305 freeze-dried vaccine powder of lactobacillus fermenti ZLT, 22 freeze-dried vaccine powder of lactobacillus plantarum ZLT, 25 freeze-dried vaccine of lactobacillus plantarum ZLT
Powder, polydextrose, oligofructose are mixed according to mass ratio 1:1:1:1:1.5:1.
4. the preparation method of humanized's probiotics as described in any one of claims 1-3, which is characterized in that including walking as follows
It is rapid:
1) by lactobacillus fermenti ZLT 11, lactobacillus fermenti ZLT 305, lactobacillus plantarum ZLT 22, lactobacillus plantarum ZLT 25
Bacterial strain is activated respectively, amplifies culture, and above-mentioned four kinds of bacterium solutions are respectively obtained；
2) above-mentioned four kinds of bacterium solutions are centrifugated respectively and collect wet thallus, then be put into freeze-drying in freeze-dryer after washing respectively and receive
Collection, the 11 bacterium powder of lactobacillus fermenti ZLT after being lyophilized, 305 bacterium powder of lactobacillus fermenti ZLT, 22 bacterium of lactobacillus plantarum ZLT
25 bacterium powder of powder and lactobacillus plantarum ZLT；
3) by 11 bacterium powder of lactobacillus fermenti ZLT, 305 bacterium powder of lactobacillus fermenti ZLT, 22 bacterium powder of lactobacillus plantarum ZLT and plant
25 bacterium powder of lactobacillus ZLT mixes in proportion.
5. the preparation method of humanized's probiotics as claimed in claim 4, which is characterized in that further comprise: by poly- Portugal
Grape sugar and oligofructose are proportionally added into step 3).
6. the preparation method of humanized's probiotics as described in claim 4 or 5, which is characterized in that the activation method
Are as follows: aforementioned four bacterial strain is inoculated into respectively in MRS broth bouillon, 37 DEG C of stationary culture 12h；The side of the expansion culture
Method are as follows: be inoculated into aforementioned four bacterial strain in MRS broth bouillon with 5% inoculum concentration respectively, 37 DEG C of stationary culture 12h.
7. the food and health care product of humanized's probiotics as described in any one of claims 1-3 as assisting in reducing blood sugar
Using.
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