CN116875502A - Composite microbial preparation and application thereof - Google Patents
Composite microbial preparation and application thereof Download PDFInfo
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- CN116875502A CN116875502A CN202310856237.9A CN202310856237A CN116875502A CN 116875502 A CN116875502 A CN 116875502A CN 202310856237 A CN202310856237 A CN 202310856237A CN 116875502 A CN116875502 A CN 116875502A
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- lactobacillus plantarum
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- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims abstract description 46
- 229940072205 lactobacillus plantarum Drugs 0.000 claims abstract description 46
- 150000001875 compounds Chemical class 0.000 claims abstract description 35
- 235000015140 cultured milk Nutrition 0.000 claims abstract description 21
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 102000057297 Pepsin A Human genes 0.000 description 3
- 108090000284 Pepsin A Proteins 0.000 description 3
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- 241000196324 Embryophyta Species 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
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- 229910021538 borax Inorganic materials 0.000 description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 2
- 239000004327 boric acid Substances 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 235000013365 dairy product Nutrition 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 235000021001 fermented dairy product Nutrition 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 229960003692 gamma aminobutyric acid Drugs 0.000 description 2
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- 238000004128 high performance liquid chromatography Methods 0.000 description 2
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- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- 208000031648 Body Weight Changes Diseases 0.000 description 1
- KKUKTXOBAWVSHC-UHFFFAOYSA-N Dimethylphosphate Chemical compound COP(O)(=O)OC KKUKTXOBAWVSHC-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
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- 239000001888 Peptone Substances 0.000 description 1
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- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
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- 206010070863 Toxicity to various agents Diseases 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
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- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
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- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 229940040526 anhydrous sodium acetate Drugs 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
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- 239000007853 buffer solution Substances 0.000 description 1
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- YXVFQADLFFNVDS-UHFFFAOYSA-N diammonium citrate Chemical compound [NH4+].[NH4+].[O-]C(=O)CC(O)(C(=O)O)CC([O-])=O YXVFQADLFFNVDS-UHFFFAOYSA-N 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
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- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000012417 linear regression Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
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- 210000004798 organs belonging to the digestive system Anatomy 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
Abstract
The invention provides a compound microecological preparation, and belongs to the technical field of compound microecological preparations. The composite microecological preparation comprises: lactobacillus plantarum 2-33, lactobacillus plantarum 43 and lactobacillus plantarum 10 are compounded according to the mass ratio of 1-3:1:1. The probiotics number of the composite microecological preparation is 5.32 multiplied by 10 9 CFU/g, the survival rate of probiotics is highest. The fermented milk supernatant prepared by the compound microecological preparation has good inhibition effect on the activity of angiotensin converting enzyme. The composite microecological preparation provided by the invention has good application prospect.
Description
Technical Field
The invention belongs to the technical field of microbial preparations, and particularly relates to a composite microbial preparation and application thereof.
Background
Microbial agents are also known as probiotic agents or live bacterial agents. The microbial preparation can effectively stimulate the intestinal development of animals and has the function of regulating the immunity of organisms; meanwhile, as a beneficial flora, the microecological preparation can be rapidly amplified in the intestinal tract to maintain the balance of the intestinal flora, and plays roles in inhibiting intestinal inflammation, promoting absorption of important nutrients and the like. The quality of the microbial preparation includes the number of viable microbial populations in the microbial preparation and the length of time that can be stored. Therefore, in preparing microbial preparations, screening of the protectant is required to ensure the protective function against probiotics in order to obtain a high quality product. The current research level of the domestic microbial preparation is still in a relatively lagging state, and the quality of the produced product is limited.
The fermented dairy product is an acidic and condensed dairy product formed by the fermentation of specific bacteria, and the fermented dairy product not only contains all nutrient components in fresh dairy bodies, but also is rich in a plurality of active probiotics, corresponding organic acids and other nutrient elements. With the increasing level of economic income and the popularization of related nutritional health knowledge, the presentation of the milk products and their diversified and functional needs by the masses is becoming more apparent. It was found that the metabolites produced by certain specific lactic acid bacteria used in fermented milk have an inhibitory effect on angiotensin converting enzyme, thereby regulating and controlling blood pressure.
In general, most of the treatments for hypertension are to regulate blood pressure by using drugs, but this causes a lot of side effects. Therefore, the development of the composite microecological preparation health care product with good storability, better functions in the common environment and has the effect of improving the hypertension provides more choices and safer improvement methods for the hypertension patients.
Disclosure of Invention
Accordingly, the present invention has an object to provide a high-quality complex microbial preparation capable of inhibiting angiotensin converting enzyme activity and maintaining blood pressure health level.
In order to achieve the above object, the present invention provides the following technical solutions:
a complex microbial formulation, the complex microbial formulation comprising: lactobacillus plantarum 2-33, lactobacillus plantarum 43 and lactobacillus plantarum 10 are compounded according to the mass ratio of 1-3:1:1.
Preferably, the lactobacillus plantarum 2-33, the lactobacillus plantarum 43 and the lactobacillus plantarum 10 are compounded according to the mass ratio of 1:1:1.
The invention further aims at providing a product containing the compound microbial preparation, and the product is added with a freeze-drying protective agent to prepare the compound microbial freeze-dried powder preparation.
Preferably, the freeze-drying protective agent comprises the following components in percentage by mass: 10.0-14.0% skim milk, 5.0-7.0% sodium glutamate and 2.4-3.0% tween-80.
The invention also aims to provide fermented milk for maintaining the healthy level of blood pressure, which is fermented by using the compound microbial preparation.
Preferably, the fermented milk is prepared by the following steps: mixing fresh milk with white granulated sugar, sterilizing, adding the compound microorganism preparation into the sterilized liquid, mixing uniformly, and culturing in a constant temperature incubator at 37-42 ℃.
Preferably, the feed liquid ratio of the white granulated sugar to the fresh milk is 6-8:100g/mL.
Preferably, the feed liquid ratio of the compound microbial preparation to the sterilized liquid is 5-8:100g/mL.
It is another object of the present invention to provide the use of the complex microbial preparation, the product containing the complex microbial preparation and the fermented milk for the preparation of a product for maintaining blood pressure health level.
Preferably, the product for maintaining a healthy blood pressure level is capable of inhibiting angiotensin converting enzyme activity.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a compound microbial preparation, which has high viable count of probiotics, strong tolerance in strong acid environment and bile salt environment, smooth passage through gastrointestinal tract, higher viable count, capability of exerting probiotics in vivo and good storability. The fermented lactic acid prepared by the compound microbial preparation has the advantages of proper range of lactic acid degree, proper viscosity range, smooth texture and stable structure. The fermented milk supernatant has good inhibiting effect on Angiotensin Converting Enzyme (ACE). Provides more choices and safer improvement methods for patients with hypertension.
Drawings
Fig. 1: the test result of the tolerance of the compound microecological preparation to pH 2.0;
fig. 2: the test result of the compound microecological preparation on the resistance of bile salt;
fig. 3: simulating an artificial gastric juice test by using the composite microecological preparation;
fig. 4: hippuric acid standard curve graph;
fig. 5: HPLC profile of hippuric acid standard;
fig. 6: HPLC profile of fermented milk supernatant.
Detailed Description
The invention provides a compound microbial preparation, which comprises the following components: lactobacillus plantarum 2-33, lactobacillus plantarum 43 and lactobacillus plantarum 10 are compounded according to the mass ratio of 1-3:1:1, and preferably according to the mass ratio of 1:1:1.
The invention combines the lactobacillus plantarum 2-33, the lactobacillus plantarum 43 and the lactobacillus plantarum 10, can play a probiotic role in vivo, can inhibit the activity of angiotensin converting enzyme and maintain the healthy level of blood pressure.
In the invention, the lactobacillus plantarum 2-33 is a strain disclosed in the literature (He Yuxing and the like: modern food science and technology, 2021,37 (11): 50-57+67.); the lactobacillus plantarum 43 is a strain disclosed in the literature (screening and identification of gamma-aminobutyric acid-producing lactobacillus and ultraviolet mutagenesis research (Tian Rui, etc. food industry science and technology, 2018,39 (17): 128-132.); the lactobacillus plantarum 10 is a strain disclosed in literature (high-cellulo Yao. Inner Mongolian agricultural university, 2017.) of lactobacillus plantarum fermentation characteristics and functional yogurt application research of producing gamma-aminobutyric acid.
The lactobacillus plantarum 2-33, the lactobacillus plantarum 43 and the lactobacillus plantarum 10 are provided by food quality and safety teaching and research rooms of the inner Mongolian agricultural university.
Activating the strain before compounding, and placing strain freezing pipes of lactobacillus plantarum 2-33, lactobacillus plantarum 43 and lactobacillus plantarum 10 into a water bath kettle for melting, wherein the water bath temperature is 37 ℃ as an implementation mode; adding melted bacterial liquid into an MRS liquid culture medium, wherein the volume ratio of the bacterial liquid to the culture medium is preferably 2:100; placing the culture medium at 36-38deg.C for expansion culture, preferably for expansion culture to 100mL; after the cultivation is finished, the mixture is placed at 4 ℃ for standby.
As an implementation mode, the invention centrifugates the activated 3 strains, collects bacterial sludge after removing supernatant, and mixes the bacterial sludge according to a proportion.
Wherein the centrifugal rotating speed is 4000-8000r/min, and the centrifugal rotating speed is 5500-6500r/min preferably; centrifuging for 10-15min, preferably 12-14min.
Wherein, MRS liquid medium is: 10.0g of peptone, 5.0g of yeast powder, 20.0g of glucose, 5.0g of anhydrous sodium acetate, 10.0g of beef extract, 2.0g of diammonium citrate, 2.0g of dimethyl hydrogen phosphate, 1.0mL of tween-80, 0.58g of magnesium sulfate, 0.25g of manganese sulfate and 1.0L of distilled water, and regulating the pH to 6.2-6.4. Sterilizing at 121deg.C for 15min.
The invention provides a product containing the compound microbial preparation, and the product is added with a freeze-drying protective agent to prepare the compound microbial freeze-dried powder preparation.
In the invention, the freeze-drying protective agent comprises the following components in percentage by mass: 10.0-14.0% skimmed milk, 5.0-7.0% sodium glutamate and 2.4-3.0% tween-80, preferably 11.7% skimmed milk, 5.60% sodium glutamate and 2.66% tween-80.
The added freeze-drying protective agent has good protective effect on the composite microbial ecological agent in the freeze-drying process, and the obtained composite microbial freeze-drying powder preparation has high survival rate of probiotics and can be stored at normal temperature and low temperature (-20 ℃, -40 ℃ and-80 ℃). Can be used by activating.
As an implementation mode, after the activation culture of the lactobacillus plantarum 2-33, the lactobacillus plantarum 43 and the lactobacillus plantarum 10 is finished, the bacterial sludge is obtained by centrifuging and discarding the supernatant, the bacterial sludge is uniformly mixed according to a proportion, the bacterial sludge is uniformly mixed with a freeze-drying protective agent, the mixture is frozen after quick-frozen by liquid nitrogen, and is freeze-dried by a freeze dryer.
Wherein the centrifugal rotating speed is 4000-8000r/min, and the centrifugal rotating speed is 5500-6500r/min preferably; centrifuging for 10-15min, preferably 12-14min.
As an embodiment, the mixture is frozen at-80 ℃ after being frozen using liquid nitrogen, and the mixture is frozen for 6-12 hours; the mixture is lyophilized for 24-36h by using a freeze dryer.
In the invention, the bacterial sludge and the freeze-drying protective agent are mixed according to the mass ratio of 1:3-1:4, preferably according to the mass ratio of 1:3.
The invention provides fermented milk for maintaining blood pressure health level, which is fermented by using the compound microbial preparation.
In the invention, the fermented milk is prepared by the following steps: mixing fresh milk with white granulated sugar, sterilizing, adding the compound microorganism preparation into the sterilized liquid, mixing uniformly, and culturing in a constant temperature incubator at 37-42 ℃.
As an implementation mode, the invention mixes fresh milk with white granulated sugar and then sterilizes in a water bath at 95 ℃, wherein the water bath time is 10-12min; cooling to 37-42 deg.C for use after sterilization.
In the invention, the fresh milk and the white granulated sugar can be mixed and sterilized at the high temperature of 121 ℃ for 15-20s.
As one embodiment, the sterilized liquid is mixed with the composite microbial preparation for 60-180s.
In the invention, the feed liquid ratio of the white granulated sugar to the fresh milk is 6-8:100g/mL.
In the invention, the feed liquid ratio of the compound microbial preparation to the sterilized liquid is 5-8:100g/mL, and the preferred feed liquid ratio is 6-7:100g/mL.
The fermented milk prepared by the compound microbial preparation can inhibit the activity of angiotensin converting enzyme and maintain the healthy level of blood pressure.
The invention provides the compound microbial preparation, the product containing the compound microbial preparation and the application of the fermented milk in preparing the product for maintaining the blood pressure health level; the product for maintaining blood pressure health level can inhibit angiotensin converting enzyme activity.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
A composite microbial preparation: lactobacillus plantarum 2-33, lactobacillus plantarum 43 and lactobacillus plantarum 10 are compounded according to the mass ratio of 1:1:1.
Placing the strain freezing tubes of the lactobacillus plantarum 2-33, the lactobacillus plantarum 43 and the lactobacillus plantarum 10 in a water bath kettle at 37 ℃ for melting, taking 20 mu L of melted bacterial liquid, adding a 10mLMRS liquid culture medium, placing the culture medium in the 37 ℃ for culturing for 16 hours, performing expansion culture to 100mL, and placing the culture medium at 4 ℃ for standby.
Centrifuging the activated 3 strains at 6000r/min for 10min, discarding the supernatant, collecting bacterial mud, and mixing the bacterial mud according to a certain proportion.
Example 2
This embodiment differs from embodiment 1 in that: lactobacillus plantarum 2-33, lactobacillus plantarum 43 and lactobacillus plantarum 10 are compounded according to the mass ratio of 2:1:1.
Example 3
This embodiment differs from embodiment 1 in that: lactobacillus plantarum 2-33, lactobacillus plantarum 43 and lactobacillus plantarum 10 are compounded according to the mass ratio of 3:1:1.
Example 4
Compound microorganism freeze-dried powder preparation
The freeze-dried powder protective agent is as follows: 11.7% skim milk, 5.60% sodium glutamate and 2.66% tween-80.
After 3 strains are activated in the example 1, the bacterial sludge collected by centrifugation is mixed in proportion. And uniformly mixing the mixed bacterial mud and the freeze-drying protective agent according to the mass ratio of 1:3, quick-freezing the mixture by using liquid nitrogen, freezing at-80 ℃ for 8 hours, and freeze-drying by using a freeze dryer for 30 hours to obtain the composite microbial freeze-dried powder.
Example 5
Compound microorganism freeze-dried powder preparation
The freeze-dried powder protective agent is as follows: 12% skim milk, 6% sodium glutamate and 3% tween-80.
In example 2, 3 strains were activated, and the bacterial sludge collected by centrifugation was mixed in proportion. And uniformly mixing the mixed bacterial mud and the freeze-drying protective agent according to the mass ratio of 1:3, quick-freezing the mixture by using liquid nitrogen, freezing at-80 ℃ for 10 hours, and freeze-drying by using a freeze dryer for 32 hours to obtain the composite microbial freeze-dried powder.
Example 6
Fermented milk for maintaining healthy blood pressure level
100mL of milk and 8g of white granulated sugar are uniformly mixed, sterilized in a water bath at 95 ℃ for 10min, cooled to 37 ℃ after sterilization, added with 5g of the compound microorganism freeze-dried powder preparation prepared in the example 4, mixed for 60s and cultured in a constant temperature incubator at 37 ℃.
Example 7
Fermented milk for maintaining healthy blood pressure level
100mL of milk and 6g of white granulated sugar are uniformly mixed, sterilized in a water bath at 95 ℃ for 12min, cooled to 37 ℃ after sterilization, added with 7g of the compound microorganism freeze-dried powder preparation prepared in the example 4, mixed for 120s and cultured in a constant temperature incubator at 40 ℃.
Comparative example 1
The difference between this comparative example and example 4 is that the lyoprotectant is: 4.0% glucose, 1.6% glycerol, 4.0% trehalose.
Comparative example 2
The difference between this comparative example and example 4 is that the lyoprotectant is: 15.0% skim milk, 6.0% sodium glutamate and 2.0% tween-80.
Example 8
This example demonstrates the tolerance of a complex microbial formulation
1. Oral acute toxicity test of compound microecological preparation
The adapted mice were randomly divided into 2 groups, 5/group, respectively female and male groups of the complex microecological preparation. The complex microecological formulation of example 1 was brought to a concentration of 1X 10 with sterile PBS buffer 8 CFU/mL is used for gastric lavage, and the control group adopts sterile PBS buffer solution for gastric lavage. Each mouse was gavaged 1 time per day at a dose of 20mL/kg, fed on time per day, with pad replacement, and body weight changes were recorded 1,3, 5, 7d after gavage. During feeding, the mice were observed for mental status, excretion status, respiration and sudden death, and after 1 week of administration, all mice were sacrificed, organs were dissected, and abnormal conditions were observed, and the results are shown in table 1.
TABLE 1 results of oral acute toxicity test of mice with composite microecologics
From Table 1, it can be seen that the weight of the mice showed a stable upward trend after 7 days of continuous gastric lavage, and 0 mice died within 7 days without any toxic symptoms; the anatomical result is not obvious abnormal, and the oral acute toxicity test result is safe and nontoxic.
2. Test of tolerance of composite microecological preparation to pH 2.0
The pH of the solution was adjusted to 2.0 with hydrochloric acid in MRS liquid medium, the composite micro-ecological preparation of example 1 was added to MRS liquid medium with pH of 2.0, incubated in a constant temperature incubator at 37℃and sampled at 0h, 3h, 6h, 9h and 12h, viable count was calculated by plate counting method, three parallel tests were performed, and the tolerance of the composite micro-ecological preparation to low acid environment was evaluated with the survival rate as a measurement index, and the results are shown in FIG. 1.
The primary condition for probiotics to function is to enter the gastrointestinal tract in a viable form. The probiotics contained are required to be resistant to gastric acid before reaching the intestinal tract for action, typically the gastric juice pH is maintained in the range of 2.0-3.0 and the food residence time is about 2 hours. The survival rate of the composite microbial preparation is gradually reduced along with the time in the environment with the pH value of 2.0, but the survival rate of the composite microbial preparation in 3 hours is more than 90 percent; the survival rate in 6 hours is also above 85%, and the composite microecological preparation has good tolerance under a strong acid environment.
3. Test of resistance of composite microecological preparation to bile salts
Adding 0.3% of bile salt into MRS liquid culture medium, adding the compound microecological preparation of example 1 into MRS culture medium, culturing in a constant temperature incubator at 37 ℃, sampling at 0h, 3h, 6h, 9h and 12h respectively, calculating viable count by adopting a plate counting method, performing three parallel experiments, and evaluating the tolerance of the compound microecological preparation to bile salt environment by taking the survival rate as a measurement index, wherein the result is shown in figure 2.
After digestion and absorption in the stomach, food enters the small intestine, but bile salts in the small intestine affect cell membrane permeability, thereby reducing the survival rate of microorganisms. The content of bile salt in the small intestine of the human body is within the range of 0.03-0.30%, and the survival rate of microorganisms in the compound microecological preparation is gradually reduced along with the time extension in the bile salt of 0.30%. However, when the plant leaves stay in bile salt for 3 hours, the survival rate is about 90 percent; even after the survival rate is kept for 12 hours, the survival rate can still reach more than 70 percent, and the number of living bacteria playing a probiotic role can be reached. The compound microecological preparation can survive in a high-bile-salt environment.
4. Test for simulating artificial gastric juice tolerance by composite microecological preparation
Preparing artificial gastric juice: taking 20mL of 1.0mol/LHCL, regulating the pH to 2.0 by using distilled water and 1.0mol/L NaOH solution, then adding pepsin according to 1.0g/100mL to completely dissolve the pepsin, and filtering the solution by using a sterile filter membrane with the concentration of 0.22 mu m to obtain the artificial gastric juice which is prepared for use at present.
1mL of the composite microbial preparation bacterial liquid in example 1 is inoculated into 5mL of artificial simulated gastric fluid with pH of 2.0, the culture is carried out at 37 ℃, sampling is carried out at 0h, 1h, 2h, 3h and 4h, dilution of the bacterial liquid is carried out by using a gradient dilution method, and the bacterial liquid is cultured on an MRS solid culture medium. Colony forming units (CFU/g) were then calculated using the colony counting method described in GB 4789.35-2016. To ensure accuracy of the results, three parallel experiments were performed. The artificial gastric juice tolerance of the strains was measured by the above method, and the survival rate was calculated from the formula (1), and the result is shown in fig. 3.
Survival (%) = N1/N0; wherein: n0 represents the number of viable bacteria for 0 h; n1 represents the number of viable bacteria after 1-4 hours of exposure to artificial simulated gastric fluid.
The digestive tract is a defense line for human body to block external invaders, the stomach serves as an important digestive organ of human body, and most microorganisms are blocked from entering the intestinal tract by pepsin and strong acid environment in gastric juice. But when the mixture stays in gastric juice for 2 hours, the survival rate is more than 85 percent; even if the plant leaves for 4 hours, the survival rate is still about 80 percent, and the number of living bacteria which can play a probiotic role can be reached. The compound microecological preparation can survive in simulated artificial gastric juice environment.
Example 9
This example compares the effects of different lyoprotectants
The viability of the microorganisms in the formulations before and after lyophilization of the composite microorganism preparations of examples 4 to 5 and comparative examples 1 to 2, respectively, was examined, and the results are shown in Table 2.
TABLE 2 viable count of Compound microbial preparation before and after lyophilization
After the composite microbial preparation is freeze-dried, the viable count of the probiotics can reach 5.32 multiplied by 10 9 The survival rate of CFU/g reaches 88.70%, the composite microbial preparation can be preserved at normal temperature and low temperature (-20 ℃, -40 ℃, -80 ℃), the number of viable bacteria in the composite microbial freeze-dried preparation is effectively increased, the composite microbial preparation can be preserved for a long time, and the quality of the composite microbial preparation is improved.
Example 10
This example compares the inhibition of ACE by a complex microbial preparation in fermented milk
1. Borate buffer: 19.07g of borax was dissolved in ultrapure water to a volume of 1.0L.12.37g boric acid was dissolved in ultrapure water to a volume of 1.0L. 325mL of boric acid solution and 175mL of borax solution were taken, the two solutions were mixed together, the pH of the mixed solution was adjusted to 8.3 using sodium hydroxide and hydrochloric acid, then 17.532g of sodium chloride was added, and the volume was fixed to 1.0L using a volumetric flask.
HHL solution: HHT was dissolved in borate buffer to give a solution with a concentration of 5.0 mmol/L.
ACE solution: to 0.1UACE, 0.5mL of borate buffer was added and mixed well to give an ACE solution at a concentration of 200 mU/mL. Split into 5 tubes, 0.1mL per tube. In use, 0.7mL of borate buffer was added per tube, i.e., diluted 8-fold per tube, to give an ACE solution at a concentration of 25 mU/mL.
2. Drawing of hippuric acid standard curve
The hippuric acid standard is dissolved and diluted to 0.2, 0.4, 0.6, 0.8 and 1.0 mug/mL, the loading amount is 10 mug, the peak area is taken as an ordinate, the hippuric acid concentration is taken as an abscissa, and a standard curve is drawn.
3. Measurement of ACE inhibition Rate
In a 2mL centrifuge tube, 40. Mu.L of the supernatant of the fermented milk of example 6 was added, incubated at 37℃for 5min, 50L of HHT solution was added to carry out the reaction, 200. Mu.L of 0.1mol/mL HCl was added to terminate the reaction after 60min, borate buffer was used as a blank, and the mixture was filtered through a 0.45 μm filter membrane after the completion of the reaction. Mobile phase: 25% acetonitrile (containing 0.1% trifluoroacetic acid), 75% ultrapure water (containing 0.1% trifluoroacetic acid); flow rate: 1.0mL/min; detection wavelength: 228nm; column temperature: 30 ℃; sample injection amount: 10 mu L.
4. Data analysis
All experiments were repeated 3 times and the data results are expressed as "mean ± standard deviation". Response surface analysis was performed using Design Expert 8.0.5 and graphically plotted using origin8.6, the results of which are shown in fig. 4-6.
5. Results
As can be seen from fig. 4, the peak area value of the hippuric acid standard solution increases with the increase in concentration. The linear regression equation is y=32901x+45.5, the correlation coefficient R 2 0.999. The concentration of the hippuric acid standard substance is in the range of 0-1.0 mug/mL, and the concentration and the absorbance have good linear relation. The peak time of hippuric acid is 2.317minThe peak 2.670min in FIG. 5 can be determined to be the hippuric acid level.
As can be seen from FIG. 6, peak I is the content of hippuric acid in the sample, and the peak time is 2.769min. The peak area of the blank group is 227.8, the peak area of the sample group is 65.30, and the peak area of the sample group is obviously reduced, which indicates that the sample has good inhibition effect on the activity of the angiotensin converting enzyme. Substituting the standard curve formula to calculate and obtaining the sample inhibition rate of 66.50%. The fermented milk supernatant prepared by the compound microecological preparation has good ACE inhibition performance.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A complex microbial formulation, wherein the complex microbial formulation comprises: lactobacillus plantarum 2-33, lactobacillus plantarum 43 and lactobacillus plantarum 10 are compounded according to the mass ratio of 1-3:1:1.
2. The compound microbial preparation according to claim 1, wherein the lactobacillus plantarum 2-33, the lactobacillus plantarum 43 and the lactobacillus plantarum 10 are compounded according to a mass ratio of 1:1:1.
3. A product containing the composite microbial preparation of claim 1 or 2, wherein the product is prepared into a composite microbial lyophilized powder preparation by adding a lyoprotectant.
4. A product according to claim 3, wherein the lyoprotectant comprises the following components in mass percent: 10.0-14.0% skim milk, 5.0-7.0% sodium glutamate and 2.4-3.0% tween-80.
5. A fermented milk for maintaining a healthy blood pressure level, characterized by being fermented using the complex microbial preparation according to claim 1 or 2.
6. The fermented milk according to claim 5, which is prepared by the steps of: mixing fresh milk with white granulated sugar, sterilizing, adding the compound microorganism preparation into the sterilized liquid, mixing uniformly, and culturing in a constant temperature incubator at 37-42 ℃.
7. The fermented milk according to claim 6, wherein the feed ratio of white granulated sugar to fresh milk is 6-8:100g/mL.
8. The fermented milk of claim 6, wherein the ratio of feed liquid of the complex microbial formulation to the sterilized liquid is 5-8:100g/mL.
9. Use of a complex microbial preparation according to claim 1 or 2, a product according to claim 3 or 4, or a fermented milk according to any one of claims 5-8 for the preparation of a product for maintaining blood pressure health levels.
10. The use according to claim 9, wherein the product for maintaining a healthy blood pressure level is capable of inhibiting angiotensin converting enzyme activity.
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