CN114437989A - Lactobacillus fermentum LF028 with blood sugar reducing effect and application thereof - Google Patents
Lactobacillus fermentum LF028 with blood sugar reducing effect and application thereof Download PDFInfo
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- CN114437989A CN114437989A CN202210216462.1A CN202210216462A CN114437989A CN 114437989 A CN114437989 A CN 114437989A CN 202210216462 A CN202210216462 A CN 202210216462A CN 114437989 A CN114437989 A CN 114437989A
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- lactobacillus fermentum
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Classifications
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23C11/10—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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- A23C11/00—Milk substitutes, e.g. coffee whitener compositions
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- A23C11/10—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins
- A23C11/103—Milk substitutes, e.g. coffee whitener compositions containing at least one non-milk component as source of fats or proteins containing or not lactose but no other milk components as source of fats, carbohydrates or proteins containing only proteins from pulses, oilseeds or nuts, e.g. nut milk
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- 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
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- A—HUMAN NECESSITIES
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- 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
- A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
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- 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
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- A—HUMAN NECESSITIES
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- A—HUMAN NECESSITIES
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- Chemical & Material Sciences (AREA)
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- Chemical Kinetics & Catalysis (AREA)
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- Oil, Petroleum & Natural Gas (AREA)
- Mycology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Dairy Products (AREA)
Abstract
The invention discloses a lactobacillus fermentum LF028 with hypoglycemic effect and application thereof, wherein the lactobacillus fermentum is preserved in China general microbiological culture Collection center of the Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 22833; the invention also discloses an application of the lactobacillus fermentum LF 028. The lactobacillus fermentum LF028 provided by the invention is a probiotic with a hypoglycemic effect, and can be used for preparing a fermentation inoculant, wherein the pure fermentation inoculant can be used for further preparing fermented milk and plant yogurt. The lactobacillus fermentum LF028 provided by the invention is a probiotic with a hypoglycemic effect, can be used for preparing a fermentation inoculant, and is further applied to preparing functional fermented milk and plant yogurt.
Description
Technical Field
The invention belongs to the field of microbial engineering, relates to lactic acid bacteria, and particularly relates to lactobacillus fermentum LF028 with a blood sugar reducing effect and application thereof.
Background
According to the data published by the IDF, the number of diabetic patients in the world in 2019 is 4.63 hundred million, and the number of diabetic patients in 2045 is increased to 7 hundred million. In China, the number of people with diabetes is nearly 1.3 hundred million, and the number of people with diabetes at the early stage is about 2 hundred million. Currently, the drug treatment of diabetes still faces the challenges of high price, high treatment cost, strong toxic and side effects and the like. At present, the intervention of probiotics taking intestinal flora as a target point is more and more valued by people. Lactobacillus fermentum, a typical probiotic, has a variety of functionalities, of which research and development on the hypoglycemic effects of Lactobacillus fermentum and its health products, especially fermented dairy products, has become an ongoing focus.
The fermentation inoculum is the key point for ensuring stable quality, good sensory quality and excellent tissue state of fermented milk, at present, most of the fermentation inoculants used for producing fermented dairy products in China are complex fermentation inoculants, and the complex fermentation inoculants have the advantage of short fermentation production period; however, when the compound fermentation inoculum is prepared, in addition to selecting strains with excellent fermentation characteristics, the matching and proportion of different strains are also required, the preparation process is complex to operate, and the contamination probability is greatly higher than that of a pure strain fermentation inoculum.
According to domestic and overseas reports, when the set-style yogurt with the hypoglycemic effect is prepared, most of the applied fermentation bacteria are prepared by compounding common yogurt fermentation bacteria and probiotics (such as lactobacillus fermentum) with the hypoglycemic effect, and because the existing lactobacillus fermentum is difficult to have the characteristics of acid production, viscosity production, flavor production and hypoglycemic effect, the set-style yogurt with the hypoglycemic effect is prepared by using pure lactobacillus fermentum as the fermentation bacteria has a fresh report.
With the increasing importance of people on healthy life, foods mainly comprising plants are greatly welcomed, and under the new thought wave, the plant yoghourt is produced. Compared with common animal yogurt, the plant yogurt has the advantages of unique nutritional structure, environmental protection, wide raw material sources and the like, and is a hot direction for the development of plant-based food in the future. At present, the smell and the taste of the plant yoghourt are a great difficulty to be broken through. However, the plant yogurt using plant base as raw material generally has the problems of rough mouthfeel, serious powdery texture, bitter taste or fishy smell and the like, which are still inseparable in relation to the composition of the plant base raw material, and more importantly, the research on the aspect of the fermentation bacteria agent in the field of plant yogurt is relatively lacked.
Disclosure of Invention
The invention aims to solve the technical problem of providing lactobacillus fermentum LF028 with the function of reducing blood sugar, which is separated and screened from farmhouse sour soup in Guiyang city of Guizhou province.
The lactobacillus fermentum LF028 of the invention has been preserved in China general microbiological culture Collection center (CGMCC No. 22833) at 6.7.2021, and the preservation unit address is No. 3 Siro-1 Hospital of Beijing Korean.
The invention also aims to provide application of the lactobacillus fermentum LF028 in preparing a fermentation inoculant, and the lactobacillus fermentum can be used for preparing fermented milk and plant yogurt so as to achieve industrial application of the lactobacillus fermentum.
In order to achieve the purpose, the invention adopts the technical scheme that:
a Lactobacillus fermentum LF028 with hypoglycemic effect is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.22833, and the Latin with the name of Lactobacillus fermentum.
As a limitation of the invention, the 16S rDNA gene sequence of lactobacillus fermentum LF028 is as follows:
ACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGCAGGCGAGTTGCAGCCTGCAGTCCGAACTGAGAACGGTTTTAAGAGATTTGCTTGCCCTCGCGAGTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATCTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAACTAGTAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTCATTGCGTTCCCGAAGGAAACGCCCTATCTCTAGGGTTGGCGCAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTCCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGTCTCAGCGTCAGTTGCAGACCAGGTAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTACCCTCTTCTGCACTCAAGTTATCCAGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACTTAGAAAACCGCCTGCACTCTCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTAAATACCGTCAACGTATGAACAGTTACTCTCATACGTGTTCTTCTTTAACAACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCACGCGGTGTTGCTCCATCAGGCTTGCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTATGGGCCGTGTCTCAGTCCCATTGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATCGCCTTGGTAGGCCGTTACCCCACCAACAAGCTAATGCACCGCAGGTCCATCCAGAAGTGATAGCGAGAAGCCATCTTTTAAGCGTTGTTCATGCGAACAACGTTGTTATGCGGTATTAGCATCTGTTTCCAAATGTTGTCCCCCGCTTCTGGGCAGGTTACCTACGTGTTACTCACCCGTCCGCCACTCGTTGGCGACCAAAATCAATCAGGTGCAAGCACCATCAATCAATGGGCCAACGCGTTC。
the invention also provides an application of the lactobacillus fermentum LF028, and the strain can be used for preparing a fermentation inoculant.
As a limitation to the above mentioned fermentation inoculum, the fermentation inoculum may be a pure fermentation inoculum.
The invention also provides an application of the pure fermentation inoculant, and the pure fermentation inoculant can be used for preparing fermented milk.
As a limitation to one application of the pure strain of the fermentation inoculum, the method for preparing the fermented milk comprises the following steps in sequence:
s1, taking 80-100 parts of raw milk, uniformly mixing with 7-11 parts of arabinose, homogenizing, and sterilizing to obtain a material A;
s2, cooling the material A, inoculating a pure fermentation bacterium agent, filling, covering, and fermenting to curd to obtain a material B;
and S3, refrigerating the material B, and after-ripening to obtain the fermented milk.
In the step S1, the homogenizing temperature is 65-75 ℃, the pressure is 18.5-21.5 MPa, the sterilizing temperature is 90-95 ℃, and the time is 5-10 min;
in the step S2, the temperature of the material A is reduced to 35-39 ℃, the inoculation amount of the pure fermentation inoculant is 2-4%, the fermentation temperature is 34-40 ℃, the time is 14-18 h, and the pH value of curd is 4.2-4.5;
in the step S3, the refrigeration temperature is 2-6 ℃, and the after-ripening time is 10-14 h.
As another limitation on the application of the pure fermentation inoculum, the fermentation inoculum is applied to the preparation of the plant yoghourt.
As a further limitation, the plant-based raw materials selected for preparing the plant yogurt comprise at least one of soybean, mung bean, corn, coconut, almond, walnut, black bean and peanut.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the technical progress that:
the lactobacillus fermentum LF028 provided by the invention has an inhibiting effect on alpha-amylase and alpha-glucosidase, so that the lactobacillus fermentum has a hypoglycemic effect, has good acid and bile salt resistance, stable production performance and good fermentation performance, and is a probiotic strain with industrial production prospect;
secondly, the lactobacillus fermentum LF028 provided by the invention has the characteristics of acid production, viscosity production, flavor production, blood sugar reduction and the like, and single strain fermentation of the lactobacillus fermentum LF028 can prepare set yoghurt with good sense and blood sugar reduction effect;
the lactobacillus fermentum LF028 provided by the invention can produce extracellular polysaccharide, and can maintain good stability and texture of the product without adding a stabilizer or a thickener when fermenting the yoghourt;
the lactobacillus fermentum LF028 provided by the invention can be used for preparing a fermentation inoculum, and the prepared fermentation inoculum has the characteristics of high activity, small volume, convenience in carrying and use and the like, is beneficial to maintaining stable product quality, and can prevent degeneration and pollution of strains;
the lactobacillus fermentum LF028 pure strain is simple and convenient to prepare and operate;
the fermented milk prepared by the invention has the advantages of excellent flavor, high viscosity, strong water holding capacity, stable texture, uniform and fine tissue and wiredrawing characteristic; meanwhile, the health-care tea also has the function of reducing blood sugar;
the invention provides high-quality plant yoghourt for people by using the plant base as the raw material and applying single strain fermentation of the lactobacillus fermentum LF028, and also provides a probiotic strain with strong practicability and large market potential for the field of plant yoghourt lacking of plant base fermentation strains.
In conclusion, the lactobacillus fermentum LF028 with the blood sugar reducing function and the application thereof have the advantages of good acid resistance, bile salt resistance, stable production performance and good fermentation performance; the lactobacillus fermentum LF028 can be used for preparing a lactobacillus fermentum with high activity, small volume and convenient carrying and use, and the pure lactobacillus fermentum can be used for preparing the plant yoghourt with excellent sense and the set yoghourt with the effect of reducing blood sugar.
The lactobacillus fermentum LF028 provided by the invention is a probiotic with a hypoglycemic effect, can be used for preparing a fermentation inoculant, and can also be further applied to producing fermented milk and plant yogurt.
Detailed Description
The present invention will be described in further detail with reference to specific examples. It should be understood that the described embodiments are only for illustrating the invention and do not limit the invention.
The materials, reagents and the like used in the examples of the present invention are commercially available unless otherwise specified. The experimental procedures, in which specific conditions are not indicated in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturer.
Example 1 a strain of lactobacillus fermentum LF028
The lactobacillus fermentum LF028 is separated and screened from farmhouse sour soup in Guiyang city, Guizhou province, and the lactobacillus fermentum has been preserved in China general microbiological culture Collection center (CGMCC No. 22833) at 7/6 th 2021.
The 16S rDNA gene sequence is as follows:
ACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGCAGGCGAGTTGCAGCCTGCAGTCCGAACTGAGAACGGTTTTAAGAGATTTGCTTGCCCTCGCGAGTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATCTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAACTAGTAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTCATTGCGTTCCCGAAGGAAACGCCCTATCTCTAGGGTTGGCGCAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTCCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGTCTCAGCGTCAGTTGCAGACCAGGTAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTACCCTCTTCTGCACTCAAGTTATCCAGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACTTAGAAAACCGCCTGCACTCTCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTAAATACCGTCAACGTATGAACAGTTACTCTCATACGTGTTCTTCTTTAACAACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCACGCGGTGTTGCTCCATCAGGCTTGCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTATGGGCCGTGTCTCAGTCCCATTGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATCGCCTTGGTAGGCCGTTACCCCACCAACAAGCTAATGCACCGCAGGTCCATCCAGAAGTGATAGCGAGAAGCCATCTTTTAAGCGTTGTTCATGCGAACAACGTTGTTATGCGGTATTAGCATCTGTTTCCAAATGTTGTCCCCCGCTTCTGGGCAGGTTACCTACGTGTTACTCACCCGTCCGCCACTCGTTGGCGACCAAAATCAATCAGGTGCAAGCACCATCAATCAATGGGCCAACGCGTTC。
example 2 acid and bile salt resistance test of Lactobacillus fermentum LF028
The present example is an acid-resistant and bile salt-resistant property experiment of lactobacillus fermentum LF028, and the acid-resistant experiment and the bile salt-resistant experiment of the strain were respectively performed, and the experiments include the following contents:
(1) acid resistance test
S1, preparing artificial gastric juice
Diluting 10% hydrochloric acid with deionized water, adjusting pH to 2.0, 3.0, and 4.0, respectively, adding pepsin to make the final concentration 1%, and adjusting pH of the solution to 2.0, 3.0, and 4.0 with hydrochloric acid. Filtering with 0.22 μm microporous membrane in sterile operating platform;
s2, probiotic activation
S21, taking out the frozen tube of the lactobacillus fermentum LF028 from a refrigerator, melting at room temperature, inoculating 100 mu L of the frozen tube into 10mL of sterilized MRS culture medium, and culturing at 37 ℃ for 24 hours to obtain a first generation bacterial liquid;
s22, inoculating one-generation bacterial liquid into a sterilized MRS culture medium in an inoculation amount of 3%, and culturing at 37 ℃ for 12h to obtain the second-generation bacterial liquid;
s23, taking 10mL of second-generation bacterium liquid, centrifuging for 10min at 8000r/min, and separating thalli and supernate;
s3, preparation of bacterial suspension
The cells prepared in step S23 were washed 3 times with 0.85% physiological saline to prepare a bacterial suspension.
S4. acid resistance experiment
Mixing 0.5mL of bacterial suspension with 4.5mL of artificial gastric juice with different pH values, standing and culturing in an incubator at 37 ℃, sampling after 2h and 4h respectively, diluting and coating a flat plate, culturing at 37 ℃ for 48h, and determining the colony count. The number of colonies which were not treated with artificial gastric juice was used as a control to calculate the survival rate, and the results of acid resistance test of Lactobacillus fermentum LF028 are shown in Table 1 below.
TABLE 1 acid resistance test results of Lactobacillus fermentum LF028
Note: colony number pairs without artificial gastric juice treatment 5.61 x 107CFU/mL。
The experimental results in table 1 show that the survival rate of lactobacillus fermentum LF028 reaches 99.11% in 2 hours and 98.4% in 4 hours when the pH of gastric acid is 4, the gastric acid can reach 3.5-4.0 after meals, and the gastric emptying time is about 4 hours, so that the probiotics can effectively assist the human body to pass through the gastric juice to reach the intestinal tract after eating the human body after meals.
(2) Bile salt resistance test
S1. preparation of bile salt solution
Preparing MRS culture medium containing bile salt with concentration of 0.1%, 0.2% and 0.3% respectively;
s2, probiotic activation
Taking out the preserved lactobacillus fermentum LF028 from the glycerol tube for resuscitation, taking 100 mu L of lactobacillus fermentum to be inoculated into 10mL of MRS liquid culture medium, and obtaining a first-generation bacterium liquid after culturing for 24h at 37 ℃; inoculating the first generation bacterial liquid into a sterilized MRS culture medium by 3 percent of inoculation amount, and culturing for 12 hours at 37 ℃ to obtain the second generation bacterial liquid.
S3. bile salt resistance experiment
Respectively inoculating the cultured second generation bacterium liquid into 50mL culture media containing bile salts with different concentrations according to the inoculation amount of 2%, and culturing for 2h and 4h at 37 ℃. Sampling, gradient dilution, coating MRS plate, culturing at 37 deg.C for 48h, and determining colony number. The number of colonies not treated with the bile salt solution was used as a control to calculate the survival rate, and the results of the bile salt resistance test of Lactobacillus fermentum LF028 are shown in Table 2 below.
TABLE 2 Lactobacillus fermentum LF028 bile salt resistant test results
Note: colony number without treatment with bile salt solution was 3.35X 107CFU/mL。
The experimental results in table 2 show that the survival rate of lactobacillus fermentum LF028 is good at 0.1% bile salt concentration, reaching more than 90%; the 0.2% bile salt concentration has strong inhibition effect; 0.3% bile salt has obvious inhibiting effect, but can survive and proliferate slowly.
Example 3 in vitro experiment of hypoglycemic Effect of Lactobacillus fermentum LF028
This example is an in vitro test of the hypoglycemic effect of lactobacillus fermentum LF028, comprising the following:
(1) preparation of fermentation broths and suspensions
S1, taking out a cryopreservation tube of lactobacillus fermentum LF028 from a refrigerator, naturally dissolving, inoculating 100 mu L of lactobacillus fermentum to 10mL of sterilized MRS culture medium, and culturing at 37 ℃ for 24h to obtain a generation of seed bacterial liquid; inoculating the first-generation seed bacterial liquid into a sterilized MRS culture medium in an inoculation amount of 3%, and culturing at 37 ℃ for 12h to obtain second-generation bacterial liquid;
s2, taking 10mL of second-generation bacterium liquid, centrifuging for 10min at 8000r/min, and separating thalli and fermentation supernatant;
s3, passing the fermentation supernatant through a 0.22-micron microporous filter membrane to obtain a supernatant;
s4, washing the thalli for 3 times by using PBS buffer solution, and adjusting the concentration of the thalli to be 1 multiplied by 108And (5) CFU/mL to obtain the bacterial suspension.
(2) Inhibition of alpha-amylase
S1, respectively sucking 200 mu L of supernatant and bacterial suspension of lactobacillus fermentum LF028, adding 100 mu L of alpha-amylase of 0.05mg/ml, mixing the two, and keeping the temperature at 37 ℃ for 10 min;
s2, adding 625 mu L of 0.1% starch solution, and reacting for 15min at 37 ℃;
s3, adding 1.0mol/L HCl 300 mu L to terminate the reaction, and then adding 0.01mol/L iodine solution 200 mu L to develop color;
s4, adding distilled water to a constant volume of 5mL, measuring the absorbance A at a wavelength of 660nm, calculating the alpha-amylase inhibition rate, and showing the results of the alpha-amylase inhibition effect of the fermentation liquor and the bacterial suspension in Table 3.
Note: in formula 1, ODA-absorbance values containing the sample and the alpha-amylase; ODB-no alpha-amylase and only the absorbance of the sample; ODC-absorbance of a sample containing alpha-amylase; ODDNeither alpha-amylase nor absorbance of the sample.
TABLE 3 results of alpha-amylase inhibition by fermentation broth and supernatant
| Supernatant fluid | Bacterial suspension | |
| ODA | 0.2372 | 0.3997 |
| ODB | 0.1872 | 0.1322 |
| ODC | 0.6010 | 0.6010 |
| ODD | 0.2210 | 0.2210 |
| Inhibition ratio (%) | 86.84±0.02 | 29.61±0.01 |
It can be seen from table 3 that the supernatant of lactobacillus fermentum LF028 inhibits alpha-amylase more than the suspension, probably because the viscous metabolites produced by lactobacillus fermentum LF028 effectively inhibit the activity of alpha-amylase and prevent starch degradation.
(3) Inhibition of alpha-glucosidase
S1, respectively sucking 100 mu L of supernatant and bacterial suspension of lactobacillus fermentum LF028, adding 100 mu L of alpha-glucosidase solution of 20U/mL, placing the two in a test tube, mixing, and keeping the temperature at 37 ℃ for 10 min;
s2, adding 100 mu L of p-nitrophenol alpha-D-glucopyranoside solution with the concentration of 20mmol/L into each test tube, and reacting for 20min at 37 ℃;
s3, adding 0.2mol/L NaCO into each test tube31mL, terminate the reaction;
s4.96 pore plates are spotted, the light absorption value is measured at 405nm, the alpha-glucosidase inhibition rate is calculated, and the results of the inhibition effect of the supernate and the bacterial suspension on the alpha-glucosidase are shown in Table 4.
Note: OD in equation 2A-adding the absorbance of the sample to be tested and the alpha-glucosidase; ODB-adding a light absorption value of the sample to be tested which does not contain alpha-glucosidase; ODC-absorbance values without the sample to be tested with the addition of alpha-glucosidase; ODD-absorbance values that contain neither the sample to be tested nor the alpha-glucosidase.
TABLE 4 results of fermentation broth and supernatant on alpha-glucosidase inhibition
| Supernatant fluid | Bacterial suspension | |
| ODA | 0.4790 | 0.3582 |
| ODB | 0.4513 | 0.3236 |
| ODC | 0.3658 | 0.3658 |
| ODD | 0.2636 | 0.2636 |
| Inhibition ratio (%) | 72.89±0.04 | 66.14±0.03 |
As can be seen from the experimental results in Table 4, the supernatant and the bacterial suspension of the Lactobacillus fermentum LF028 can effectively inhibit alpha-glucosidase, inhibit starch hydrolysis, reduce glucose increase and reduce sugar absorption of human body.
Examples 4-9 Complex fermentation inoculum containing Lactobacillus fermentum LF028
Examples 4 to 9 are complex fermentation inoculants containing lactobacillus fermentum LF028, and the raw materials for preparing the effective components are the same, but the different raw materials are different in dosage in different examples, and the specific proportions of the raw materials in different examples are shown in table 5.
TABLE 5 raw material proportioning table in examples 4-9
Example 10 preparation of pure fermentation inoculum of Lactobacillus fermentum LF028
The embodiment is a preparation method of a pure fermentation inoculant of lactobacillus fermentum LF028, which specifically comprises the following steps that:
s1, activating lactobacillus fermentum LF028 in an MRS liquid culture medium for two generations, inoculating the lactobacillus fermentum LF028 into the MRS liquid culture medium according to the inoculum size of 2-4%, and performing constant-temperature amplification culture at 37 ℃ for 18 h;
s2, centrifuging the activated and expanded seed solution for 20min at 8000rpm in an environment of 4 ℃, removing supernatant, and collecting thalli;
s3, respectively adding 5-20% of skim milk powder, 5-25% of trehalose and 1-10% of sucrose into the aqueous solution, and sterilizing to prepare a freeze-drying protective agent;
s3, washing the thalli collected in the step S2 for 2 times by using sterile water, centrifuging, discarding supernatant, and suspending the obtained thalli precipitate in 2-6 times of freeze-drying protective agent to obtain 1.0 multiplied by 1010～1.0×1011CFU/mL of bacterial suspension;
s4, subpackaging the prepared bacterial suspension into sterile containers, pre-freezing for 3 hours, and then carrying out freeze drying, wherein the temperature of a cold trap is-50 ℃, and the freeze drying is carried out for 46-50 hours.
S5, collecting freeze-dried bacterial powder, and counting viable bacteria, wherein the viable bacteria count of the bacterial powder is 1.0 multiplied by 1011～3×1011CFU/g。
Example 11-15 method for preparing solidified fermented milk by pure Lactobacillus fermentum LF028
Examples 11 to 15 are solidified fermented milks prepared from pure culture of lactobacillus fermentum LF028, and the active ingredients are prepared from the same raw materials, except that the amounts of the raw materials used in the examples are different, and the specific ratios of the raw materials in the examples are shown in table 6.
TABLE 6 raw material proportioning table in examples 11 to 15
Examples 11-15 preparation of solidified fermented milk prepared from the pure culture starter of lactobacillus fermentum LF028 was carried out according to the following steps:
s1, weighing raw milk, standardizing, uniformly mixing the raw milk with arabinose, and putting the mixture into a blending tank to obtain a material A;
s2, homogenizing the material A, and sterilizing to obtain a material B;
s3, rapidly cooling the material B to 35-39 ℃, inoculating a fermentation microbial inoculum, filling, covering, fermenting at a proper fermentation temperature until curd is formed, stopping fermentation when the curd state is good and the pH value is 4.2-4.5 to obtain a material C;
s4, quickly putting the material C into a refrigerating chamber at the temperature of 2-6 ℃, and after-ripening for 10-14 h to obtain the solidified fermented milk prepared by the lactobacillus ferments LF 028.
In the process of preparing the fermented milk by using the lactobacillus fermentum LF028 pure strain, various process parameters of different preparation steps are shown in the following table 7.
TABLE 7 Process parameters of different preparation steps
Example 16 Effect of fermented Lactobacillus fermentum LF028 and fermented milks prepared therefrom on STZ-induced diabetic mice
In this example, the effect of the fermentation broth of lactobacillus fermentum LF028 and the fermented milk prepared in example 15 on STZ-induced diabetic mice was examined, specifically as follows:
preparation of fermentation broth of lactobacillus fermentum LF 028: taking viable count as 1.0 × 108～3×109CFU/mL lactobacillus fermentum LF0283mL was inoculated into 100mL of sterilized MRS medium at 3% inoculum size and cultured at 37 ℃ for 12h to obtain a fermentation broth.
Selecting 50 male Kunming mice with the weight of 18-20 g, randomly dividing the mice into 5 groups, namely a blank control group, a model group, a positive control group, a fermentation liquor group and a fermented milk group, wherein each group comprises 10 mice, feeding each mouse in a single cage, and filling filter paper and wire netting at the bottom of the cage.
Blank control group: normally feeding under normal environment; the model group, positive control group, fermentation broth group, and fermented milk group were treated with commercially available Streptozotocin (STZ) at 200 mg/kg-1And (5) performing intraperitoneal injection to mold, and detecting the serum glucose level for 72 hours, wherein the blood sugar is higher than 18mmol/L to meet the mold-making requirement. Blank control and model groups: feeding under normal environmentCulturing for 24 days; positive control group: the intragastric administration is carried out for 24 days 1 time per day according to the intragastric administration dosage of the metformin hydrochloride tablets of 30 g/kg; and (3) fermentation liquor group: 5mL of fermentation product of lactobacillus fermentum LF028 is perfused for 24 days after being perfused for 1 time every day; 5mL of lactobacillus fermentum LF028 fermented milk is perfused into each fermented milk group every day, 1 time every day, and the stomach is perfused for 24 days.
Measurement of Fasting Blood Glucose (FBG) index of mice: the fasting blood glucose of mice on day 0, day 4, day 8, day 12, day 16, day 20 and day 24 was measured, 10. mu.L of whole blood was collected from the tail of the mice on the day of fasting blood glucose measurement, and the measurement results were recorded by a glucometer and shown in Table 8.
TABLE 8 Effect of Lactobacillus fermentum LF028 and fermented milks prepared therefrom on diabetic mice
As can be seen from Table 8, the fermentation broth of Lactobacillus fermentum LF028 and fermented milk positively affect blood glucose levels of diabetic mice and effectively lower blood glucose levels of the mice.
Example 17 sensory evaluation of Lactobacillus fermentum LF028 compared with commercially available yogurt ferments and three commercially available Lactobacillus fermentum set-style fermented milks
In this example, the physical and chemical indexes and sensory indexes of the prepared fermented milk were compared with those of the solidified fermented milk fermented by lactobacillus fermentum LF028, commercially available yogurt starter, and commercially available three strains of lactobacillus fermentum, respectively, and the specific method was as follows:
s1, standardizing 10kg of raw milk in each portion, uniformly mixing the raw milk with 1kg of arabinose, and putting the mixture into a preparation tank to obtain a material A;
s2, homogenizing the material A at 70 ℃ and 20MPa, and sterilizing at 90-95 ℃ for 5-10 min to obtain a material B;
s3, rapidly cooling the material B to 37 ℃;
s4, inoculating a commercially available yoghourt fermentation microbial agent MG03 consisting of Lactobacillus delbrueckii subspecies bulgaricus and streptococcus thermophilus into the cooled sterilized milk according to the inoculation amount of 3%, filling, covering, fermenting for about 5 hours in a 42 ℃ fermentation chamber to detect the pH and the curd state, rapidly cooling to 4 ℃ after the fermentation is finished, and keeping for about 12 hours after the fermentation is finished to obtain the fermented milk prepared by the commercially available yoghourt fermentation microbial agent;
s5, respectively preparing pure fermentation inoculants prepared from lactobacillus fermentum LF028, lactobacillus fermentum CECT5716, lactobacillus fermentum LF-G89 and lactobacillus fermentum JL-3 according to the inoculation amount of about 3%, keeping the number of inoculated viable bacteria consistent, inoculating into the cooled sterilized milk, filling, covering, fermenting in a 37 ℃ fermentation chamber for about 16 hours to detect the pH value and the curd state, rapidly cooling after the fermentation is finished, cooling to 4 ℃, and then maturing for about 12 hours to obtain the fermented milk prepared from different lactobacillus fermentum pure fermentation inoculants.
The physical and chemical indexes of acidity, pH value and hardness of the five coagulated fermented milks were measured, and the results are shown in Table 9.
TABLE 9 measurement results of physical and chemical indexes of five set-style yoghurts
The differences of acidity, pH value and hardness of products prepared by the lactobacillus fermentum group LF028 and the commercially available yoghurt starter MG03 are small; the lactobacillus fermentum CECT5716 group and the lactobacillus fermentum JL-3 group have stronger acidity, lower pH value and higher hardness than the lactobacillus fermentum LF028 group and the commercially available yoghourt starter MG03 group; the pH value of the lactobacillus fermentum LF-G89 group was slightly higher than that of the lactobacillus fermentum LF028 group and the commercially available yogurt starter MG03 group, and the hardness was significantly lower.
Sensory evaluation is carried out on the five prepared set fermented milks, a sensory evaluation group comprises 10 yoghourt professional trainers and 20 common consumers, the sensory evaluation group scores the preference degree and various sensory indexes of the product, each full evaluation is 10 points, and the final result is averaged. The scoring standard is a ten-degree standard: 1-extremely dislike; 2-very dislike; 3-dislike; 4-slightly disliked; 5-dislike and no-nuisance; 6-slight liking; 7-liking; 8-very favorite; 9-favorite; 10-perfection. Sensory evaluation results data analysis was performed using IBM SPSS 20 software, and the sensory evaluation results are shown in table 10.
TABLE 10 sensory evaluation results of five set yoghurts
Note: the same letter designation in the same column did not differ significantly at the p ═ 0.05 level, and the different letters designated differences were significant.
The general description is described by combining the experimental results of the table 10 and a sensory evaluation group, the fermented milk prepared by the commercially available yogurt fermentation inoculant MG03 group is good in sour and sweet taste, low in viscosity, and good in public acceptance, and a little whey is separated out, the overall preference score is 7.08 points; the fermented lactic acid prepared by the lactobacillus fermentum CECT5716 group has heavy taste, bad sweet taste, uncomfortable viscosity, poor fineness and whey separation, the score of the overall preference degree is only 5.48 points, and the popular acceptance degree is not good; the fermented milk prepared by the lactobacillus fermentum LF-G89 group has heavy sour taste, poor sweet taste, low viscosity, poor fineness, separated whey, and poor popular acceptance, and the overall preference score is 5.68; the fermented milk prepared by the lactobacillus fermentum JL-3 group is slightly sour, has slightly good sweet taste, low viscosity and poor fineness, has a little whey precipitation, has the overall preference score of 6.27, and has less ideal popular acceptance, but the overall result is better than the lactobacillus fermentum CECT5716 group and the lactobacillus fermentum LF-G89 group; the fermented milk prepared by the lactobacillus fermentum LF028 group has the advantages of palatable sour and sweet taste, fine and smooth mouthfeel, moderate viscosity, no whey precipitation, good uniformity, wire drawing characteristic and 8.32 points of integral preference score.
In conclusion, compared with the commercial yogurt fermentation inoculant MG03 group, the lactobacillus fermentum LF028 group has higher overall preference score, and is obviously better than the commercial yogurt fermentation inoculant MG03 group in terms of total aroma, sour taste, sweet taste, viscosity in mouth, fineness, uniformity and the like; the lactobacillus fermentum group LF028 is significantly superior to the other three commercially available lactobacillus fermentum groups in overall preference, overall aroma, sourness, sweetness, viscosity in the mouth, fineness and homogeneity. The lactobacillus fermentum LF028 can be used as a leaven of probiotic fermented milk to improve the quality of the fermented milk.
Example 18 preparation of plant yogurt with Lactobacillus fermentum LF028 pure fermentation inoculum
In the embodiment, four kinds of plant-based materials, namely soybean milk powder, corn juice powder, coconut milk powder and badam protein powder, are used as raw materials, a pure fermentation inoculant of lactobacillus fermentum LF028 is used to prepare four kinds of plant yoghurts, and the physicochemical indexes and sensory indexes of the four kinds of prepared plant yoghurts are further determined, and the specific method is as follows:
s1, taking 2.3kg of plant-based raw materials per part, adding 7.7kg of purified water per part, and uniformly mixing with 1.0kg of arabinose to obtain a material A;
s2, homogenizing the material A at 70 ℃ and 20MPa, and sterilizing at 90-95 ℃ for 5-10 min to obtain a material B;
s3, rapidly cooling the material B to about 37 ℃ to obtain a material C;
s4, inoculating the pure fermentation microbial inoculum of the lactobacillus fermentum LF028 to the material C according to the inoculation amount of about 3%, filling, covering, fermenting for 16h in a 37 ℃ fermentation chamber, rapidly cooling to 4 ℃, and keeping for about 12h after ripening to prepare the plant yoghourt.
The physical and chemical indexes of the four prepared plant yoghurts, such as acidity, pH value, hardness and the like, are measured, and the measurement results are shown in table 11.
TABLE 11 measurement results of physical and chemical indexes of four set-style yoghurts
The experimental results in Table 11 show that the soybean milk powder has moderate acidity, pH value and hardness and good taste; the coconut milk powder has low acidity, pH value and hardness and low solidification strength; the corn juice powder and the badam protein powder have higher fermentation hardness, lower pH value and high acidity. This is related to the different protein and fat contents of the different raw materials. The product made of the soybean milk powder has good state from acidity, pH value and state.
And performing sensory evaluation on the prepared four kinds of plant yogurt, wherein a sensory evaluation group comprises 10 professional trainers and 20 common consumers, the sensory evaluation group scores the preference degree and various sensory indexes of the product, each sensory index is 10 points, and the average value of the results is taken finally. The scoring criteria were in tenths: 1-extremely dislike; 2-very dislike; 3-dislike; 4-slightly disliked; 5-dislike and no-nuisance; 6-slight liking; 7-liking; 8-very much like; 9-favorite; 10-perfection. Sensory evaluation results data analysis was performed using IBM SPSS 20 software, and the sensory evaluation results are shown in Table 12
TABLE 12 four vegetable yogurt sensory evaluation results
The general summary is described by combining the experimental results of the table 12 and a sensory evaluation group, the soybean milk powder has better sourness and sweetness, high viscosity and small beany flavor, and the integral preference score is 8.55 points; the corn juice powder group has heavy sour taste, coarse mouthfeel, low viscosity and corn fragrance, and the whole preference score is 5.21 points; the coconut milk powder group has higher sweet taste, proper sweet and sour taste, stronger coconut flavor, low viscosity and 7.23 points of integral preference score; the badam protein powder group is moderate in sweetness and sourness, has the peculiar fragrance of nuts, is slightly low in viscosity and heavy in oil taste, and the overall preference score is 6.43.
In conclusion, the plant yoghourt prepared from the soybean milk powder group and the coconut milk powder group has good overall acceptance, the lactobacillus fermentum LF028 can ferment various plant-based raw materials, and the prepared products have different flavors due to different contents of protein, fat and the like in the components of the plant-based raw materials.
SEQUENCE LISTING
<110> Tianjin Xiaowei Biotechnology Ltd
<120> lactobacillus fermentum LF028 with hypoglycemic function and application thereof
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1389
<212> DNA
<213> Lactobacillus fermentum LF028 (Lactobacillus fermentum)
<400> 1
actctcatgg tgtgacgggc ggtgtgtaca aggcccggga acgtattcac cgcggcatgc 60
tgatccgcga ttactagcga ttccgacttc gtgcaggcga gttgcagcct gcagtccgaa 120
ctgagaacgg ttttaagaga tttgcttgcc ctcgcgagtt cgcgactcgt tgtaccgtcc 180
attgtagcac gtgtgtagcc caggtcataa ggggcatgat gatctgacgt cgtccccacc 240
ttcctccggt ttgtcaccgg cagtctcact agagtgccca acttaatgct ggcaactagt 300
aacaagggtt gcgctcgttg cgggacttaa cccaacatct cacgacacga gctgacgacg 360
accatgcacc acctgtcatt gcgttcccga aggaaacgcc ctatctctag ggttggcgca 420
agatgtcaag acctggtaag gttcttcgcg tagcttcgaa ttaaaccaca tgctccaccg 480
cttgtgcggg cccccgtcaa ttcctttgag tttcaacctt gcggtcgtac tccccaggcg 540
gagtgcttaa tgcgttagct ccggcactga agggcggaaa ccctccaaca cctagcactc 600
atcgtttacg gcatggacta ccagggtatc taatcctgtt cgctacccat gctttcgagt 660
ctcagcgtca gttgcagacc aggtagccgc cttcgccact ggtgttcttc catatatcta 720
cgcattccac cgctacacat ggagttccac taccctcttc tgcactcaag ttatccagtt 780
tccgatgcac ttctccggtt aagccgaagg ctttcacatc agacttagaa aaccgcctgc 840
actctcttta cgcccaataa atccggataa cgcttgccac ctacgtatta ccgcggctgc 900
tggcacgtag ttagccgtga ctttctggtt aaataccgtc aacgtatgaa cagttactct 960
catacgtgtt cttctttaac aacagagctt tacgagccga aacccttctt cactcacgcg 1020
gtgttgctcc atcaggcttg cgcccattgt ggaagattcc ctactgctgc ctcccgtagg 1080
agtatgggcc gtgtctcagt cccattgtgg ccgatcagtc tctcaactcg gctatgcatc 1140
atcgccttgg taggccgtta ccccaccaac aagctaatgc accgcaggtc catccagaag 1200
tgatagcgag aagccatctt ttaagcgttg ttcatgcgaa caacgttgtt atgcggtatt 1260
agcatctgtt tccaaatgtt gtcccccgct tctgggcagg ttacctacgt gttactcacc 1320
cgtccgccac tcgttggcga ccaaaatcaa tcaggtgcaa gcaccatcaa tcaatgggcc 1380
aacgcgttc 1389
Claims (9)
1. The Lactobacillus fermentum LF028 with the hypoglycemic effect is characterized in that the Lactobacillus fermentum is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.22833 and is named as Lactobacillus fermentum in Latin.
2. The Lactobacillus fermentum LF028 for reducing blood glucose according to claim 1, wherein 16S rDNA has the following gene sequence:
ACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGCAGGCGAGTTGCAGCCTGCAGTCCGAACTGAGAACGGTTTTAAGAGATTTGCTTGCCCTCGCGAGTTCGCGACTCGTTGTACCGTCCATTGTAGCACGTGTGTAGCC
CAGGTCATAAGGGGCATGATGATCTGACGTCGTCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACTAGAGTGCCCAACTTAATGCTGGCAACTAGTAACAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTCATTGCGTTCCCGAAGGAAACGCCCTATCTCTAGGGTTGGCGCAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTCCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCACTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGTCTCAGCGTCAGTTGCAGACCAGGTAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTCCACCGCTACACATGGAGTTCCACTACCCTCTTCTGCACTCAAGTTATCCAGTTTCCGATGCACTTCTCCGGTTAAGCCGAAGGCTTTCACATCAGACTTAGAAAACCGCCTGCACTCTCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGACTTTCTGGTTAAATACCGTCAACGTATGAACAGTTACTCTCATACGTGTTCTTCTTTAACAACAGAGCTTTACGAGCCGAAACCCTTCTTCACTCACGCGGTGTTGCTCCATCAGGCTTGCGCCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTATGGGCCGTGTCTCAGTCCCATTGTGGCCGATCAGTCTCTCAACTCGGCTATGCATCATCGCCTTGGTAGGCCGTTACCCCACCAACAAGCTAATGCACCGCAGGTCCATCCAGAAGTGATAGCGAGAAGCCATCTTTTAAGCGTTGTTCATGCGAACAACGTTGTTATGCGGTATTAGCATCTGTTTCCAAATGTTGTCCCCCGCTTCTGGGCAGGTTACCTACGTGTTACTCACCCGTCCGCCACTCGTTGGCGACCAAAATCAATCAGGTGCAAGCACCATCAATCAATGGGCCAACGCGTTC。
3. use of a lactobacillus fermentum LF028 with hypoglycemic effect according to claim 1 or 2, for the preparation of a fermentation inoculant.
4. The use of Lactobacillus fermentum LF028 having hypoglycemic effect according to claim 3, wherein the said Lactobacillus fermentum is a pure Lactobacillus fermentum.
5. The use of the pure fermentation inoculum according to claim 4, wherein the pure fermentation inoculum is used for preparing fermented milk.
6. The use of the pure fermentation inoculum according to claim 5, wherein the preparation method of the fermented milk comprises the following steps in sequence:
s1, taking 80-100 parts of raw milk, uniformly mixing with 7-11 parts of arabinose, homogenizing, and sterilizing to obtain a material A;
s2, cooling the material A, inoculating a pure fermentation microbial inoculum, filling, covering, and fermenting until curd is formed to obtain a material B;
and S3, refrigerating the material B, and after-ripening to obtain the fermented milk.
7. The use of the pure fermentation inoculum according to claim 6, wherein in step S1, the homogenization temperature is 65-75 ℃, the pressure is 18.5-21.5 MPa, the sterilization temperature is 90-95 ℃, and the sterilization time is 5-10 min;
in the step S2, the temperature of the material A is reduced to 35-39 ℃, the inoculation amount of the pure fermentation inoculant is 2-4%, the fermentation temperature is 34-40 ℃, the time is 14-18 h, and the pH value of curd is 4.2-4.5;
in the step S3, the refrigeration temperature is 2-6 ℃, and the after-ripening time is 10-14 h.
8. The use of the pure fermentation inoculum according to claim 4, wherein the pure fermentation inoculum is used for preparing plant yogurt.
9. The use of the pure fermentation inoculum according to claim 8, wherein the plant-based raw materials selected for preparing the plant yogurt comprise at least one of soybean, mung bean, corn, coconut, almond, walnut, black bean and peanut.
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