CN114456977A - Lactobacillus delbrueckii subspecies bulgaricus SF-L-18 and fermented beverage and application thereof - Google Patents
Lactobacillus delbrueckii subspecies bulgaricus SF-L-18 and fermented beverage and application thereof Download PDFInfo
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Classifications
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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
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/38—Other non-alcoholic beverages
- A23L2/382—Other non-alcoholic beverages fermented
-
- 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
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
-
- 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
-
- 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
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
-
- 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
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- 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
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- 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
- A23V2400/137—Delbrueckii
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
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- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
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- Biomedical Technology (AREA)
- Virology (AREA)
- Microbiology (AREA)
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- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Non-Alcoholic Beverages (AREA)
Abstract
The invention relates to lactobacillus delbrueckii subspecies bulgaricus SF-L-18 and a fermented beverage and application thereof, belonging to the technical field of microorganisms. The Lactobacillus delbrueckii subsp. bulgaricus is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC NO.20928, the preservation date of 2020, 10 and 21 days, the address of the preservation organization: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North. The plant beverage prepared by fermenting the lactobacillus delbrueckii subspecies bulgaricus SF-L-18 can effectively improve bleeding and pain of hemorrhoids, and can play a significant role in relieving and assisting in treatment.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to lactobacillus delbrueckii subsp bulgaricus SF-L-18 and a fermented beverage and application thereof.
Background
With the improvement of economic level, the life and dietary habits of people are changed greatly, and the incidence of hemorrhoids is increased year by year. The national professional anorectal epidemiological survey shows that the prevalence rate of anorectal diseases reaches 50.1%, and 98.09% of anorectal diseases have symptoms related to hemorrhoids. The focus of the hemorrhoid is positioned in the anus and rectum, and because the vein has no valve, the blood does not flow back or flows back smoothly, and then the mechanical extrusion and the blood deposition are carried out during defecation, the blood backflow difficulty is increased, and the hemorrhoid is finally formed. Hemorrhoids can be attacked at any age, are mostly attacked by middle-aged and elderly people, surgical treatment is the final choice, and complications such as incision edema pain, urinary retention, anal stenosis and the like after surgery and postoperative hemorrhoid regeneration are often overlooked by patients, so how to control hemorrhoid development or conservative treatment becomes a great problem in anorectal departments. Therefore, it is desirable to provide an effective product for the improvement of hemorrhoids.
Disclosure of Invention
Aiming at the defect that the existing product is not used for improving the hemorrhoids, the invention provides a Lactobacillus delbrueckii subsp bulgaricus SF-L-18 and a fermented beverage and application thereof, so as to solve the technical problems.
Lactobacillus delbrueckii subsp. bulgaricus SF-L-18 is preserved in China general microbiological culture Collection center with the preservation number of CGMCC NO.20928, the preservation date of 2020, 10 and 21 days, the preservation organization address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North.
Preferably, the Lactobacillus delbrueckii subspecies bulgaricus SF-L-18 is obtained from the feces of a hemorrhoid rehabilitee.
The application of the lactobacillus delbrueckii subspecies bulgaricus SF-L-18 is to prepare the plant fermented beverage.
A culture method of Lactobacillus delbrueckii subspecies bulgaricus SF-L-18 comprises the steps of inoculating Lactobacillus delbrueckii subspecies bulgaricus SF-L-18 into a sterilized MRS liquid culture medium, wherein the inoculation amount is 2%, and culturing for 24 hours at 37 ℃ to obtain a culture solution of Lactobacillus delbrueckii subspecies bulgaricus SF-L-18.
Preferably, the MRS medium comprises the following components: 10.0g of beef extract, 5.0g of yeast extract, 10.0g of peptone, 20.0g of glucose and K2HPO42.0g, sodium acetate 5.0g, MgSO4·7H2O 0.2g，MnSO4·4H2O0.05 g, Tween 801.0 g, triammonium citrate 2.0g, and distilled water 1000 ml.
A preparation method of a plant fermentation beverage comprises the following steps:
(1) mixing honeysuckle, sophora flower, angelica dahurica, gorgon fruit, orange peel, coix seed, lily, Chinese yam, rhizoma polygonati, ginseng, dark plum powder and purslane, adding water, decocting for four times, and filtering through 200 meshes to obtain a mixed solution.
(2) Adding elastic collagen peptide into the mixed solution, sterilizing for 30min under high pressure, and cooling to 30-40 ℃ to obtain a sterilized solution;
(3) inoculating Lactobacillus delbrueckii subspecies Bulgaria SF-L-18 into MRS liquid culture medium, and culturing at 37 deg.C to prepare inoculant;
(4) inoculating the inoculant into a sterilization solution, wherein the inoculation amount is 500-1000 ten thousand cfu/mL, and fermenting for 24-48 h at 37 ℃ to obtain fermentation liquor;
(5) and adding carrot concentrated juice, prebiotic syrup and juicy peach concentrated juice into the fermentation liquor, seasoning, performing wall breaking and pasteurization to obtain the probiotic plant beverage, wherein the wall breaking pressure is 20-60 MPa, and thus obtaining the plant fermented beverage.
Preferably, the plant fermented beverage comprises the following raw materials in parts by weight: 50-80 parts of purified water, 5-10 parts of carrot concentrated juice and 2-5 parts of prebiotics syrup; 2-5 parts of juicy peach concentrated juice, 1.5-2 parts of honeysuckle, 2-4 parts of sophora flower, 0.1-1 part of angelica dahurica, 0.5-1 part of gordon euryale seed, 2-5 parts of orange peel, 2-6 parts of coix seed, 3-6 parts of lily, 0.5-1 part of Chinese yam, 0.5-1 part of rhizoma polygonati, 0.1-2 parts of ginseng, 0.5-2 parts of dark plum powder, 0.1-2 parts of elastic collagen peptide and 0.2-2 parts of purslane.
Preferably, the product comprises the following raw materials in parts by weight: 73 parts of purified water, 6 parts of carrot concentrated juice, 3 parts of prebiotics syrup, 3 parts of juicy peach concentrated juice, 2 parts of honeysuckle, 2 parts of sophora flower, 0.5 part of angelica dahurica, 0.5 part of gordon euryale seed, 2 parts of orange peel, 2 parts of coix seed, 3 parts of lily, 1 part of Chinese yam, 0.5 part of rhizoma polygonati, 0.5 part of ginseng, 0.5 part of dark plum powder, 0.15 part of elastic collagen peptide and 0.35 part of purslane.
The Lactobacillus delbrueckii subspecies bulgaricus SF-L-18 is applied to preparing the plant fermented beverage for improving bleeding and pain of hemorrhoids.
The invention has the beneficial effects that:
the plant beverage prepared by fermenting the lactobacillus delbrueckii subspecies bulgaricus SF-L-18 can effectively improve bleeding and pain of hemorrhoids, and can play a significant role in relieving and assisting in treatment.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Screening of Lactobacillus delbrueckii subspecies bulgaricus SF-L-18
(1) The strain source is as follows: feces obtained from patients with hemorrhoid rehabilitation. Collecting time: year 2018, month 01; the collection place comprises: the inner Mongolia autonomous region of China is called Haote City.
(2) Plate culture medium: 10.0g of beef extract, 5.0g of yeast extract, 10.0g of peptone, 20.0g of glucose and K2HPO42.0g, sodium acetate 5.0g, MgSO4·7H2O 0.2g，MnSO4·4H20.05g of O, 801.0 g of Tween, 2.0g of triammonium citrate, 15.0g of agar and distilled water added to the solution until the volume is 1000 ml.
(3) Isolation of the Strain
Ten-fold gradient dilution is carried out on the sample liquid, 100 mu L of each gradient is uniformly coated on MRS solid plate culture medium, and then the sample liquid is taken out and placed in an anaerobic tank to be anaerobically cultured overnight at 37 ℃. The next day, colonies with different shapes and sizes grow on the plate, a plurality of characteristic colonies of lactic acid bacteria are selected for the second-generation streak purification treatment, the plate is subjected to anaerobic activation for 2 days to obtain a culture, and then the strain identification is carried out.
(4) The 16S rDNA amplification primers used were 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3' are provided. The product was commissioned for Sanger sequencing in a qualified third party laboratory. Sequences were compared to the 16SrDNA gene in the GenBank database using the BLAST algorithm. BLAST analysis showed that the strain SF-L-18 is very close to Lactobacillus delbrueckii subspecies Bulgaria (99% relatedness).
And (3) detection results:
TCAGGACGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAGCTGAATTCAAAGATCCCTTCGGGGTGATTTGTTGGACGCTAGCGGCGGATGGGTGAGTAACACGTGGGCAATCTGCCCTAAAGACTGGGATACCACTTGGAAACAGGTGCTAATACCGGATAACAACATGAATCGCATGATTCAAGTTTGAAAGGCGGCGCAAGCTGTCACTTTAGGATGAGCCCGCGGCGCATTAGCTAGTTGGTGGGGTAAAGGCCTACCAAGGCAATGATGCGTAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTCTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGCAGTAACTGGTCTTTATTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGAATGATAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAACTGCATCGGAAACTGTCATTCTTGAGTGCAGAAGAGGAGAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTCTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGCGCTAGGTGTTGGGGACTTTCCGGTCCTCAGTGCCGCAGCAAACGCATTAAGCGCTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTGCGCTACACCTAGAGATAGGTGGTTCCCTTCGGGGACGCAGAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTTTAGTTGCCATCATTAAGTTGGGCACTCTAAAGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGTACAACGAGAAGCGAACCCGCGAGGGTAAGCGGATCTCTTAAAGCTGCTCTCAGTTCGGACTGCAGGCTGCAACTCGCCTGCACGAAGCTGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGAAGTCTGCAATGCCCAAAGTCGGTGGGATAACCTTTATAGGAGTCAGCCGCCTAAGGCAGGGCAGATGACTGGGGTGAAGTC。
example 2
A preparation method of a plant fermented beverage comprises the following steps:
(1) mixing 2 parts of honeysuckle, 2 parts of sophora flower, 0.5 part of angelica dahurica, 0.5 part of gordon euryale seed, 2 parts of orange peel, 2 parts of coix seed, 3 parts of lily, 1 part of Chinese yam, 0.5 part of sealwort, 0.5 part of ginseng, 0.5 part of dark plum powder and 0.35 part of purslane, adding 73 parts of water, decocting for four times, and filtering through 200 meshes to obtain a mixed solution.
(2) Adding 0.15 part of elastic collagen peptide into the mixed solution, sterilizing the mixed solution for 30 minutes at 121 ℃ under high pressure, and cooling the mixed solution to 30-40 ℃ to obtain a sterilized solution;
(3) inoculating lactobacillus delbrueckii subspecies bulgaricus SF-L-18 into a sterilized MRS liquid culture medium, wherein the inoculation amount is 2%, and culturing at 37 ℃ for 24 hours to obtain a lactobacillus delbrueckii subspecies bulgaricus SF-L-18 culture solution; the MRS culture medium comprises the following components: 10.0g of beef extract, 5.0g of yeast extract, 10.0g of peptone, 20.0g of glucose and K2HPO42.0g, sodium acetate 5.0g, MgSO4·7H2O 0.2g，MnSO4·4H20.05g of O, 801.0 g of Tween, 2.0g of triammonium citrate and distilled water to 1000 ml; autoclaving at 121 deg.C for 20 min;
(4) inoculating a lactobacillus delbrueckii subspecies bulgaricus SF-L-18 culture solution into a sterilization solution, wherein the inoculation amount is 500-1000 ten thousand cfu/mL, and fermenting for 24 hours at 37 ℃ to obtain a fermentation solution;
(5) 6 parts of carrot concentrated juice, 3 parts of prebiotics syrup and 3 parts of juicy peach concentrated juice are added into the fermentation liquor, and then wall breaking and pasteurization are carried out to obtain the probiotic plant beverage, the wall breaking pressure is 20-60 Mpa, and the plant fermented beverage is obtained, is brown liquid and can be directly drunk.
Example 3
A preparation method of a plant fermentation beverage comprises the following steps:
(1) mixing 1.8 parts of honeysuckle, 3 parts of sophora flower, 0.1 part of angelica dahurica, 0.7 part of gordon euryale seed, 4 parts of orange peel, 4 parts of coix seed, 5 parts of lily, 0.8 part of Chinese yam, 0.7 part of rhizoma polygonati, 0.1 part of ginseng, 1 part of dark plum powder and 0.2 part of purslane, adding 50 parts of water, decocting for four times, and filtering through 200 meshes to obtain a mixed solution.
(2) Adding 0.1 part of elastic collagen peptide into the mixed solution, sterilizing the mixed solution for 30 minutes at 121 ℃ under high pressure, and cooling the mixed solution to 30-40 ℃ to obtain a sterilized solution;
(3) inoculating lactobacillus delbrueckii subspecies bulgaricus SF-L-18 into a sterilized MRS liquid culture medium, wherein the inoculation amount is 2%, and culturing at 37 ℃ for 24h to obtain a lactobacillus delbrueckii subspecies bulgaricus SF-L-18 culture solution;
(4) inoculating a lactobacillus delbrueckii subspecies bulgaricus SF-L-18 culture solution into a sterilization solution, wherein the inoculation amount is 500-1000 ten thousand cfu/mL, and fermenting for 24 hours at 37 ℃ to obtain a fermentation solution;
(5) and adding 5 parts of carrot concentrated juice, 2 parts of prebiotic syrup and 2 parts of juicy peach concentrated juice into the fermentation liquor, seasoning, and performing wall breaking and pasteurization to obtain a probiotic plant beverage, wherein the wall breaking pressure is 20-60 Mpa, so that the plant fermented beverage is obtained, and the prepared plant fermented beverage is brown liquid and can be directly drunk.
Example 4
A preparation method of a plant fermentation beverage comprises the following steps:
(1) mixing 2 parts of honeysuckle, 4 parts of sophora flower, 1 part of angelica dahurica, 1 part of gordon euryale seed, 5 parts of orange peel, 6 parts of coix seed, 6 parts of lily, 0.5 part of Chinese yam, 1 part of rhizoma polygonati, 2 parts of ginseng, 2 parts of dark plum powder and 2 parts of purslane, adding 80 parts of water, decocting for four times, and filtering through 200 meshes to obtain a mixed solution.
(2) Adding 2 parts of elastic collagen peptide into the mixed solution, sterilizing the mixed solution for 30 minutes at the temperature of 121 ℃, and cooling the mixed solution to 30-40 ℃ to obtain a sterilized solution;
(3) inoculating lactobacillus delbrueckii subspecies bulgaricus SF-L-18 into a sterilized MRS liquid culture medium, wherein the inoculation amount is 2%, and culturing at 37 ℃ for 24h to obtain a lactobacillus delbrueckii subspecies bulgaricus SF-L-18 culture solution;
(4) inoculating a lactobacillus delbrueckii subspecies bulgaricus SF-L-18 culture solution into a sterilization solution, wherein the inoculation amount is 500-1000 ten thousand cfu/mL, and fermenting for 48 hours at 37 ℃ to obtain a fermentation solution;
(5) 10 parts of carrot concentrated juice, 5 parts of prebiotics syrup and 5 parts of juicy peach concentrated juice are added into the fermentation liquor, and then wall breaking and pasteurization are carried out to obtain the probiotic plant beverage, the wall breaking pressure is 20-60 Mpa, and the plant fermented beverage is obtained, is brown liquid and can be directly drunk.
Example 5
Experiment of mouse model of hemorrhoid with plant beverage
80 ICR mice were purchased, wherein the mice were randomly modeled with 60 mice, 0.05mL of 20% acetic acid solution was injected 0.5cm into the anus of the mice in the modeling group, the solution was kept for 1min, and the normal group was replaced with the same amount of physiological saline. After 24h of molding, the mold has a white ulcer surface, perianal swelling and inflammation exudation which are accompanied, indicating that the molding is successful. After the modeling is successful, the modeling group is randomly divided into a model group, a plant beverage group and a Chinese herbal medicine group according to the size of swelling, and each group contains 20 animals.
Mixing 2 parts of honeysuckle, 2 parts of sophora flower, 0.5 part of angelica dahurica, 0.5 part of gordon euryale seed, 2 parts of orange peel, 2 parts of coix seed, 3 parts of lily, 1 part of Chinese yam, 0.5 part of sealwort, 0.5 part of ginseng, 0.5 part of dark plum powder and 0.35 part of purslane, and adding 8 times of water to decoct for 3 times to obtain Chinese herbal medicine juice as a Chinese herbal medicine for administration (1 ml/kg); the fermented plant drink obtained in example 2 was administered as a group of plant drinks (1 ml/kg); the model group and the normal group were fed with the same dose of sterile water.
The detection items comprise: perianal score, serum inflammatory cytokines and nitric oxide detection.
Perianal scoring: the 4 groups of mice were given a continuous dose of 15d according to the experimental protocol, and perianal was scored by washing perianal 12h after the last dose. The scoring criteria were: the scoring items comprise dry stool, hematochezia, anus moisture, perianal swelling, perianal ulcer and perianal ecchymosis; the method comprises the following steps of: score 1 is none, score 2 is visible, score 3 is more evident, and score 4 is evident.
Serum inflammatory cytokine detection: the animals were anesthetized 12h after the last administration, the ventral main vein was bled, the serum was separated, and the peripheral blood IL-6, IL-1 β and TNF- α content was detected according to the CBA kit instructions.
Detecting the content of Nitric Oxide (NO) in serum: according to the instruction, the content of the NO in the serum is detected by using a Greiss kit.
Data were processed using SPSS 18.0 software, and experimental results were expressed as mean. + -. standard deviation (x. + -.s), and comparisons between groups were performed using one-way analysis of variance and t-test, and Ridit test for grade data. P <0.05 indicates that the difference is statistically significant. The specific experimental results are shown in the following tables 1-3:
TABLE 1 perianal scores of mice from different experimental groups
| Grouping | Dry stool | Hematochezia | Anus moisture | Swelling of the perianal area | Perianal ulcer | Ecchymosis of anus |
| Normal group | 0.66±0.23 | 0.00±0.00 | 0.59±0.13 | 0.41±0.11 | 0.00±0.00 | 0.00±0.00 |
| Model set | 2.63±0.49A | 3.16±0.52A | 3.45±0.62A | 3.54±0.43A | 3.57±0.74A | 3.18±0.65A |
| Plant beverage group | 1.63±0.62B | 1.66±0.29B | 2.06±0.54b | 1.65±0.32B | 1.33±0.51B | 1.39±0.48B |
| Chinese herbal medicine | 1.98±0.56bc | 1.87±0.54Bc | 2.07±0.47b | 1.81±0.67B | 2.04±0.23bc | 2.01±0.74bc |
Note: p <0.01 for a compared to normal; b <0.05 for P compared to model group, B <0.01 for P compared to model group; c P <0.05 compared to the plant drink group.
As can be seen from the experimental data in Table 1, compared with the normal group, all mice in the model group have obviously increased scores (P <0.01) such as dry stool, hematochezia, anus dampness, perianal swelling, perianal ulcer and anal ecchymosis, which indicates that the success rate of the model of the experimental modeling method is 100%; compared with the model group, the scores of the plant beverage group and the Chinese herbal medicine group for mice are obviously reduced (P <0.05), and the scores of the plant beverage group and the Chinese herbal medicine group for mice for dry stool, bloody stool, moist anus, perianal swelling, perianal ulcer and anal ecchymosis are obviously lower than the scores of the Chinese herbal medicine group for dry stool, bloody stool, perianal ulcer, anal ecchymosis and the like (P < 0.05).
TABLE 2-serum IL-6, IL-1. beta. and TNF-alpha. levels in mice of different experimental groups
| Grouping | IL-6(pg/ml) | IL-1β(pg/ml) | TNF-ɑ(pg/ml) |
| Normal group | 211.64±28.74 | 118.22±24.15 | 191.14±31.25 |
| Model set | 343.14±33.19A | 165.88±26.87A | 275.11±28.99A |
| Plant beverage group | 255.11±24.55B | 124.12±24.91b | 221.61±21.43b |
| Chinese herbal medicine | 296.19±32.87bc | 136.41±24.66b | 234.14±25.47b |
Note: p <0.01 for a compared to normal; b <0.05 for P compared to model group, B <0.01 for P compared to model group; c P <0.05 compared to the plant drink group.
An important cause of the onset of hemorrhoids is an abnormal level of inflammation, in which cytokines play a central role. The expression of IL-6, IL-1 beta and TNF-alpha in serum is increased and is positively correlated with the severity of inflammatory destruction and inflammatory reaction; these inflammatory cytokines can induce local inflammatory cell infiltration and tissue damage. Inflammation is the basic defense reaction after body trauma, and the content of inflammatory factors can reflect the severity of inflammation. IL-6, IL-1 beta and TNF-alpha are all clinical common inflammation indexes and are commonly used as important indexes for evaluating the degree of inflammatory response after the treatment of hemorrhoids. As can be seen from the experimental data in Table 2, the contents of the serum inflammatory cytokines IL-6, IL-1 beta and TNF-alpha of the model group mice are all obviously increased (P is less than 0.05) compared with the normal group; compared with the model group, the contents of IL-6, IL-1 beta and TNF-alpha in the serum of mice in the plant drink group and the Chinese herbal medicine group are all obviously reduced (P is less than 0.05); compared with the Chinese herbal medicine, the content of IL-1 beta and TNF-alpha in the serum of the mice in the plant drink group is lower, but no significant statistical difference exists. But the IL-6 content of the plant drink group is obviously lower than that of the Chinese herbal medicine group.
TABLE 3 serum nitric oxide levels in mice of different experimental groups
| Grouping | NO(μmol/L) |
| Normal group | 79.84±8.74 |
| Model set | 128.44±9.19A |
| Plant beverage group | 86.15±7.65B |
| Chinese herbal medicine | 105.19±6.33bc |
Note: p <0.01 for a compared to normal; b <0.05 for P compared to model group, B <0.01 for P compared to model group; c P <0.05 compared to the plant drink group.
Nitric Oxide (NO), an important second messenger, is an endogenous angiotensin factor and is widely involved in regulating numerous pathophysiological processes such as blood circulation, platelet activity, neurotransmission, immunity and inflammation. As can be seen from the experimental data in table 3, the serum NO content of the model group mice is significantly increased (P <0.05) compared with the normal group; compared with the model group, the serum NO content of the plant beverage group and the Chinese herbal medicine mouse is obviously reduced (P is less than 0.05); compared with the Chinese herbal medicine, the serum NO content of the mice in the plant drink group is lower, and the statistical difference is significant (P < 0.05).
As can be seen by the test of example 5, the fermented plant beverage prepared in example 2 can obviously reduce the levels of IL-6, IL-1 beta, TNF-alpha and nitric oxide in the serum of the hemorrhoid mice molded by 20% acetic acid, and the perianal score is obviously reduced; compared with Chinese herbal medicines, the plant fermented drink has better effect.
Example 6
1. Subject screening
1.1 participating test subjects
1) Patients who meet the first and second degree diagnosis standard of hemorrhoids (hematochezia, can stop after defecation; if the stool is separated, the stool can be returned automatically); there is a history of hemorrhoids and occasionally recurrences.
2) Age 18 to 60 years, sex randomized.
3) Voluntarily participate in clinical trials.
1.2 non-participatory tests in which the following events occurred
1) Pregnancy and lactation.
2) Other pathological changes (such as injury, operation, inflammation, and tumor) in the rectal anal canal.
3) The patients can not follow up on time.
4) Patients are taking antibiotics, glucocorticoids.
1.3 required termination of the test
1) Serious adverse reactions appear, and the research and treatment are difficult to be continued after judgment.
2) Other drugs were used ad libitum because the patients did not work cooperatively.
3) The patient is allergic or intolerant to the plant drink.
4) Abnormal exacerbations and other reasons may make it difficult for the investigator to continue study treatment.
5) Pregnancy occurred during the test.
6) The patient is asked to exit the trial.
7) Those who are lost of visit.
8) No plant beverage is used.
9) The associated auxiliary checks are not completed as required.
10) Misdiagnosis and mistaking.
11) In the test process, the majority of plant drinks have no curative effect or serious safety problems, and have no clinical value.
12) In the test process, a determined test scheme has major errors, or major deviation occurs in the implementation process, so that the effect of the microbial inoculum is difficult to evaluate.
2. Test method
2.1 selecting the population meeting the test, and taking the product by the subject half an hour in the morning and at night every day for 2 times a day, one bag (30 g/bag) each time, and continuously taking for 28 days. During the trial period, the subject is required to fill out a trial condition record table every day according to the self condition. Taking symptoms before and after the patient takes the test as a contrast, and comparing the symptoms of bleeding, prolapse, pain and pruritus of the patient and the improvement degree of defecation; detecting the content of short-chain fatty acid in feces before and after use by a subject, detecting by gas chromatography, and collecting and detecting signals by an N3000 double-channel chromatographic workstation. And meanwhile, detecting the intestinal flora according to technical specifications for health food inspection and evaluation (2003), and counting the bifidobacteria, the lactobacilli, the enterococci, the enterobacteriums, the bacteroides and the clostridium perfringens in the excrement of the subject before and after use.
3. Observation index
The standard of curative effect is formulated according to the diagnosis standard of hemorrhoid diseases formulated by anorectal surgery in China, and is divided into three conditions of healing, effectiveness and ineffectiveness.
And (3) healing: the symptoms of perianal pain, edema and the like of the patient disappear completely or basically.
The method has the following advantages: the perianal pain of the patient is obviously improved, and edema is relieved.
And (4) invalidation: the symptoms of perianal pain, edema and the like of the patients are not obviously improved.
And (4) comparing the symptom scores of bleeding, prolapse, pain and pruritus before and after treatment of the patients.
Bleeding: the score of 0-3 is 0, the score of 0 indicates that the patient has no bleeding symptoms, the score of 1 indicates that the patient bleeds slightly and is stained with blood after defecation, the score of 2 indicates that the patient drips and carries blood when defecating, the blood stops automatically after defecation, and the score of 3 indicates that the patient bleeds in a jet shape and cannot stop automatically in a short time.
Removing: 0-3 points, wherein 0 point indicates that the patient does not prolapse, 1 point indicates that the patient automatically receives the excrement after having hemorrhoid prolapse, 2 points indicate that the patient needs to receive the excrement by hands after having hemorrhoid prolapse, and 3 points indicate that the patient continuously prolapse.
Pain: the score of 0-9 indicates that the patient has no pain, the score of 4 indicates that the patient has mild pain and can endure while defecation, and the score of 9 indicates that the patient has severe pain and can not normally relax the bowels.
Pruritus: the score of 0-9 indicates that the patient has no pruritus, the score of 4 indicates that the patient has mild pruritus and does not need intervention, and the score of 9 indicates that the patient has severe pruritus and seriously affects daily life.
4. Results of taking test of the product
(1) The number of people who finish trying to dress this time is 36 in total.
(2) The results of the tests are shown in tables 4 to 6 below.
TABLE 4 comparison of the integral Total sign changes before and after administration
| Number of human subjects | Before taking | After taking | P value |
| 36 | 15.460±3.845 | 6.127±1.875 | P＜0.05 |
As shown in Table 4, the total score of symptom signs and the quantitative score of symptom signs before and after the trial had significant difference (P <0.05), the total score of symptom before treatment was 15.460 + -3.845, and the score after treatment was 6.127 + -1.875.
TABLE 5 quantitative integral value change table for symptom and sign before and after treatment
| Symptomatic signs | Before taking | After taking |
| Bleeding conditions | 1.025±0.545 | 0.012±0.033 |
| Prolapse of lumbar intervertebral disc | 1.225±0.875 | 0.123±0.845 |
| Pain (due to cold or dampness) | 3.243±1.042 | 0.502±0.223 |
| Itching (pruritus) | 2.985±0.988 | 0.745±0.575 |
Note: p <0.05
As shown in Table 5, the quantitative integral value changes of symptom signs before and after the test taking are significantly different (P is less than 0.05), and the bleeding, the prolapse, the pain and the pruritus of the patient after taking the product are obviously improved.
TABLE 6 Experimental administration curative effect statistics table
| Number of test persons | Recovery method | Is effective | Invalidation | Total effective rate |
| 36 | 21(58.33％) | 13(36.11％) | 2(5.56％) | 94.44％ |
As shown in Table 6, according to the different disease conditions of the test-taker, the curative effect analysis is respectively carried out, the Lactobacillus delbrueckii subsp bulgaricus SF-L-18 plant beverage has better effect in the aspects of treating and relieving mild and moderate hemorrhoids, the cure rate is 58.33%, the effective rate is 36.11%, and the total effective rate is 94.44%.
5. Analysis of results
The number of trial patients completed in this trial was 36, and 34 patients had no effective improvement in symptoms except for 2 patients who took the trial. Of the 34 patients, 12 were patients with degree I, 15 were patients with degree II, and the remaining 7 were patients with degree III.
The symptoms before and after the patient takes the test are taken as a contrast, and the quantitative scoring is carried out according to the symptoms of bleeding, prolapse, pain and pruritus of the patient and the defecation condition. The total symptom score before treatment was 15.460 + -3.845, and after treatment was 6.127 + -1.875, which was a 60.37% reduction. The four main symptoms of bleeding, prolapse, pain and pruritus are obviously reduced, wherein the bleeding symptom integral is reduced from 1.025 to 0.012, the pain symptom integral is reduced from 3.243 to 0.502, and the quantitative integral value changes with significant difference (P is less than 0.05), so that the plant drink can improve the symptoms of the hemorrhoids and relieve the pain of patients suffering from the hemorrhoids. After two weeks of trial administration, the hemorrhoid symptoms of 21 of 36 subjects completely disappeared, part of 13 subjects were relieved, and 2 subjects were not improved, i.e., cured 58.33%, effective 36.11%, and the total effective rate was 94.44%. The plant beverage can play a significant role in relieving and assisting in treating mild and moderate hemorrhoids.
Although the present invention has been described in detail by way of preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
SEQUENCE LISTING
<110> Shandong sunflower bioengineering Co., Ltd
<120> Lactobacillus delbrueckii subspecies bulgaricus SF-L-18 and fermented beverage and application thereof
<130> 2022.01.24
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1495
<212> DNA
<213> Lactobacillus delbrueckii subsp
<400> 1
tcaggacgaa cgctggcggc gtgcctaata catgcaagtc gagcgagctg aattcaaaga 60
tcccttcggg gtgatttgtt ggacgctagc ggcggatggg tgagtaacac gtgggcaatc 120
tgccctaaag actgggatac cacttggaaa caggtgctaa taccggataa caacatgaat 180
cgcatgattc aagtttgaaa ggcggcgcaa gctgtcactt taggatgagc ccgcggcgca 240
ttagctagtt ggtggggtaa aggcctacca aggcaatgat gcgtagccga gttgagagac 300
tgatcggcca cattgggact gagacacggc ccaaactcct acgggaggca gcagtaggga 360
atcttccaca atggacgcaa gtctgatgga gcaacgccgc gtgagtgaag aaggtcttcg 420
gatcgtaaag ctctgttgtt ggtgaagaag gatagaggca gtaactggtc tttatttgac 480
ggtaatcaac cagaaagtca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg 540
gcaagcgttg tccggattta ttgggcgtaa agcgagcgca ggcggaatga taagtctgat 600
gtgaaagccc acggctcaac cgtggaactg catcggaaac tgtcattctt gagtgcagaa 660
gaggagagtg gaactccatg tgtagcggtg gaatgcgtag atatatggaa gaacaccagt 720
ggcgaaggcg gctctctggt ctgcaactga cgctgaggct cgaaagcatg ggtagcgaac 780
aggattagat accctggtag tccatgccgt aaacgatgag cgctaggtgt tggggacttt 840
ccggtcctca gtgccgcagc aaacgcatta agcgctccgc ctggggagta cgaccgcaag 900
gttgaaactc aaaggaattg acgggggccc gcacaagcgg tggagcatgt ggtttaattc 960
gaagcaacgc gaagaacctt accaggtctt gacatcctgc gctacaccta gagataggtg 1020
gttcccttcg gggacgcaga gacaggtggt gcatggctgt cgtcagctcg tgtcgtgaga 1080
tgttgggtta agtcccgcaa cgagcgcaac ccttgtcttt agttgccatc attaagttgg 1140
gcactctaaa gagactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaagtcatc 1200
atgcccctta tgacctgggc tacacacgtg ctacaatggg cagtacaacg agaagcgaac 1260
ccgcgagggt aagcggatct cttaaagctg ctctcagttc ggactgcagg ctgcaactcg 1320
cctgcacgaa gctggaatcg ctagtaatcg cggatcagca cgccgcggtg aatacgttcc 1380
cgggccttgt acacaccgcc cgtcacacca tggaagtctg caatgcccaa agtcggtggg 1440
ataaccttta taggagtcag ccgcctaagg cagggcagat gactggggtg aagtc 1495
<210> 2
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 2
agagtttgat cmtggctcag 20
<210> 3
<211> 19
<212> DNA
<213> Artificial Synthesis
<400> 3
ggttaccttg ttacgactt 19
Claims (8)
1. The Lactobacillus delbrueckii subsp. bulgaricus SF-L-18 is characterized in that the Lactobacillus delbrueckii subsp. bulgaricus is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC NO.20928, the preservation date of 2020, 10 and 21 days, and the preservation organization address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, Beicheng.
2. The method for culturing Lactobacillus delbrueckii subsp bulgaricus SF-L-18 according to claim 1, comprising inoculating Lactobacillus delbrueckii subsp bulgaricus SF-L-18 into sterilized MRS liquid medium in an amount of 2%, and culturing at 37 ℃ for 24 hours to obtain Lactobacillus delbrueckii subsp bulgaricus SF-L-18 culture solution.
3. The culture method according to claim 2, wherein the MRS liquid medium comprises the following components: 10.0g of beef extract, 5.0g of yeast extract, 10.0g of peptone, 20.0g of glucose and K2HPO42.0g, sodium acetate 5.0g, MgSO4·7H2O 0.2g，MnSO4·4H2O0.05 g, Tween 801.0 g, triammonium citrate 2.0g, and distilled water 1000 ml.
4. Use of lactobacillus delbrueckii subsp bulgaricus SF-L-18 according to claim 1, in the manufacture of a product for improving bleeding and pain in hemorrhoids.
5. Use according to claim 4, wherein the product is a fermented plant beverage.
6. A method for preparing a fermented plant drink using lactobacillus delbrueckii subsp bulgaricus SF-L-18 of claim 1, comprising the steps of:
(1) mixing flos Lonicerae, flos Sophorae Immaturus, radix Angelicae Dahuricae, semen euryales, pericarpium Citri Tangerinae, Coicis semen, Bulbus Lilii, rhizoma Dioscoreae, rhizoma Polygonati, Ginseng radix, mume fructus powder, and herba Portulacae, adding water, decocting for four times, and filtering with 200 mesh to obtain a mixed solution;
(2) adding elastic collagen peptide into the mixed solution, sterilizing for 30min under high pressure, and cooling to 30-40 ℃ to obtain a sterilized solution;
(3) inoculating lactobacillus delbrueckii subspecies bulgaricus SF-L-18 into a sterilized MRS liquid culture medium, wherein the inoculation amount is 2%, and culturing at 37 ℃ for 24h to obtain a lactobacillus delbrueckii subspecies bulgaricus SF-L-18 culture solution;
(4) inoculating a lactobacillus delbrueckii subspecies bulgaricus SF-L-18 culture solution into a sterilization solution, wherein the inoculation amount is 500-1000 ten thousand cfu/mL, and fermenting at 37 ℃ for 24-48 h to obtain a fermentation solution;
(5) and adding carrot concentrated juice, prebiotic syrup and juicy peach concentrated juice into the fermentation liquor, seasoning, performing wall breaking and pasteurization to obtain the probiotic plant beverage, wherein the wall breaking pressure is 20-60 MPa, and thus obtaining the plant fermented beverage.
7. The method of claim 6, wherein the plant fermented beverage comprises the following raw materials in parts by weight: 50-80 parts of purified water, 5-10 parts of carrot concentrated juice and 2-5 parts of prebiotics syrup; 2-5 parts of juicy peach concentrated juice, 1.8-2 parts of honeysuckle, 2-4 parts of sophora flower, 0.1-1 part of angelica dahurica, 0.5-1 part of gordon euryale seed, 2-5 parts of orange peel, 2-6 parts of coix seed, 3-6 parts of lily, 0.5-1 part of Chinese yam, 0.5-1 part of rhizoma polygonati, 0.1-2 parts of ginseng, 0.5-2 parts of dark plum powder, 0.1-2 parts of elastic collagen peptide and 0.2-2 parts of purslane.
8. The method of claim 6, wherein the plant fermented beverage comprises the following raw materials in parts by weight: 73 parts of purified water, 6 parts of carrot concentrated juice, 3 parts of prebiotics syrup, 3 parts of juicy peach concentrated juice, 2 parts of honeysuckle, 2 parts of sophora flower, 0.5 part of angelica dahurica, 0.5 part of gordon euryale seed, 2 parts of orange peel, 2 parts of coix seed, 3 parts of lily, 1 part of Chinese yam, 0.5 part of rhizoma polygonati, 0.5 part of ginseng, 0.5 part of dark plum powder, 0.15 part of elastic collagen peptide and 0.35 part of purslane.
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| CN116286544A (en) * | 2023-04-14 | 2023-06-23 | 山东向日葵生物工程有限公司 | Application of lactobacillus delbrueckii subspecies bulgaricus SF-L-18 in preparation of hair loss prevention and hair quality improvement fermented product |
| CN116420831A (en) * | 2023-06-01 | 2023-07-14 | 山东向日葵生物工程有限公司 | Application of lactobacillus rhamnosus SF-L30 in preparation of fermented beverage for improving body serotonin level |
| CN116420831B (en) * | 2023-06-01 | 2023-08-22 | 山东向日葵生物工程有限公司 | Application of lactobacillus rhamnosus SF-L30 in preparation of fermented beverage for improving body serotonin level |
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