CN112458004B - Bifidobacterium animalis subsp lactis BP026 and application thereof - Google Patents
Bifidobacterium animalis subsp lactis BP026 and application thereof Download PDFInfo
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- CN112458004B CN112458004B CN202011222978.4A CN202011222978A CN112458004B CN 112458004 B CN112458004 B CN 112458004B CN 202011222978 A CN202011222978 A CN 202011222978A CN 112458004 B CN112458004 B CN 112458004B
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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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/745—Bifidobacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/20—Hypnotics; Sedatives
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- A23V2400/51—Bifidobacterium
- A23V2400/515—Animalis
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Abstract
The invention discloses a Bifidobacterium animalis subsp.lactis BP026 and application thereof, wherein the Bifidobacterium animalis subsp.lactis BP026 is preserved in China general microbiological culture Collection center (CGMCC) with the preservation time of 8-6 days at 2020 year and the preservation number of CGMCC No. 20495. The strain has sleep improving or sleep promoting effects, and can be used for preparing sleep improving food, sleep improving health food and tranquilizing medicine. In animal experiments, the method can obviously shorten the sleep latency and prolong the sleep time, improve the sleep quality of mice, obviously shorten the sleep latency and prolong the sleep time, improve the sleep quality of the mice, has obvious effect and wide application prospect.
Description
Technical Field
The invention relates to the technical field of microbial products, in particular to Bifidobacterium animalis subsp.
Background
Sleep problems are important problems affecting human health, mainly manifested by difficulty in falling asleep, short sleep time, easy waking up, and early waking up. According to incomplete statistics, sleep problems affect approximately 15% of the chinese population, a proportion that is higher in the united states, exceeding 30%. The sleep deficiency can have negative influence on the work and life of people in the daytime, such as fatigue, somnolence, headache and the like, and long-term sleep problems can also cause depression, obesity, diabetes, heart diseases and other diseases, and even cause social problems such as suicide and the like. At present, the treatment of insomnia is mainly to take artificially synthesized medicaments, but the synthesized medicaments have serious adverse reactions such as medicament dependence, cognitive and motor damage, medicament withdrawal and rebound insomnia and the like. Therefore, there is a need to find a safe and effective sleep-promoting drug to break through the development bottleneck of sleep-promoting drugs and dietary supplements.
In recent years, more and more animal experiments show that the intestinal flora plays an important role in the sleep-wake mechanism, and the internal environment of the organism is stably balanced through a microbe-gut-brain axis (MGBA). Therefore, the intestinal flora is improved from the aspects of diet, probiotics and prebiotics, the sleep related gene is regulated, the symptoms of the primary insomnia patient can be improved, and the traditional Chinese medicine composition can be used for treating the sleep disorder patient.
The bifidobacteria is an important beneficial bacterium in intestinal tracts, the abundance of the bifidobacteria is influenced by factors such as age, health condition, mental stress and the like, the abundance of the bifidobacteria accounts for more than 90% of intestinal microorganisms in the intestinal tracts of healthy infants, the species and the abundance of the bifidobacteria in human bodies are gradually reduced along with the increase of the age, and the abundance of the bifidobacteria in the old people is very low. Research shows that the content of bifidobacteria in intestinal tracts of the elderly with long life at home and abroad is equivalent to that of young people, and the bifidobacteria has a potential anti-aging effect. In addition, the bifidobacterium can promote the absorption of nutrient substances such as protein, vitamin D, iron, calcium, magnesium and the like, generate a large amount of beneficial substances such as vitamin B group and the like, and prevent and treat lactose intolerance; regulating the immunity of organisms, improving the immunity of intestinal mucosa, and simultaneously preventing harmful microorganisms from infecting hosts by competitively inhibiting the harmful microorganisms. Part of the bifidobacteria also have the effects of reducing blood sugar, blood fat and cholesterol, resisting tumors and the like. However, different strains of bifidobacterium, even different strains of the same species, have slight differences in biological properties, catabolic pathways and functions, and thus it is important to screen for bifidobacterium having special excellent properties and biological functions.
Although intestinal flora can absorb and utilize nutrients ingested by the body, generate metabolic small molecules such as 5-HT and GABA, and feed back to central nervous system and biological clock genes through vagus nerve to further realize intestinal brain communication, no report related to sleep improvement of animal-derived bifidobacterium species is found so far.
Disclosure of Invention
The invention provides Bifidobacterium animalis subsp.lactis BP026 and application thereof, which has the functions of improving sleep or promoting sleep and can be used for preparing sleep-improving food, sleep-improving health-care food and sedative and tranquilization medicines.
The Bifidobacterium animalis subsp. lactis BP026 provided by the invention is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, the preservation time is 2020, 8 and 6 days, the preservation number is CGMCC No.20495, the address is No. 3 of Xilu No. 1 of the morning of the Yangzhou city, and the address is 100101.
The morphological characteristics of Bifidobacterium animalis subsp lactis BP026(Bifidobacterium animalis subsp. lactis BP026) provided by the invention are as follows: gram-positive, non-sporulating, non-motile, obligate anaerobic bacilli.
After the Bifidobacterium animalis subsp. lactis BP026 provided by the invention is treated for 4 hours at pH3.0, the survival rate reaches more than 50%.
The survival rates of the Bifidobacterium animalis subsp.lactis BP026(Bifidobacterium animalis subsp.lactis BP026) which acts for 1h in bovine bile with the concentration of 0.3%, 0.6% and 1.0% are respectively 25%, 85% and 100%.
The Bifidobacterium animalis subsp. lactis BP026 provided by the invention can obviously shorten the sleep latency and prolong the sleep time in a pentobarbital sodium hypnotic mouse, and improve the sleep quality of the mouse.
The inactivated bacterial product of Bifidobacterium animalis subsp.lactis BP026 provided by the invention can obviously shorten the sleep latency and prolong the sleep time in pentobarbital sodium hypnotic mice and improve the sleep quality of the mice.
In the invention, the microbial product containing the bifidobacterium animalis subsp lactis BP026 is in all dosage forms containing live bacteria and dead bacteria of the bifidobacterium animalis subsp lactis BP 026.
The application of the bifidobacterium animalis subsp lactis BP026 in preparing food can be used for preparing all common foods, health foods and medicines such as dairy products, tablet candies, beverages, desserts, candies, sauce and the like.
The application of the bifidobacterium animalis subsp lactis BP026 in preparing health food and medicine for improving sleep and promoting sleep, and in preparing health food and medicine for improving sleep or promoting sleep.
The application of the bifidobacterium animalis subsp lactis BP026 in preparing medicines for treating sleep disorder and in preparing medicines for treating sleep disorder is provided.
The bifidobacterium animalis subsp lactis BP026 can be used for preparing food for improving sleep, health-care food for improving sleep and medicines for tranquilizing and allaying excitement, and the application specifically comprises the following steps:
fermenting Bifidobacterium animalis subsp lactis BP026, vacuum freeze drying to obtain lyophilized powder, and adding the lyophilized powder into food, health food and medicine to obtain sleep improving food, sleep improving health food and tranquilizing medicine.
Compared with the prior art, the invention has the following advantages:
the invention discloses a novel Bifidobacterium animalis subsp.lactis BP026, which has the function of improving sleep or promoting sleep and can be used for preparing sleep-improving food, sleep-improving health-care food and sedative and tranquilization medicines. In animal experiments, the sleep latency period can be obviously shortened, the sleep duration can be prolonged, the sleep quality of mice can be improved, the effect is obvious, the marketization popularization and utilization are facilitated, and the application prospect is wide.
Drawings
FIG. 1 is a graph comparing the influence of BP026 on sleep latency (A) and sleep duration (B) of a sodium pentobarbital sleeping mouse, wherein A in FIG. 1 is a graph comparing the influence of BP026 on sleep latency (A) and B in FIG. 1 is a graph comparing the influence of BP026 on sleep duration (B) of a sodium pentobarbital sleeping mouse;
fig. 2 is a graph comparing the influence of BP026 on sleep latency (a) and sleep duration (B) of a pentobarbital sodium sleeping mouse after heat inactivation, wherein a in fig. 2 is a graph comparing the influence of BP026 on sleep latency of a pentobarbital sodium sleeping mouse after heat inactivation, and B in fig. 2 is a graph comparing the influence of BP026 on sleep duration of a pentobarbital sodium sleeping mouse after heat inactivation.
Detailed Description
The following examples are illustrative only, and the present invention is not limited by these examples.
In the following examples, unless otherwise specified, the experimental methods used were all conventional.
In the following examples, reagents, materials, instruments and the like used are commercially available.
The bifidobacterium animalis subsp lactis BP026 can be inoculated into a fixed culture medium suitable for the growth of bifidobacterium for anaerobic culture to obtain seeds, and then the seeds are inoculated into a liquid culture medium suitable for the growth of the bifidobacterium.
The liquid culture medium suitable for the growth of bifidobacterium animalis subsp lactis BP026 can be a culture medium suitable for the culture of bifidobacterium in the prior art, which is known by the technical personnel in the field, and specifically can be: 1-10g/L hydrolyzed casein, 1-5g/L phytone, 1-2g/L yeast powder, 1-5g/L glucose, 0.2-0.5 g/L-cysteine, 0.5-2.0g/L dipotassium hydrogen phosphate, 0.2-0.5g/L magnesium chloride, 0.10-0.25g/L zinc sulfate, 0.05-0.15g/L calcium chloride, 0.0001-0.0005g/L ferric chloride, 801.0-2.0g/L Tween, and pH 6.5-7.4.
The fixed culture medium suitable for growth of Bifidobacterium animalis subsp lactis BP026 can be supplemented with agar 10-30g/L based on liquid culture medium.
The culture condition suitable for bifidobacterium animalis subsp lactis BP026 can be any condition suitable for bifidobacterium culture in the prior art, and can be at the temperature of 35-37 ℃ for 12-72 h.
Example 1 isolation and purification of Bifidobacterium animalis subsp
Taking feces of healthy people as a sample, inoculating the feces into a solid culture medium suitable for the growth of bifidobacteria, and carrying out anaerobic culture at 37 ℃ for 48 hours; selecting white colonies with smooth surfaces, inoculating the white colonies into a liquid culture medium suitable for the growth of bifidobacteria, and carrying out anaerobic culture at 37 ℃ for 48 hours; extracting genome, amplifying 16S rDNA by PCR, sequencing and comparing, finding and storing animal bifidobacterium subspecies lactis BP 026.
Example 2 acid resistance Properties of Bifidobacterium animalis subsp lactis BP026
Suspending Bifidobacterium animalis subsp lactis BP026 in PBS with pH of 1.0, 2.0, 3.0, 4.0, and 7.0, culturing at 37 deg.C for 1 hr, 2 hr, 3 hr, and 4 hr, and measuring viable bacteria rate by plate counting method. The viable bacteria rate of bifidobacterium animalis subsp lactis BP026 under different pH conditions and at different time points is shown in Table 1.
TABLE 1 viable bacteria rate of Bifidobacterium animalis subsp lactis BP026 under acidic condition
| Percent viable cell | 1h | 2h | 3h | 4h |
| pH＝1 | 0 | 0 | 0 | 0 |
| pH＝2 | 12.00±1.61 | 0.01±0.00 | 0.01±0.00 | 0.01±0.00 |
| pH＝3 | 94.42±4.03 | 86.05±24.42 | 88.37±21.31 | 61.86±4.49 |
| pH＝4 | 90.70±6.08 | 92.56±21.36 | 86.05±3.51 | 76.74±13.31 |
| pH＝7 | 98.60±8.53 | 99.53±7.68 | 85.27±6.35 | 79.53±9.77 |
Note: 3 replicates were set for this experiment and the data were mean + -SD.
Example 3 bile resistance of Bifidobacterium animalis subsp lactis BP026
Suspending Bifidobacterium animalis subsp lactis BP026 in PBS containing 0%, 0.3%, 0.6% and 1% bilis Bovina, culturing at 37 deg.C for 1 hr, 2 hr, 3 hr, and 4 hr, and measuring viable bacteria rate by plate counting method. The viable bacteria rate of bifidobacterium animalis subsp lactis BP026 at different bile concentrations and different time points is shown in Table 2.
TABLE 2 viable bacteria rate after treatment of Bifidobacterium animalis subsp lactis BP026 bile
| Percent viable cell | 1h | 2h | 3h | 4h |
| 0% bile | 94.09±19.73 | 87.50±30.31 | 83.99±10.02 | 75.07±11.42 |
| 0.3% of bile | 24.75±1.77 | 2.13±0.26 | 1.27±0.42 | 1.77±0.26 |
| 0.6% of bile | 85.91±6.13 | 8.49±1.56 | 6.87±3.01 | 2.09±0.36 |
| 1% bile | 99.45±24.23 | 36.52±21.44 | 4.60±0.35 | 1.01±0.22 |
Note: 3 replicates were set for this experiment and the data were mean + -SD.
Example 4 Bifidobacterium animalis subsp lactis BP026 Properties to promote sleep in mice
First, a 24h cultured bifidobacterium animalis subsp lactis BP026 was taken, centrifuged, washed 3 times with PBS, and diluted to 109CFU/mL or so;
extracting 20-24g of ICR mice according to the proportion of 1-3 multiplied by 10 per one mouse per day9Continuously intragastrically administering the amount of Bifidobacterium lactis of CFU for 14 days; placebo mice were given the same volume of PBS for 14 consecutive days; positive control mice were given the same volume of PBS for 13 consecutive days and 2.5mg/kg diazepam on the fourteenth day.
Thirdly, injecting 48mg/kg of pentobarbital sodium into the abdominal cavity, and calculating the sleep latency and the sleep duration of the mouse. The sleep latency period is the time when the mice turn over the normal reflex to disappear by injecting the sodium pentobarbital, and the sleep time is the time when the mice turn over the normal reflex to disappear to recover to sleep.
As a result, it was found that: bifidobacterium animalis subsp lactis BP026 can significantly shorten the sleep latency of mice and prolong the sleep duration of mice (fig. 1). FIG. 1 is a graph showing the effect of BP026 on sleep latency (A) and sleep duration (B) of sodium pentobarbital; 9-11 mice per group. *: p < 0.05; **: p < 0.01; ***: p < 0.001.
Example 5 sleep-promoting Properties of killed Bifidobacterium animalis subsp lactis BP026
First, a 24h cultured bifidobacterium animalis subsp lactis BP026 was taken, centrifuged, washed 3 times with PBS, and diluted to 109CFU/mL, then placing in boiling water to boil for 15 minutes;
extracting 20-24g of ICR mice according to the proportion of 1-3 multiplied by 10 per one mouse per day9Continuously intragastrically administering the amount of Bifidobacterium lactis of CFU for 14 days; placebo mice were given the same volume of PBS for 14 consecutive days; positive control mice were given the same volume of PBS for 13 consecutive days and 2.5mg/kg diazepam on the fourteenth day.
Thirdly, injecting 48mg/kg of pentobarbital sodium into the abdominal cavity, and calculating the sleep latency and the sleep duration of the mouse. The sleep latency period is the time when the mice turn over the normal reflex to disappear by injecting the sodium pentobarbital, and the sleep time is the time when the mice turn over the normal reflex to disappear to recover to sleep.
As a result, it was found that: after the bifidobacterium animalis subsp lactis BP026 is thermally extinguished, the sleep latency of a mouse can be still obviously shortened, and the sleep time of the mouse is prolonged (figure 2), wherein the influence of the BP026 on the sleep latency (A) and the sleep time (B) of the pentobarbital sodium sleep-induced mouse after thermal inactivation is realized; 9-11 mice per group. *: p < 0.05; ***: p < 0.001.
Example 6 preparation of bacterial liquid
The bifidobacterium animalis subsp lactis BP026 can be inoculated into a fixed culture medium suitable for bifidobacterium growth for anaerobic culture to obtain seeds, and then inoculated into a fermentation liquid suitable for bifidobacterium growth for fermentation, and the fermented supernatant is a bacterial liquid.
EXAMPLE 7 preparation of lyophilized bacterial powder
The bifidobacterium animalis subsp lactis BP026 can be inoculated into a fixed culture medium suitable for bifidobacterium growth for anaerobic culture to obtain seeds, then the seeds are inoculated into a fermentation liquid suitable for bifidobacterium growth for fermentation, and then the seeds are dried by a low-temperature vacuum freeze dryer to obtain freeze-dried bacterium powder.
EXAMPLE 8 preparation of capsules
The freeze-dried powder prepared from the bifidobacterium animalis subsp lactis BP026 is put into capsules to obtain the capsules.
EXAMPLE 9 preparation of tablets
The freeze-dried powder prepared from the bifidobacterium animalis subsp lactis BP026 is tableted to obtain tablets.
EXAMPLE 10 preparation of granules
The freeze-dried powder prepared from bifidobacterium animalis subsp lactis BP026 is uniformly mixed with a proper amount of sugar powder, and then the mixture is granulated, dried, granulated and subpackaged with a proper amount of ethanol to obtain the bifidobacterium animalis.
Claims (7)
1. A Bifidobacterium animalis subsp lactis BP026, which is preserved in China general microbiological culture Collection center (CGMCC) with a preservation number of CGMCC No. 20495.
2. Use of bifidobacterium animalis subsp lactis BP026 according to claim 1 in the preparation of a food for improving sleep.
3. The application according to claim 2, characterized in that it specifically comprises:
fermenting Bifidobacterium animalis subsp lactis BP026, vacuum freeze drying to obtain lyophilized powder, and adding the lyophilized powder into food to obtain sleep improving food.
4. Use of bifidobacterium animalis subsp lactis BP026 according to claim 1 in the preparation of a health food for improving sleep.
5. The application according to claim 4, specifically comprising:
fermenting Bifidobacterium animalis subsp lactis BP026, vacuum freeze drying to obtain lyophilized powder, and adding the lyophilized powder into health food to obtain sleep improving health food.
6. Use of bifidobacterium animalis subsp lactis BP026 according to claim 1 in the preparation of a medicament for improving sleep and tranquilizing the mind.
7. The application according to claim 6, specifically comprising:
fermenting Bifidobacterium animalis subsp lactis BP026, vacuum freeze drying to obtain lyophilized powder, and adding the lyophilized powder into the medicine to obtain the medicine with sleep improving and tranquilizing effects.
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