CN111110705A - Application of lactobacillus plantarum LP45 in preparation of products for improving memory, relieving anxiety and relieving depression - Google Patents
Application of lactobacillus plantarum LP45 in preparation of products for improving memory, relieving anxiety and relieving depression Download PDFInfo
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- CN111110705A CN111110705A CN201911376513.1A CN201911376513A CN111110705A CN 111110705 A CN111110705 A CN 111110705A CN 201911376513 A CN201911376513 A CN 201911376513A CN 111110705 A CN111110705 A CN 111110705A
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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
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
- A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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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
- A23L2/52—Adding ingredients
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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
- 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
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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
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Abstract
The invention relates to an application of lactobacillus plantarum LP45 in products with functions of memory, anxiety alleviation and depression alleviation, wherein the lactobacillus plantarum LP45 is preserved in China general microbiological culture Collection center (CGMCC) in 2013, 8 and 26 months, and the preservation number is CGMCC NO: 8072 (c); the preservation address is No. 3 Xilu No. 1 of Beijing, Chaoyang, the district of rising Yang. The strain can relieve abnormal expression of BDNF and GABA, improve memory of brain, and relieve anxiety and depression-like behavior, and can be used for preparing products for improving memory, relieving anxiety and relieving depression.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to application of lactobacillus plantarum LP45 in preparation of products for improving memory, relieving anxiety and relieving depression.
Background
With the acceleration of modern life rhythm, modern people often bear higher working intensity and social pressure, so that anxiety, depression and other conditions of different degrees can be induced. The stimulation of bad living habits and external factors may further lead to the aggravation of anxiety and depression. Anxiety can stress the human mood, which in turn leads to a more or less reduced memory. Depression can cause feelings of depression and inattention, and also can lead to memory impairment. The memory deterioration brings about the decline of life quality for people of any age group, and particularly for the elderly, the memory deterioration is one of the important symptoms of the senile dementia.
Lactobacillus plantarum LP45 (LP 45) is a novel patent strain obtained by separating and screening from traditional inner Mongolia yogurt, and has the functions of enhancing intestinal barrier, inhibiting gastrointestinal pathogenic bacteria, improving immunity and regulating fat metabolism. However, no document in the prior art reports the effects of Lactobacillus plantarum LP45 in improving memory, relieving anxiety and relieving depression.
Disclosure of Invention
Aiming at the technical problems that anxiety and depression can damage memory and reduce life quality, the invention provides application of lactobacillus plantarum LP45 in preparing products for improving memory, relieving anxiety and relieving depression.
In order to achieve the purpose of the invention, the embodiment of the invention adopts the following technical scheme:
the application of lactobacillus plantarum LP45 in preparing products for improving memory, relieving anxiety and relieving depression is characterized in that the preservation number of lactobacillus plantarum LP45 is CGMCC NO: 8072.
the research shows that the strain can improve anxiety and depressive behavior, relieve abnormal expression of BDNF and GABA, and improve memory. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 26.8.2013, and the preservation address is No. 3 Xilu No. 1 Beijing of the Kyowa sunward.
Preferably, the product is for improving memory.
Preferably, the product is for use in the relief of anxiety.
Preferably, the product is for use in alleviating depression.
Preferably, the product is a food product. The strain is prepared into food, can be conveniently eaten by a user in daily diet, is beneficial to keeping the health state of brain function and keeping good memory and emotion. It is especially suitable for people who want to improve or maintain memory, and avoid or relieve anxiety and depression.
Preferably, the food product comprises lactobacillus plantarum LP45, a main ingredient, and other additives.
Preferably, the preparation method of the food comprises the following steps: the lactobacillus plantarum LP45 and the excipient and/or additive are prepared into food, and the lactobacillus plantarum LP45 is not heated above 40 ℃ in the production process of the food, so that the activity of the lactobacillus plantarum LP45 is prevented from being reduced. Such food products include, but are not limited to, beverages, yogurt, and the like.
Preferably, the product is an oral pharmaceutical preparation or a health food. The strain is prepared into an oral medicinal preparation or a health-care food, can be conveniently taken by patients with dysmnesia, anxiety and depression caused by brain dysfunction or brain function decline, or is convenient for users to eat for the purpose of health care, is used for keeping good memory and mood, and slowing or avoiding the situations of memory decline, anxiety, depression and the like.
Preferably, the oral pharmaceutical preparation or health food comprises lactobacillus plantarum LP45 and acceptable excipients and additives in pharmacy or health food.
Preferably, the preparation method of the health food comprises the following steps: the lactobacillus plantarum LP45, the excipient and the additive are prepared into a conventional oral preparation or health-care food, and the lactobacillus plantarum LP45 is not heated to more than 40 ℃ in the preparation process, so that the activity of the lactobacillus plantarum LP45 is prevented from being reduced to influence the use effect. The conventional oral dosage forms include but are not limited to oral liquid, granules, tablets, capsules, powder and the like, and the health food includes but is not limited to oral liquid, granules, tablets, capsules and the like.
Drawings
FIG. 1 shows the results of the darkening test in example 1 of the present invention;
FIG. 2 shows the results of the Morris water maze test in example 1 of the present invention;
FIG. 3 shows the results of the tail overhang test in example 1 of the present invention;
FIG. 4 shows the expression of mRNA in example 1 of the present invention;
FIG. 5 is a graph showing the body weight gain factor in example 1 of the present invention;
FIG. 6 shows organ coefficients in example 1 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
The embodiment of the invention provides application of lactobacillus plantarum LP45 in preparing products for improving memory, relieving anxiety and relieving depression.
1. Subjects and groups:
the new SPF male Kunming mice are taken as experimental objects and are randomly divided into three groups, namely a control group, an antibiotic group and an LP45 group, wherein each group comprises 15 mice.
The antibiotic gavage is carried out for 1 time per day for mice in antibiotic group and LP45 group according to the dosage of ampicillin sodium (100mg/kg) + neomycin sulfate (100mg/kg) + bacitracin (100mg/kg) + vancomycin hydrochloride (50mg/kg) + imipenem/cilastatin sodium (50mg/kg) + water-soluble amphotericin B (1mg/kg), and each LP45 group of mice is filled with antibiotic for 1 time per day according to the dosage of more than or equal to 109And (4) performing primary intragastric perfusion of the lactobacillus plantarum LP45 again by using the CFU/d dosage, wherein the interval between intragastric perfusion of LP45 and intragastric perfusion of antibiotics is more than 2 h. The control group was gavaged with distilled water 1 time per day. The intragastric volumes for each group are shown in table 1.
TABLE 1 intragastric volume conditions for each group
After 21d, the groups were randomly divided into 5 cages each.
2. Open field experiment
The open field reaction box of the mouse has the specification of 625mm (length) multiplied by 740mm (width) multiplied by 510mm (height), the inner wall is painted black, and the bottom surface (software) is averagely divided into 25 small squares. A digital camera is arranged 2m above the field of view, and the field of view can cover the inside of the whole open field. The experimental procedure was as follows:
1) the experiments are all carried out in a quiet environment in the morning;
2) grabbing a third of the root of the mouse tail, lifting, gently placing into a middle lattice of an open field experimental box, and simultaneously carrying out camera shooting and timing;
3) stopping shooting after 5min of observation;
4) the inner wall and the bottom surface of the box are cleaned, so that the information (such as the excrement, urine and smell of the animal) remained by the animal at the last time is prevented from influencing the next test result. Animals were replaced and the experiment continued.
5) Observation indexes are as follows: the residence time of the animal in the central compartment in unit time, and the number of compartments crossed by a certain limb is the horizontal score; total movement distance, total movement time, etc. The results are shown in Table 2.
TABLE 2 open field Experimental conditions for groups of mice
Denotes P <0.001 compared to the control group, denotes P <0.01 compared to the control group, denotes P <0.05 compared to the control group, and denotes P <0.05 compared to the antibiotic group.
As can be seen from the results in table 2, the center activity time (P <0.001), total locomotor distance (P <0.01), total locomotor time (P <0.05) and level score (P <0.05) were all decreased and fecal grain number was increased (P <0.01) in the antibiotic group compared to the control group. Compared with the antibiotic group, the LP45 group showed an increased trend in the score of central activity time and level and a decreased number of fecal pellets (P < 0.05). The results indicate that lactobacillus plantarum LP45 was able to improve anxiety in mice.
3. Dark avoidance experiment
The external dimension of the movable box is 640mm (length) multiplied by 340mm (width) multiplied by 290mm (height), and the dimension of the single-space movable space is 155mm (length) multiplied by 140mm (width) multiplied by 210mm (height). The movable box is divided into a bright room and a dark room, and a gate is arranged in the middle of the movable box for connection. When the animal enters the dark room and is shocked, the time (latency period) that the mouse enters the dark room from the bright room for the first time is recorded, and the animal entering the dark room is electrically stimulated to return to the bright room, and if the animal enters the dark room again, the animal is marked as an error. The specific experimental steps are as follows:
1) the method is suitable for the following steps: mice were placed in a dark room for 15min and then returned to the mouse cage.
2) Training: the mouse was placed in the light room with the arch facing away, at which time the gate was closed. After all mice entered the light room, the gate was opened and the dark room started the electrical stimulator (24V). The mice were observed for 300s for latency and number of dark chambers. If the animal does not enter the dark room for more than 180s at the first training, it is either eliminated or gently driven into the dark room by hand.
3) And (3) testing: after 24h of training, the animals were again placed in the light room for testing. The mice were observed for latency and number of errors into the dark room in 300 s. The results are shown in FIG. 1: the difference between the two-day latency of the antibiotic group (P <0.001) and the LP45 group (P <0.01) is smaller than that of the control group, while the difference between the two-day latency of the LP45 group is not statistically different (P >0.05) but has an increasing trend compared with the antibiotic group; the difference between the number of errors in the antibiotic group in two days tended to decrease compared to the control group, while the difference between the number of errors in the LP45 group in two days tended to increase compared to the antibiotic group. The results indicate that Lactobacillus plantarum LP45 was able to improve memory in mice.
4. Morris water maze
Morris water maze reaction barrel specification: diameter 800mm, height 400mm, circular platform specification: diameter 65mm, height 150 mm. The interior of the pool is free of any markings. Proper amount of titanium dioxide is added into water to make the water pool become opaque milky white. E, S, W and N4 marking points are equidistantly arranged on the upper edge of the barrel, and the projection points of the 4 points on the water surface and the bottom of the barrel divide the water surface and the barrel into 4 quadrants. The platform was placed in the center of SW as required for the experiment. The position of the reaction barrel is fixed during the experiment, and the water temperature is kept at 21-22 ℃. The experimental procedure was as follows:
1) positioning navigation experiment: the training was continued for 5 days, 4 times per day. During training, the mice are respectively placed into the pool from four water entry points of N, E, SE and NW (see table 3 in detail) facing the pool wall, and the time from water entry to finding an underwater hidden platform (located at 1cm underwater) and standing on the platform is recorded as a latency period. After the mouse found the platform, it was allowed to stand on the platform for 15 s. If the mice failed to find the platform 60s after entering the water, the latency was recorded as 60s, and the mice were placed on the platform for rest for 15 s. After training, the rats were wiped dry with a towel and placed in a cage. Training is carried out on 4 water entry points 1 time each day, and all mice are trained on the next water entry point after training on 1 water entry point. The statistical analysis was performed with the arithmetic mean of the four latencies as the performance of the day.
2) Spatial search experiments: 6d for measuring the ability of the mouse to learn to find the platform and then to retain the memory of the spatial position of the platform. After the navigation experiment is positioned, the platform is removed, the mouse is put into water from the NE, and the time for the mouse to reach the original platform position for the first time is measured.
3) Visual platform experiment: in order to eliminate the influence of sensory, visual or motor dysfunction on spatial learning and memory, a visual platform experiment is finally carried out in 1 d. And (3) exposing the platform position to 1cm above the water surface, sticking a yellow adhesive tape, and performing navigation experiments in the same positioning mode with other operations.
TABLE 3 Water maze training procedure
The results are shown in FIG. 2: the 1 st to 3 rd latent period of the positioning training of the antibiotic group is consistent with that of the control group, and the 4 th d (P <0.05) and the 5 th d (P <0.01) of the antibiotic group are higher than that of the control group; latency was not different between 1d and 2d, with a downward trend from 3-5d, but not statistically different (P >0.05) in LP45 group compared to antibiotic group; the latency was higher in both antibiotic (P <0.001) and LP45 (P <0.001) groups than in the control group at the time of the 6d search test; there was no statistical difference in latency between the antibiotic group and the LP45 group (P >0.05), but there was a trend towards a decrease in the LP45 group compared to the antibiotic group. The results indicate that Lactobacillus plantarum LP45 was able to improve memory in mice.
5. Tail suspension experiment
The mouse tail tip portion 1/3 is fixed by adhesive tape along the direction of the fixed rod, and then the fixed magnet head is adsorbed on the top of the mouse movable box channel, so that the mouse is inversely hung on a cross rod 15cm away from the ground. After the mice are hung upside down for 2min, the duration time of the immobile state of the mice within 4min is recorded, namely, the mice abandon actively struggling and the bodies are in the state of suspension and no twisting. The results are shown in FIG. 3: the immobility time was longer in the antibiotic group compared to the control group (P < 0.01); compared with the antibiotic group, the immobility time of the LP45 group was reduced (P < 0.05). The results indicate that lactobacillus plantarum LP45 was able to alleviate depression in mice.
6. Brain mRNA expression
Using GENEOUTTMExtracting mouse hippocampus and prefrontal cortex respectively by TRIzol total RNA extraction kitTotal RNA, using iScriptTMgDNA Clear cDNA Synthesis Kit reverse transcribing RNA into cDNA Using β -actin as reference Gene, brain-derived neurotrophic factor (BDNF), Gamma-aminobutyric acid (GABA) A receptor α 1 subtype (GABA)Aα1) And GABA B receptor subtype 1 (GABA)B1) β -actin primer was purchased from Biotechnology engineering (Shanghai) Ltd, product primer (cat number B661302), BDNF primer was designed by Biotechnology engineering (Shanghai) Ltd, GABAAα1And GABAB1The primers of (a) were obtained from an on-line primer library of Harvard medical college. The primer sequences of the genes are shown in Table 4. The primers were synthesized by Biotechnology engineering (Shanghai) Inc. The qPCR reaction system is shown in table 5. qPCR amplification procedure: 30s at 95 ℃; 5s, T at 95 DEG Cm5s at 40 cycles; read the dissolution curve (T) from 65 ℃ to 95 ℃mSee table 4). Use 2-△△CtCalculating the relative expression level of the target gene by relative quantification method (△ Ct ═ Ct)Purpose(s) to-CtInternal reference，△△Ct＝△CtTreatment of-△CtControl)。
TABLE 4 RT-qPCR primer sequences
TABLE 5 qPCR reaction System
The results are shown in FIG. 4: in the prefrontal cortex, BDNF mRNA expression was elevated in the antibiotic group compared to the control group (P)<0.01); the expression of BDNF mRNA in the LP45 group was reduced compared with that in the antibiotic group (P)<0.01), antibiotics group GABAAα1(P<0.05) and GABAB1(P<0.05) mRNA expression was elevated compared with the control group, and LP45 group GABAAα1(P<0.001) and GABAB1(P<0.05) mRNA TableThe effect is reduced compared with that of the antibiotic group; there was no statistical difference in BDNF mRNA expression among hippocampus, groups, antibiotic group GABAAα1(P<0.01) and GABAB1(P<0.01) mRNA expression is increased compared with that of a control group, and LP45 group is compared with that of an antibiotic groupAα1mRNA expression tends to decrease, GABAB1Reduced mRNA expression (P)<0.05). The lactobacillus plantarum LP45 is shown to relieve the abnormal expression of BDNF and GABA.
7. Weight gain factor and organ coefficient
After the experiment is finished, the weight gain multiple and the organ coefficient of each group of mice are inspected, and the weight gain multiple and the organ coefficient are calculated according to the following formulas:
before weaning: weight gain factor (n day body weight (g)/birth weight (g) (i.e., 1d body weight);
after weaning: the weight gain factor (n day body weight (g)/weaning body weight (g) (i.e., 21d body weight);
organ coefficient: liver body ratio (mg)/pre-mortem body weight (g) × 100%;
spleen-to-body ratio spleen weight (mg)/pre-necrotized body weight (g) × 100%;
lung mass ratio (lung weight (mg)/pre-mortem weight (g) × 100%;
thymic gland ratio (mg)/weight of premortal body (g) × 100%;
adrenal body ratio (weight of adrenal gland (mg)/weight of pre-sacrifice (g) × 100%.
The weight gain is shown in fig. 5, and the weight gain of the control group and the antibiotic group is not statistically different at 1-15 weeks after delivery (P > 0.05). The LP45 group all gained less weight than the antibiotic group at 1-3 weeks post partum, while the LP45 group gained more weight than the antibiotic group from week 4 through week 15.
As shown in fig. 6, the organ coefficients were lower in the antibiotic group than in the control group in adrenal body (P <0.01), liver body ratio (P <0.01), spleen body ratio (P <0.01), and lung body ratio (P < 0.01). Compared with the antibiotic group, the lung volume ratio of the LP45 group is increased (P <0.05), the thymus volume ratio is reduced (P <0.05), and the adrenal volume ratio tends to be increased.
The results show that the lactobacillus plantarum LP45 can relieve the adverse effects of antibiotics on body weight and organs, and indirectly reflect that the lactobacillus plantarum LP45 is safe to take orally.
Example 2
The embodiment provides application of lactobacillus plantarum in preparing oral liquid for improving memory, relieving anxiety and relieving depression. Preparing purified water, flavoring agent and thickening agent into oral liquid without active ingredient according to conventional production method of oral liquid, adding Lactobacillus plantarum LP45, and mixing.
Example 3
The embodiment provides application of lactobacillus plantarum in preparing granules for improving memory, relieving anxiety and relieving depression. Preparing microcrystalline cellulose, dextrin, sodium carboxymethylcellulose and aspartame into granules by using a conventional granule production method, adding lactobacillus plantarum LP45, and uniformly mixing to obtain the product.
Example 4
The present example provides the use of Lactobacillus plantarum for the preparation of a tablet for improving memory, relieving anxiety and relieving depression. Mixing Lactobacillus plantarum LP45 with microcrystalline cellulose, crosslinked polyvinylpyrrolidone, magnesium stearate, and silicon dioxide, and directly tabletting.
Example 5
The present example provides the use of Lactobacillus plantarum for the preparation of a powder for improving memory, relieving anxiety and relieving depression. Mixing Lactobacillus plantarum LP45 with common excipient of powder.
Example 6
The embodiment provides application of lactobacillus plantarum in preparing capsules for improving memory, relieving anxiety and relieving depression. Mixing Lactobacillus plantarum LP45 with common capsule excipient, and filling into hard capsule shell No. 4.
Example 7
The present example provides the use of Lactobacillus plantarum for the preparation of a beverage for improving memory, relieving anxiety and relieving depression. Making water, correctant, pigment, thickener, etc. into beverage by conventional method, adding Lactobacillus plantarum LP45, and mixing.
Example 8
The embodiment provides application of lactobacillus plantarum in preparing yoghourt for improving memory, relieving anxiety and relieving depression. Preparing yogurt by conventional method, adding Lactobacillus plantarum LP45, and mixing.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents or improvements made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. The application of lactobacillus plantarum LP45 in preparing products for improving memory, relieving anxiety and relieving depression is characterized in that the preservation number of lactobacillus plantarum LP45 is CGMCC NO: 8072.
2. use according to claim 1, wherein the product is used for improving memory.
3. The use according to claim 1, wherein the product is for the relief of anxiety.
4. Use according to claim 1, wherein the product is for relief of depression.
5. Use according to any one of claims 1 to 4, wherein the product is a food product.
6. Use according to claim 5, wherein the food product comprises Lactobacillus plantarum LP45, a main ingredient, and other additives.
7. The use according to claim 6, wherein the food product is prepared by a method comprising: the lactobacillus plantarum LP45 and the excipient and/or additive are prepared into food, and the lactobacillus plantarum LP45 is not heated above 40 ℃ in the production process of the food.
8. The use according to any one of claims 1 to 4, wherein the product is an oral pharmaceutical preparation or a health food.
9. The use according to claim 8, wherein the oral pharmaceutical preparation or health product comprises Lactobacillus plantarum LP45, together with pharmaceutically or health food acceptable excipients and additives.
10. The use according to claim 9, wherein the oral formulation is prepared by a process comprising: the lactobacillus plantarum LP45, the excipient and the additive are prepared into a conventional oral preparation or health-care food, and the lactobacillus plantarum LP45 is not heated to more than 40 ℃ in the preparation process.
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