Lactobacillus mucilaginosus capable of improving levels of dopamine, 5-hydroxytryptamine and glutamic acid and application of lactobacillus mucilaginosus

Abstract

The invention provides lactobacillus mucilaginosus and application thereof. The lactobacillus mucus provided by the invention has a preservation number of CGMCC (China General Microbiological Culture Collection Center) NO.35104 in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms. The invention provides a fermented lactobacillus mucus Eupro 06 as well as a fermented product, a composition and application of the fermented lactobacillus mucus Eupro 06. The fermented lactobacillus mucus Eupro 06 is used for promoting biosynthesis of neurotransmitters such as intestinal dopamine, 5-hydroxytryptamine and L-glutamic acid. The strain / fermentation product can significantly improve the emotional state and improve the pleasure, has multiple beneficial effects in the aspects of maintaining intestinal health, enhancing the immune function, assisting in improving sleep and the like, and solves the problem that existing emotion regulation probiotics are insufficient in effectiveness. The method can be used for developing micro-ecological preparations, functional foods or nutritional supplements which are mainly used for emotion adjustment and have multiple effects (one bacterium has multiple purposes), and has a good industrialization prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of probiotics, and specifically relates to a fermented Lactobacillus mucus and application thereof. BACKGROUND

[0002] In modern life, emotional regulation, gut health, immune function, and sleep quality maintenance are crucial for human health. Emotional fluctuations not only affect mental health, but also can trigger cardiovascular diseases, digestive system disorders, and other physiological problems. The balance of gut microecology is closely related to nutrient absorption, metabolic balance, and immune function. Immune system disorders can lead to frequent infections, chronic inflammation, and even autoimmune diseases. In addition, sleep disorders seriously affect the body's self-repairing ability, and long-term sleep deprivation can weaken immunity, cause emotional depression, and reduce life quality. Therefore, developing an intervention means that can comprehensively regulate emotions, gut health, immune function, and improve sleep disorders is of great significance for improving the health level of the public.

[0003] However, screening probiotic strains with the above-mentioned multiple functions faces many challenges. Although there are a variety of probiotics, most strains only play a role in addressing a specific health problem. For example, some probiotics mainly improve intestinal function, while others may have some effect on immune regulation. To screen probiotic strains that can simultaneously positively affect emotions, gut health, immune function, and sleep disorders, it is necessary to conduct in-depth research on their biological characteristics, metabolic products, and interaction mechanisms with the host. This not only involves cross-research in multiple disciplines such as microbiology, immunology, and neuroscience, but also requires a large amount of experimental verification and clinical data support. At present, although there are a variety of probiotic products on the market, there are very few probiotic products with multi-functional regulation capabilities. Most products have single functions and cannot meet the needs of consumers for comprehensive health improvement.

[0004] From a scientific research perspective, screening probiotic strains with emotional regulation, gut health regulation, immune regulation, and sleep disorder improvement functions will fill the research gap in the field of multi-system health regulation in microbiology. This research direction will provide new perspectives and theoretical basis for a deeper understanding of the complex interactions between microorganisms and the host. By studying the mechanisms of these probiotic strains, it can reveal how microorganisms affect the physiological and psychological health of the host through the gut-brain axis, immune system, and other pathways. In addition, this research direction will also promote the development of related disciplines and provide new directions and ideas for future research.

[0005] At the market level, there is great potential for developing probiotic products with the above-mentioned multiple functions. With the increasing concern about health, the demand for products that can comprehensively regulate mood, intestinal health, immune function, and improve sleep will continue to grow. Such multi-functional probiotic products not only bring significant economic benefits to enterprises, but also provide a safe and effective solution for public health, with important social value and market potential. SUMMARY

[0006] In view of the problems existing in the prior art, the purpose of the present application is to provide a fermented Limosilactobacillus and its application. The fermented Limosilactobacillus (Limosilactobacillus fermentum) has been deposited with the China General Microbiological Culture Collection Center (CGMCC) on July 4, 2025, and the deposit address is No. 3, Yitian West Road, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China, and the deposit number is CGMCC NO. 35104.

[0007] Specifically, the present application relates to the following aspects:

[0008] 1. A fermented Limosilactobacillus with a deposit number of CGMCC NO. 35104 at the China General Microbiological Culture Collection Center.

[0009] 2. A microbial agent comprising the fermented Limosilactobacillus of item 1.

[0010] 3. The microbial agent of item 2, wherein the number of viable bacteria of the fermented Limosilactobacillus in the microbial agent is 1 x 10 5 - 1 x 10 11 CFU / g, preferably 1 x 10 8 - 1 x 10 10 CFU / g.

[0011] 4. The microbial agent of item 2 or 3, wherein the microbial agent further comprises adjuvants allowed in the field of microbial preparations.

[0012] 5. A probiotic composition comprising the fermented Limosilactobacillus of item 1, or the microbial agent of any one of items 2-4.

[0013] 6. Use of the fermented Limosilactobacillus of item 1, the microbial agent of any one of items 2-4, or the probiotic composition of item 5 in the preparation of a product for promoting intestinal health.

[0014] 7. Use of the fermented Limosilactobacillus of item 1, the microbial agent of any one of items 2-4, or the probiotic composition of item 5 in the preparation of a product for improving immunity.

[0015] 8. Use of the L. muciidum of item 1, the bacterial agent of any one of items 2-4, or the probiotic composition of item 5 in the preparation of a product for improving mood.

[0016] 9. The use of item 6, wherein improving mood is manifested in one or more of the following: promoting dopamine synthesis, promoting serotonin synthesis, promoting L-glutamate synthesis, improving social interaction.

[0017] 10. Use of the L. muciidum of item 1, the bacterial agent of any one of items 2-4, or the probiotic composition of item 5 in the preparation of a product for improving sleep disorders.

[0018] Advantages of the present application

[0019] The L. muciidum Eupro 06 provided by the present application has multiple functions such as improving intestinal health, improving immunity, improving sleep rhythm disorders, improving mood, etc., and provides a high-quality strain for the application of probiotics in the fields of comprehensive health problem solving and multifunctional probiotic product development.

[0020] The L. muciidum Eupro 06 provided by the present application can promote the synthesis of dopamine, serotonin and L-glutamate, thereby improving mood, which makes up for the lack of high-quality mood-regulating probiotic strains on the market.

[0021] In addition, the L. muciidum Eupro 06 provided by the present application also has functions such as improving intestinal health, improving immunity, and improving sleep disorders, which fills the gap of multifunctional probiotic strains on the market. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is the growth curve of L. muciidum Eupro 06;

[0023] Figure 2 is the 24h test result of the gastric juice (acid) resistance of several strains;

[0024] Figure 3 is the 24h test result of the intestinal juice (bile salt) resistance of several strains;

[0025] Figure 4 shows the effect of L. muciidum Eupro 06 on the intestinal length of bees;

[0026] Figure 5 shows the effect of L. muciidum Eupro 06 on the intestinal motility of bees;

[0027] Figure 6Effects of L. fermentum Eupro 06 on the expression of three immune factors in the intestines of honeybees are shown.

[0028] Figure 7 Effects of L. fermentum Eupro 06 on the social interaction ability of honeybees are shown.

[0029] Figure 8 Effects of L. fermentum Eupro 06 on the sleep rhythm of honeybees are shown.

[0030] Figure 9 Effects of L. fermentum Eupro 06 on the dopamine level in the intestines of honeybees are shown.

[0031] Figure 10 Effects of L. fermentum Eupro 06 on the 5-hydroxytryptamine level in the intestines of honeybees are shown.

[0032] Figure 11 Effects of L. fermentum Eupro 06 on the L-glutamic acid level in the intestines of honeybees are shown. DETAILED DESCRIPTION

[0033] The present application is further illustrated by the following examples, which are not intended to limit the present application in any way.

[0034] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, illustrative methods and materials are described below. However, if there is a conflict between the definitions in this specification and those in the materials, methods, and examples, the definition in this specification controls. Unless otherwise indicated, materials, methods, and examples are illustrative only and not intended to be limiting.

[0035] The present application provides a Lactobacillus fermentum, referred to as Lactobacillus fermentum Eupro 06 in the present application, which is classified and named as Lactobacillus fermentum, and has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on July 4, 2025, at the address of No. 3, Yitianli, Beichenxili, Chaoyang District, Beijing, China, the Institute of Microbiology, Chinese Academy of Sciences, with the postal code of 100101, and the preservation number of CGMCC NO. 35104.

[0036] The fermenting L. m. of the present application has excellent acid and bile salt tolerance, wherein the acid tolerance is better than that of other similar strains that have been commercialized (such as L. m. CECT5716 and L. rhamnosus HN001), and the performance is comparable, and in terms of bile salt tolerance, especially in terms of long-term tolerance, the performance is significantly better than that of other similar strains that have been commercialized (such as L. m. CECT5716 and L. rhamnosus HN001).

[0037] The fermenting L. m. Eupro provided by the present application can improve the happy mood of the subject, specifically, can be embodied in one or more of the following: promoting dopamine synthesis, promoting 5-hydroxytryptamine synthesis, promoting L-glutamic acid synthesis, and improving social interaction ability.

[0038] Among them, existing research shows that dopamine is an important neurotransmitter and plays a key role in human physiological functions. It mainly exists in the substantia nigra-striatal pathway and the mesolimbic system of the brain, and is involved in the regulation of motor function, emotion, reward mechanism and cognitive process. Imbalance of dopamine level is closely related to many diseases, for example, Parkinson's disease is caused by the degeneration and death of dopaminergic neurons in the substantia nigra, resulting in reduced dopamine synthesis, and patients will have symptoms such as motor slowing and muscle rigidity; in the field of mental health, abnormal dopamine function is also related to the pathogenesis of diseases such as depression and schizophrenia. Normal secretion of dopamine is crucial for maintaining the stability of human mental state and physiological function. 5-hydroxytryptamine (5-HT, also known as serotonin) is an important monoamine neurotransmitter, widely exists in the central nervous system, gastrointestinal tract and platelets. It can regulate neural signal transmission and is involved in a variety of physiological and neural processes such as emotion, sleep, digestion, cognition, learning and memory; in addition, 5-HT also affects the overall function of the nervous system by regulating other neurotransmitters (such as glutamate, gamma-aminobutyric acid, dopamine, etc.) and their receptors. In terms of emotion regulation, changes in 5-HT levels are closely related to emotional disorders such as depression and anxiety, and many antidepressants work by regulating the 5-HT system. In addition, recent neurochemical research has confirmed that L-glutamic acid is the most important excitatory neurotransmitter in the central nervous system, and its moderate release can significantly improve synaptic plasticity and learning and memory efficiency; at the same time, as a precursor of gamma-aminobutyric acid, it maintains the balance of excitation and inhibition, reduces anxiety levels, and has protective significance for cognitive and emotional homeostasis.

[0039] Therefore, the fermenting L. m. Eupro provided by the present application can improve the happy mood and make up for the lack of high-quality emotion-regulating probiotic strains on the market.

[0040] In addition, the fermenting L. m. Eupro provided by the present application also has the functions of improving intestinal health, improving immunity, and improving sleep disorders.

[0041] The present application also provides a microbial agent comprising the L. fermentum Eupro with the preservation number of CGMCC NO. 35104.

[0042] The L. fermentum Eupro of the present application can be cultured using MRS liquid medium.

[0043] In a specific embodiment, the L. fermentum Eupro is cultured by selecting MRS liquid medium as the culture medium, and the fermentation conditions are 37℃, pH 6.5, rotation speed 150 rpm, and the fermentation time is 12-24 h.

[0044] The microbial agent of the present application can be obtained by culturing the L. fermentum Eupro and further processing the obtained fermentation broth. For example, the fermentation broth can be directly used as a liquid microbial agent, or the fermentation broth can be freeze-dried to obtain a solid microbial agent. Those skilled in the art can understand that, in addition to the L. fermentum Eupro, the microbial agent can also include auxiliary materials allowed in the field of microbial preparations, such as the culture medium during fermentation, freeze-drying protectants, etc.

[0045] In a specific embodiment, the number of viable cells of the L. fermentum in the microbial agent is 1×10 5 CFU / g, for example, it can be 1×10 11 CFU / g, 2×10 5 CFU / g, 5×10 5 CFU / g, 8×10 5 CFU / g, 1×10 5 CFU / g, 2×10 6 CFU / g, 5×10 6 CFU / g, 8×10 6 CFU / g, 1×10 6 CFU / g, 2×10 7 CFU / g, 5×10 7 CFU / g, 8×10 7 CFU / g, 1×10 7 CFU / g, 2×10 8 CFU / g, 5×10 8 CFU / g, 8×10 8 CFU / g, 1×10 8 CFU / g, 2×10 9 CFU / g, 5×10 9 CFU / g, 8×10 9 CFU / g, 1×10 9 CFU / g, 2×10 10 CFU / g, 5×10 10CFU / g, 5x10 10 CFU / g, 8x10 10 CFU / g, 1x10 11 CFU / g, and any value between these values.

[0046] In one embodiment, the viable cell number of the L. mesenteroides in the probiotic agent is 1x10 8 -1x10 10 CFU / g.

[0047] The present application also provides a probiotic composition comprising the above-mentioned L. mesenteroides Eupro, or the above-mentioned probiotic agent.

[0048] The present application also provides the above-mentioned L. mesenteroides Eupro, the above-mentioned probiotic agent, or the above-mentioned probiotic composition for use in improving the mood.

[0049] The present application also provides the above-mentioned L. mesenteroides Eupro, the above-mentioned probiotic agent, or the above-mentioned probiotic composition for use in the manufacture of a product for improving the mood.

[0050] The mood improvement is manifested in one or more of the following: promoting dopamine synthesis, promoting 5-hydroxytryptamine synthesis, promoting L-glutamic acid synthesis, improving social interaction.

[0051] The present application also provides the above-mentioned L. mesenteroides Eupro, the above-mentioned probiotic agent, or the above-mentioned probiotic composition for use in improving immunity.

[0052] The present application also provides the above-mentioned L. mesenteroides Eupro, the above-mentioned probiotic agent, or the above-mentioned probiotic composition for use in the manufacture of a product for improving immunity.

[0053] The present application also provides the above-mentioned L. mesenteroides Eupro, the above-mentioned probiotic agent, or the above-mentioned probiotic composition for use in promoting intestinal health.

[0054] The present application also provides the above-mentioned L. mesenteroides Eupro, the above-mentioned probiotic agent, or the above-mentioned probiotic composition for use in the manufacture of a product for promoting intestinal health.

[0055] The present application also provides the above-mentioned L. mesenteroides Eupro, the above-mentioned probiotic agent, or the above-mentioned probiotic composition for use in improving sleep disorders.

[0056] The present application also provides the above-mentioned L. mesenteroides Eupro, the above-mentioned probiotic agent, or the above-mentioned probiotic composition for use in the manufacture of a product for improving sleep disorders.

[0057] In the above uses, the product can be a food, a drug, or a health product.

[0058] Embodiment

[0059] Example 1 Isolation and purification of Lactobacillus muci genus Eupro 06 and evaluation of fermentation capacity

[0060] 1.1 Isolation and purification

[0061] A healthy human fecal sample was diluted by several times, spread on MRS flat plate, and cultured in a constant temperature incubator at 35°C for 48h to obtain single colonies. The single colonies were picked and inoculated in MRS liquid medium and placed in a constant temperature incubator at 35°C for 20h. The bacterial liquid was streaked on MRS flat plate and cultured to obtain single colonies. The single colonies were picked and re-streaked and cultured to obtain single colonies, which were the pure isolated strain Eupro 06. The pure isolated single colonies were subjected to Gram staining and specific detection.

[0062] The specific 16S rDNA sequence of the bacteria is shown as follows (SEQ ID NO: 1):

[0063] TGATTGATGGTGCTTGCACCTGATTGATTTTGGTCGCCAACGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCAGAAGCGGGGGACAACATTTGGAAACAGATGCTAATACCGCATAACAACGTTGTTCGCATGAACAACGCTTAAAAGATGGCTTCTCGCTATCACTTCTGGATGGACCTGCGGTGCATTAGCTTGTTGGTGGGGTAACGGCCTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGGACTGAGACACGGCCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACACGTATGAGAGTAACTGTTCATACGTTGACGGTATTTAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGAGAGTGCAGGCGGTTTTCTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGATAACTTGAGTGCAGAAGAGGGTAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTACCTGGTCTGCAACTGACGCTGAGACTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCAT

[0064] The identified strain Eupro 06 is Limosilactobacillus fermentum, which has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on July 4, 2025. The address of the CGMCC is No. 1, Beichen West Road, Yard 3, Beijing, China, and the postal code is 100101. The preservation number of the strain is CGMCC NO. 35104.

[0065] 1.2 Activation of the strain

[0066] -80℃ preservation of strain liquid passage→20h culture after plate streaking→single colony picking to liquid medium culture, which can be used for subsequent experiments.

[0067] 1.3 Growth curve determination

[0068] The third generation of activated strain was inoculated at 2% after 20h culture, and the OD value was determined during the growth process at 37℃ constant temperature culture to obtain the specific time of the stable phase of the strain growth (such as shown in FIG. 1), which was prepared for subsequent fermentation. Figure 1

[0069] 1.4 Fermentation conditions

[0070] Fermentation experiment was carried out in a 5L fermenter, and the medium was MRS liquid medium. The fermentation conditions were 37℃, pH 6.5, and rotation speed 150rpm. According to the strain growth curve, fermentation to the stable phase can be carried out for subsequent freeze-drying experiment.

[0071] 1.5 Freeze-dried bacterial powder

[0072] The fermented bacterial liquid was centrifuged at 6000g at 4℃ for 15min to obtain bacterial mud. 25mL of protective agent was added to the bacterial mud obtained from each 250mL of bacterial liquid. The protective agent used included 1% trehalose, 0.5% sodium glutamate, 1% skim milk, 0.25% ascorbic acid, and 0.25% dextran.

[0073] Before freeze-drying, the sample was pre-frozen at -80℃ for 3 hours in an ultra-low temperature environment. Then a three-stage freeze-drying process was adopted: the first stage was freeze-drying at 512Pa for 6 hours, the second stage was freeze-drying at 256Pa for 5 hours, and finally freeze-drying was continued at 103Pa until the sample formed a powder product.

[0074] The freeze-dried bacterial agents were stored at 4℃ and 25℃ respectively, and the viable bacterial count of each bacterial powder was measured every 2 months, with 3 parallel experiments. The results are shown in Table 1. According to the table, after 10 months of storage at 4℃ and 25℃, the viable bacterial count of the freeze-dried bacterial agents remained at a high level. Although the stability at 25℃ was lower than that at 4℃, it still maintained a survival level of more than 90%. The effective bacterial count of the freeze-dried L. mesenteroides Eupro 06 was not less than 4×10 10 cfu / g.

[0075] Table 1 Survival rate of freeze-dried bacterial powder

[0076]

[0077] Example 2. Evaluation of acid and bile salt tolerance of L. mesenteroides Eupro 06 ​

[0078] 2.1 Test strains

[0079] L. fermentum Eupro 06, L. fermentum CECT5716, L. rhamnosus HN001

[0080] 2.2 Reagents required

[0081] MRS broth, plates 100, 0.85% saline, pepsin (Amresco), trypsin (Amresco), ox bile salt (Oxiod), 0.1 mol / L HC1 20 mL, 0.1 mol / L NaOH 20 mL.

[0082] 2.3 Consumables required

[0083] White, blue, yellow tips (sterilized), pipette, Petri dishes 100, glass beads or spreader bar, 1.5 mL centrifuge tubes, 0.22 microfiltration membrane 2, 10 mL centrifuge tubes, 10 mL syringes 2, plates, 10 ml conical flasks.

[0084] 2.4 Preparation of reagents and materials used

[0085] (1) MRS liquid medium

[0086] (2) MRS solid medium

[0087] (3) PBS: 500 mL PBS Preparation method: ① 4 g Nacl ② 0.1 g Kcl ③ 1.79 g Na2HPO4 ④ 0.12 g KH2PO4 Make up to 500 mL

[0088] (4) 0.85% saline: prepare 100 mL, take 0.85 g Nacl with deionized water to make up to 100 mL (5) tip sterilization (10 μL, 200 μL, 1000 μL)

[0089] (6) centrifuge tube and conical flask sterilization (1.5 mL centrifuge tube, 10 mL centrifuge tube)

[0090] 2.5 Experimental reagent preparation and experimental process

[0091] (1) Strain activation process (20 h transmission of a generation)

[0092] ① Take the strain stored at -80 C, take an appropriate amount and add to MRS solid medium, culture medium overnight activation (24-48 h), for a generation.

[0093] ② Pick up an appropriate amount of activated first generation strain into 5 mL MRS liquid medium (reaction 20 h), for the second generation of bacterial liquid.

[0094] ③ From the activated second generation bacteria, 400 μL of bacterial solution was taken into 20 mL of MRS liquid medium (reaction for 20 h) to obtain the third generation bacterial solution.

[0095] (2) Preparation of gastric juice and intestinal juice

[0096] ④ Preparation of artificial gastric juice: 20 mL of sterilized physiological saline + 0.06 g of pepsin (3 g / L), pH 3.0 was adjusted with 0.1 mol / L HCl, and then filtered into a sterilized empty conical flask with a 0.22 micron filter membrane. It was ready for use.

[0097] ⑤ Preparation of artificial intestinal juice: 20 mL of sterilized physiological saline + 0.02 g of trypsin (1 g / L) + 0.06 g of choline salt (0.3%), pH 8.0 was adjusted with 0.1 mol / L NaOH, and then filtered into a sterilized empty conical flask with a 0.22 micron filter membrane. It was ready for use.

[0098] (3) Experimental process

[0099] ① Pretreatment: centrifuge the cultured bacterial solution (6000 g or 8000 rpm, 10 min), discard the supernatant, wash the bacteria once with 20 mL of physiological saline, discard the supernatant, add 2 mL of physiological saline, shake evenly to become the original bacterial solution. Dilute the original bacterial solution to -8, -6, -7, and -8 for counting, do two parallel, as 0 h of bacterial count, culture for 24 h.

[0100] ② Acid (gastric juice) resistance test: take 500 μL of the original solution to a test tube containing 4.5 mL of gastric juice, immediately put into a 37℃ incubator for reaction for 24 h, take the gastric juice after reaction for 24 h for gradient dilution counting to 10 -7 times, 10 -5 , 10 -6 , 10 -7 times for counting, do two parallel, put into a 37℃ incubator for culture for 24-48 h. The results are shown in Figure 2 .

[0101] ③ Intestinal juice (choline salt) resistance test: take 500 μL of the original solution to a test tube containing 4.5 mL of intestinal juice, immediately put into a 37℃ incubator for reaction for 24 h, take the intestinal juice after reaction for 24 h for gradient dilution counting to 10 -6 times, 10 -4 , 10 -5 , 10 -6 times for counting, do two parallel, put into a 37℃ incubator for culture for 24-48 h. The results are shown in Figure 3 .

[0102] The results show that the acid and bile salt tolerance of the fermented L. mesenteroides Eupro 06 is significantly better than that of the commercial strains L. mesenteroides CECT5716 and L. rhamnosus HN001 at 24 h. ** indicates p < 0.01; *** indicates p < 0.001; **** indicates p < 0.0001

[0103] Example 3. Effect of fermented L. mesenteroides Eupro 06 on intestinal health

[0104] 3.1 Preparation before experiment

[0105] 500 g of sucrose was weighed and made up to 1 L with ultrapure water to obtain a 50 w / v% sucrose solution, which was filtered with a filter membrane with a pore size of 0.22 pm to remove bacteria to obtain a 50 w / v% sterile sucrose solution required for the experiment. The purchased pollen (Qinghai Province rape pollen) was divided into self-sealing bags, ensuring strict sealing, and sterilized by electron beam with a strength of 30 kGy for 4 h to obtain sterile pollen.

[0106] 3.2 Selection and culture of experimental bees

[0107] Suitable honeycomb guts were selected, and a sterilized awl or forceps was used to gently pry off the pupa cover at several random points on the honeycomb guts. The maturity of the pupae was observed, and the pupa cover was continued to be pricked around the pupae with black color and no hair. When pricking the pupa cover, care should be taken to clean the surrounding area as much as possible to avoid carrying impurities from the honeycomb when taking the pupae. The sterilized forceps were inserted from above the pupae and then spread to both sides of the pupae, and the pupae were taken out by gently pinching the chest. Physical damage to the pupae was avoided as much as possible. In addition, pupae that have developed wings cannot be used.

[0108] The selected pupae were placed in a plastic box that had been sterilized with 84 disinfectant and alcohol in advance, and a 2 mL centrifuge tube containing sterile sucrose solution was inserted into the side or top of the box. Each box could hold 150-200 pupae. The box containing the pupae was placed in a incubator at a temperature of 35°C and a humidity of 60-80% for overnight culture.

[0109] The next day, the pupae were observed for emergence (if not emerged, they were cultured for another night), and the emerged bees were gently taken out and divided into disposable transparent culture cups. A new sterile 2 mL centrifuge tube was taken, 990 pL of sterile PBS was added, and 10 pL of normal worker bee intestinal juice was added, and mixed well. Finally, the centrifuge tube was filled with 50% (w / v) sterile sucrose solution to fill the remaining space, and the centrifuge tube was shaken evenly in the hand. Each cup of newly emerged bees was fed with the mixed solution, and was continued to be placed in a constant temperature incubator for two days.

[0110] 3.3 Animal grouping and experimental design

[0111] Take the above bees, 150, randomly divided into two groups, 75 in each group, every 25 cages. Group is control group and experimental group, control group liquid diet feeding 50% (w / v) sugar water, solid diet feeding rape pollen. The experimental group liquid diet feeding 50% (w / v) sugar water, solid diet feeding rape pollen containing Eupro 06 bacteria powder, wherein the concentration of Eupro 06 bacteria powder is 10 10 CFU / g. Solid diet needs to be replaced daily, continuous culture for 7 days.

[0112] 3.4 Experimental observation and sampling

[0113] Feed culture to the 7th day, randomly select 9 bees from each group, and detect the intestinal motility and intestinal length indicators.

[0114] (1) Intestinal motility detection method

[0115] Take 1.5ml centrifuge tube, punch a hole at the cover; take the bees to be tested into the punched centrifuge tube, starve in the incubator for 1-2h, then transfer to the test table, use a pipette to take 5μL 0.2% (w / v) coomassie brilliant blue in 50% (w / v) sugar water, feed the bees from the punched hole on the centrifuge tube or gently open the cover;

[0116] Put the fed bees into separate feeding cups for free activity, accurately time, and immediately extract the complete intestine after each bee is fed for 30 minutes, place it on the measurement paper, gently stretch the intestine into a straight line with tweezers, mark the starting point and ending point of the intestine and the length of the dye on the measurement paper with a marker pen, and take a picture with a ruler. Measure the length of the dye and the total length of the intestine with ImageJ, and the length of the dye / the total length of the intestine is the intestinal motility value.

[0117] (2) Intestinal development length detection method

[0118] Hold the bee from the chest with tweezers with the tail upwards, hold the last segment of the bee's tail with another pair of tweezers, and slowly pull out the complete intestine; place the extracted intestine neatly on the measurement paper, mark the grouping information and the ruler, and take a picture record, and measure the length with ImageJ software. The intestinal development length of the bee is shown in Figure 4 , wherein * represents a significance less than 0.05. The results show that the Lactobacillus kunkeei H09 described in the application can significantly increase the intestinal development length of the bee.

[0119] The intestinal motility results of the bee are shown in Figure 5 , and the results show that the fermented Lactobacillus mucus Eupro 06 treatment can significantly increase the intestinal motility of the bee and significantly promote the development of the intestinal length of the bee. Wherein * represents p<0.05; ** represents p<0.01; *** represents p<0.001

[0120] Example 4. The effect of fermented Lactobacillus muciadis Eupro 06 on the immune capacity

[0121] 4.1 Preparation before experiment

[0122] Same as 3.1

[0123] 4.2 Selection and cultivation of experimental bees

[0124] Same as 3.2

[0125] 4.3 Grouping of animals and experimental design

[0126] Same as 3.3

[0127] 4.4 Observation and sampling of experiment

[0128] On the 7th day of feeding and cultivation, 6 bees were randomly selected from each group for detection of intestinal motility and intestinal length indicators.

[0129] Total RNA of the intestines of bees in the control group and the fermented Lactobacillus muciadis Eupro 06 treatment group was extracted using the BTN81220 columnar insect RNA extraction kit, and the three immune genes Defensin, Abaecin and Apidaecins were quantitatively analyzed using the Applied Biosystems real-time fluorescence quantitative PCR instrument.

[0130] The three immune gene amplification primers are shown in Table 2:

[0131] Table 2: Sequences of immune gene amplification primers

[0132]

[0133] The results are shown in Figure 6 It can be seen that the fermented Lactobacillus muciadis Eupro 06 treatment can significantly increase the expression of the three immune factors of bees. Among them, * indicates p<0.05; ** indicates p<0.01

[0134] Example 5. The effect of fermented Lactobacillus muciadis Eupro 06 on social interaction ability

[0135] 5.1 Preparation before experiment

[0136] Same as 3.1

[0137] 5.2 Selection and cultivation of experimental bees

[0138] Same as 3.2

[0139] 5.3 Grouping of animals and experimental design

[0140] Same as 3.3

[0141] 5.4 Observation and sampling of experiment

[0142] The bees were fed on the 7th day of culture, and 36 bees were randomly selected from each group, 3 bees per group, placed in 90 mm culture dishes, and placed in a pre-arranged photo studio for video shooting. The collected video was analyzed for social behavior using the Ethovision XT17 animal behavior analysis system.

[0143] As shown in Figure 7 , fermentation of Lactobacillus muci de Eupro 06 can significantly increase the social interaction ability of bees. * indicates p < 0.05; ** indicates p < 0.01; **** indicates p < 0.0001.

[0144] Example 6. Effect of fermented Lactobacillus muci de Eupro 06 on sleep rhythm

[0145] 6.1 Preparation before experiment

[0146] The same as 3.1

[0147] 6.2 Selection and culture of experimental bees

[0148] The same as 3.2

[0149] 6.3 Animal grouping and experimental design

[0150] Take the above bees, 90, randomly divide them into three groups, 30 in each group, and place each bee in a 6 cm culture dish in a culture box. The groups are control group, model group and experimental group, and the model group is a sleep disorder model. The control group is fed on liquid diet 50% (w / v) sugar water and solid diet rape pollen; the model group is fed on liquid diet 50% (w / v) sugar water containing 200 mg / L concentration of caffeine and solid diet rape pollen; the experimental group is fed on liquid diet 50% (w / v) sugar water containing 200 mg / L concentration of caffeine and solid diet rape pollen containing fermented Lactobacillus muci de Eupro 06 powder, wherein the concentration of Eupro 06 powder is 10 10 CFU / g. The solid diet needs to be replaced daily, and the bees are first raised for 4 days in a 12h:12h light-dark alternating environment, and then cultured in complete darkness for 4 days, and the whole process is videoed.

[0151] 6.4 Analysis of experimental results

[0152] The collected video was analyzed for social behavior using the Ethovision XT17 animal behavior analysis system. The sleep duration and sleep frequency per 24h were statistically analyzed.

[0153] As shown in Figure 8As shown in Table 6, the treatment of fermented L. m. Eupro 06 can significantly improve the sleep frequency reduction of the honeybee sleep disorder model, and make the sleep frequency of the honeybee return to the same level as the control; and extremely significantly improve the sleep time reduction of the honeybee sleep disorder model, and make the sleep time of the honeybee return to the same level as the control. * indicates p < 0.05; **** indicates p < 0.0001.

[0154] Example 7. Effect of fermented L. m. Eupro 06 on honeybee tissue dopamine synthesis and metabolism

[0155] 7.1 Preparation before experiment

[0156] Same as 3.1

[0157] 7.2 Selection and culture of experimental honeybees

[0158] Same as 3.2

[0159] 7.3 Grouping of animals and experimental design

[0160] Same as 3.3

[0161] 7.4 Experimental observation and sampling

[0162] On the 7th day of feeding and culture, the intestines of the honeybees were dissected, and the whole intestines were collected for subsequent intestinal metabolome detection.

[0163] The results are shown in Table 7. Figure 9 As shown in Table 7, the treatment of fermented L. m. Eupro 06 can extremely significantly increase the dopamine level in the intestines of honeybees. **** indicates p < 0.0001.

[0164] Example 8. Effect of fermented L. m. Eupro 06 on honeybee tissue 5-hydroxytryptamine synthesis and metabolism

[0165] 8.1 Preparation before experiment

[0166] Same as 3.1

[0167] 8.2 Selection and culture of experimental honeybees

[0168] Same as 3.2

[0169] 8.3 Grouping of animals and experimental design

[0170] Same as 3.3

[0171] 8.4 Experimental observation and sampling

[0172] On the 7th day of feeding and culture, the intestines of the honeybees were dissected, and the whole intestines were collected for subsequent intestinal metabolome detection.

[0173] The results are shown in the figure. Treatment with fermented Lactobacillus mucin Eupro 06 significantly increased the level of 5-hydroxytryptamine in the bee gut. *** indicates p<0.001.

[0174] Example 9. Effects of fermentation of Lactobacillus mucilaginosus Eudro 06 on L-glutamate synthesis and metabolism in bee tissues

[0175] 9.1 Pre-experimental preparation

[0176] Same as 3.1

[0177] 9.2 Selection and Cultivation of Experimental Bees

[0178] Same as 3.2

[0179] 9.3 Animal grouping and experimental design

[0180] Same as 3.3

[0181] 9.4 Experimental observation and sampling

[0182] After feeding and culturing for 7 days, the bees' intestines were dissected and the whole intestines were collected for subsequent intestinal metabolomics analysis.

[0183] The results are as follows Figure 11 As shown, treatment with fermented Lactobacillus mucinus Eupro 06 significantly increased L-glutamate levels in the bee gut. **** indicates p < 0.0001.

Claims

1. A Lactobacillus muci genitalis strain, having a preservation number of CGMCC NO. 35104 in China General Microbiological Culture Collection Center. 2.A microbial agent comprising the Lactobacillus muci genitalis strain of claim 1.

3. The bacterial agent according to claim 2, wherein the number of viable cells of L. fermentum in the bacterial agent is 1 x 10 5 CFU / g, preferably 1 x 10 11 CFU / g, more preferably 1 x 10 8 CFU / g, even more preferably 1 x 10 10 CFU / g. 4.The microbial agent of claim 2 or 3, wherein the microbial agent further comprises adjuvants allowed in the field of microbial preparation. 5.A probiotic composition comprising the Lactobacillus muci genitalis strain of claim 1, or the microbial agent of any one of claims 2-4. 6.Use of the Lactobacillus muci genitalis strain of claim 1, the microbial agent of any one of claims 2-4, or the probiotic composition of claim 5 in the preparation of a product for promoting intestinal health. 7.Use of the Lactobacillus muci genitalis strain of claim 1, the microbial agent of any one of claims 2-4, or the probiotic composition of claim 5 in the preparation of a product for improving immunity. 8.Use of the Lactobacillus muci genitalis strain of claim 1, the microbial agent of any one of claims 2-4, or the probiotic composition of claim 5 in the preparation of a product for improving happy mood. 9.The use of claim 6, wherein improving happy mood is manifested in one or more than two of the following: promoting dopamine synthesis, promoting 5-hydroxytryptamine synthesis, promoting L-glutamic acid synthesis, and improving social interaction ability. 10.Use of the Lactobacillus muci genitalis strain of claim 1, the microbial agent of any one of claims 2-4, or the probiotic composition of claim 5 in the preparation of a product for improving sleep disorders.

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