A Pediococcus pentosaceus for regulating mood, improving sleep and increasing height and its application

Abstract

The present invention provides a Pediococcus pentosaceus for regulating mood, improving sleep and increasing height and its application, belonging to the technical field of probiotics. The classification name of the Pediococcus pentosaceus ZJPY017 of the present invention is Pediococcus pentosaceus, which is preserved in the General Microbiological Center of the China Microbiological Culture Collection Administration Committee, and the preservation number is CGMCC No.1.60177. The Pediococcus pentosaceus provided by the present invention has strong acid resistance and bile salt resistance, and has the effects of increasing bone density, helping the body to excrete lead, promoting height growth, regulating mood and improving sleep. It has general applicability to all kinds of people and provides new ideas for the research and development of health foods and medicines.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics, and in particular relates to Pediococcus pentosaceus for regulating mood, improving sleep and increasing height, and application thereof. Background Art

[0002] Lead is a heavy metal element widely found in nature that is not essential to the human body. If the lead content in the body is too high, it can cause many acute and chronic diseases. After lead enters the human body, in addition to some excretion through feces and sweat, the rest dissolves into the blood after a few hours, hindering the synthesis of blood, causing anemia in the human body, headaches, dizziness, fatigue, drowsiness, constipation and limb pain, etc. Some people have a metallic taste in the mouth, and symptoms such as arteriosclerosis, peptic ulcers and fundus hemorrhage are also related to lead pollution. Children in the growth and development period are particularly sensitive to lead exposure. The presence of lead in the body will hinder the growth and development of children, and the height development of children is an important issue that every parent is concerned about. Good height development has a profound impact on children's physical health, psychological development and social adaptability. For example, physical maldevelopment may lead to growth retardation, low immunity and other problems. Therefore, it is of great significance to promote the excretion of lead from the body and promote the height growth of children.

[0003] Sleep is a basic function of organisms and plays an important role in maintaining normal physiological activities. Sufficient sleep is an important part of restoring mental and physical strength, integrating and consolidating memory. Its physiological significance is second only to breathing and heartbeat, and it is indispensable for maintaining the health of the body. Studies have shown that good sleep can reduce the incidence of certain chronic diseases. Conversely, insufficient sleep affects a variety of physiological functions, such as cognition, emotion, immunity, and reproductive function. Depression is one of the common psychological problems of people, which will have a great negative impact on people's daily life, work, and study. If depression lasts for a long time, a series of consequences will occur. In terms of physiology, long-term depression will lead to decreased immunity and increased risk of infectious diseases; it will lead to increased levels of adrenaline and cortisol in the blood, increasing the risk of heart disease and stroke; it will lead to digestive system problems, such as excessive stomach acid, stomach cramps, diarrhea, etc. In terms of mental health, long-term depression is a precursor to depression or leads to the occurrence of anxiety. It can be seen that sleep conditions are closely related to good or bad moods. Insomnia and depression are very detrimental to the health of the body. At present, chemical drugs are the main means of treating insomnia clinically, but long-term use of them can easily cause some nerve damage and dependence in patients. Therefore, it is urgent to find an efficient, safe and non-toxic treatment for insomnia.

[0004] Bone is a highly active connective tissue that can repair its own micro-damage through bone metabolism and bone remodeling, maintaining the homeostatic balance of bone structure, load and calcium content. At the same time, bone also regulates metabolic processes as an endocrine organ and is an important organ for maintaining human life. Osteoporosis is a systemic bone metabolic disease caused by a variety of factors. It is characterized by deterioration of bone mass, bone density and microstructure of bone tissue, increased bone brittleness and susceptibility to fractures. Osteoporosis has seriously threatened human health and will eventually lead to reduced daily activities, reduced quality of life and increased mortality in patients. Therefore, how to effectively increase bone density is of great significance for the prevention and treatment of osteoporosis.

[0005] Pediococcus pentosaceus is a gram-positive coccus belonging to the genus Pediococcus of the family Streptococcus. It can ferment glucose to produce lactic acid and is distributed in malt wort and fermented plant materials, such as pickles, kimchi, silage, etc. The reported probiotic functions of Pediococcus pentosaceus include inhibiting inflammation, inhibiting bacterial growth, promoting digestion, and improving immunity. Currently, there are no reports on whether Pediococcus pentosaceus can also increase bone density, help the body excrete lead, promote children's height growth, regulate emotions, and improve sleep. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a Pediococcus pentosaceus that has the effects of increasing bone density, helping the body to excrete lead, promoting children's height growth, regulating emotions and improving sleep.

[0007] Another object of the present invention is to provide a Pediococcus pentosaceus bacterial liquid which has the effects of increasing bone density, helping the body to excrete lead, promoting children's height growth, regulating emotions and improving sleep.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The invention provides a Pediococcus pentosaceus. The Pediococcus pentosaceus is Pediococcus pentosaceus ZJPY017, which is classified and named as Pediococcus pentosaceus and is preserved in the General Microbiological Center of China National Committee for Microbiological Culture Collection with a preservation number of CGMCC No.1.60177.

[0010] The present invention also provides a bacterial liquid of Pediococcus pentosaceus, which comprises at least one of a live bacterial liquid and an inactivated bacterial liquid of the above-mentioned Pediococcus pentosaceus.

[0011] Preferably, the preparation method of the live bacterial liquid comprises the following steps: inoculating the above-mentioned Pediococcus pentosaceus into a liquid culture medium, fermenting and culturing at 40°C-50°C, counting the fermentation culture liquid, and adjusting the bacterial concentration to 10 with sterile water. 4CFU / mL-10 6 CFU / mL, and obtain live bacterial solution.

[0012] Preferably, the method for preparing the inactivated bacterial liquid comprises the following steps: inactivating the live bacterial liquid to obtain the inactivated bacterial liquid.

[0013] Preferably, when the Pediococcus pentosaceus bacterial liquid comprises the live bacterial liquid and the inactivated bacterial liquid of the above-mentioned Pediococcus pentosaceus, the ratio of the number of bacteria in the live bacterial liquid to the number of bacteria in the inactivated bacterial liquid is 1:2-4.

[0014] The present invention also provides the use of the above-mentioned Pediococcus pentosaceus or the above-mentioned Pediococcus pentosaceus bacterial liquid in the preparation of a product for increasing bone density.

[0015] The present invention also provides the use of the above-mentioned Pediococcus pentosaceus or the above-mentioned Pediococcus pentosaceus bacterial liquid in the preparation of a product that helps the body to excrete lead.

[0016] The present invention also provides the use of the above-mentioned Pediococcus pentosaceus or the above-mentioned Pediococcus pentosaceus bacterial liquid in the preparation of a product promoting height growth.

[0017] The present invention also provides the use of the above-mentioned Pediococcus pentosaceus or the above-mentioned Pediococcus pentosaceus bacterial liquid in the preparation of a mood regulating product.

[0018] The present invention also provides the use of the above-mentioned Pediococcus pentosaceus or the above-mentioned Pediococcus pentosaceus bacterial liquid in the preparation of a product for improving sleep.

[0019] Beneficial effects of the present invention:

[0020] The Pediococcus pentosaceus ZJPY017 provided by the present invention has strong acid and bile salt resistance, and has the functions of increasing bone density, helping the body to excrete lead, promoting height growth, regulating emotions and improving sleep. It has universal applicability to all kinds of people and provides a new idea for the research and development of health foods and medicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the growth curve of Pediococcus pentosaceus of the present invention;

[0022] Figure 2 This is the result of the lead content in feces.

[0023] Collection Instructions

[0024] The present invention's Pediococcus pentosaceus ZJPY017 is deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration Committee, and is classified and named Pediococcus pentosaceus. The preservation date is April 15, 2024, and the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 1.60177. DETAILED DESCRIPTION

[0025] The invention provides a Pediococcus pentosaceus. The Pediococcus pentosaceus is Pediococcus pentosaceus ZJPY017, which is classified and named as Pediococcus pentosaceus and is preserved in the General Microbiological Center of China National Committee for Microbiological Culture Collection with a preservation number of CGMCC No.1.60177.

[0026] The Pediococcus pentosaceus of the present invention is obtained by screening from fermented old dough of a farmer in Cicheng, Ningbo City, Zhejiang Province. The strain obtained by screening is identified as Pediococcus pentosaceus through morphological identification and 16S rRNA sequencing analysis. The separation and purification method of the Pediococcus pentosaceus of the present invention is specifically as follows: a loop of fermented old dough is taken with a bacterial inoculation loop and inoculated into an MRS solid culture medium, and anaerobically cultured for 30 hours at 40°C; a white colony with a smooth surface is selected for Gram staining, and the bacteria are observed to be Gram-positive cocci under a microscope. The colony is inoculated into an MRS liquid culture medium, and anaerobically cultured for 30 hours at 40°C; the genome is extracted, 16S rDNA is amplified by PCR, and the sequencing results are compared in the NCBI database, and the sample obtained by screening is finally identified as Pediococcus pentosaceus.

[0027] The present invention also provides a bacterial liquid of Pediococcus pentosaceus, which comprises at least one of a live bacterial liquid and an inactivated bacterial liquid of the above-mentioned Pediococcus pentosaceus.

[0028] In the present invention, the preparation method of the live bacterial liquid preferably comprises the following steps: inoculating the above-mentioned Pediococcus pentosaceus into a liquid culture medium, fermenting and culturing at 40°C-50°C, counting the fermentation culture liquid, and adjusting the bacterial concentration to 10 with sterile water. 4 CFU / mL-10 6 CFU / mL, and a live bacterial liquid is obtained. In the present invention, the liquid culture medium preferably includes the following raw materials: protein powder 20g / L-30g / L, glucose 20g / L-30g / L, dipotassium hydrogen phosphate 2.1g / L-2.4g / L, magnesium chloride 0.2g / L-0.4g / L, zinc sulfate 0.3g / L-0.35g / L, calcium carbonate 0.16g / L-0.2g / L, and the pH value of the liquid culture medium is preferably 5.5-6.5. In the present invention, the temperature of the fermentation culture is preferably 43°C-47°C, more preferably 45°C-46°C. The present invention has no special limitation on the specific fermentation time, as long as the bacterial concentration in the culture solution obtained by fermentation is higher than 10 4 CFU / mL or higher than 10 6 CFU / mL is enough.

[0029] In the present invention, the preparation method of the inactivated bacterial solution preferably comprises the following steps: inactivating the active bacterial solution to obtain the inactivated bacterial solution. The inactivation method is preferably high-temperature steam sterilization, the temperature of the high-temperature steam sterilization is preferably 140°C-150°C, more preferably 144°C-148°C, and the time of the high-temperature steam sterilization is preferably 20min-30min, more preferably 23min-26min.

[0030] In the Pediococcus pentosaceus bacterial solution of the present invention, when the Pediococcus pentosaceus bacterial solution is only the live bacterial solution of the above-mentioned Pediococcus pentosaceus, the bacterial concentration in the live bacterial solution is preferably 10 5 CFU / mL; when the pentosaceae bacterial solution is only the inactivated bacterial solution of the above-mentioned pentosaceae, the bacterial concentration in the inactivated bacterial solution is preferably 10 5 CFU / mL; when the Pediococcus pentosaceus bacterial solution includes both the live bacterial solution and the inactivated bacterial solution of the above-mentioned Pediococcus pentosaceus, the ratio of the number of bacteria in the live bacterial solution to the number of bacteria in the inactivated bacterial solution is preferably 1:2-4, more preferably 1:3.

[0031] The present invention also provides the use of the above-mentioned Pediococcus pentosaceus or the above-mentioned Pediococcus pentosaceus bacterial liquid in the preparation of products that increase bone density, help the body to excrete lead, promote height growth, regulate emotions or improve sleep.

[0032] In the present invention, the types of products preferably include food and medicine.

[0033] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0035] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0036] Example 1

[0037] The fermented dough of a farmer in Cicheng, Ningbo City, Zhejiang Province was used as a sample. A loop of fermented dough was inoculated into MRS solid medium with a bacterial inoculation loop and cultured anaerobically at 40°C for 30 hours. The white colonies with smooth surface were selected for Gram staining, and the bacteria were observed to be Gram-positive cocci under a microscope. The colonies were inoculated into MRS liquid medium and cultured anaerobically at 40°C for 30 hours. The genome was extracted, 16S rDNA was amplified by PCR, and the sequencing results were compared in the NCBI database. The sample obtained by the final identification and screening was Pediococcus pentosaceus ZJPY017.

[0038] Example 2

[0039] Study on the growth curve and probiotic characteristics of Pediococcus pentosaceus ZJPY017 obtained in Example 1

[0040] Growth curve of Pediococcus pentosaceus ZJPY017

[0041] Pediococcus pentosaceus ZJPY017 was inoculated into MRS liquid medium and cultured in a shaking incubator at 40°C (anaerobic) for 1 day. The OD was measured every 2 hours. 595 Value, with time as the horizontal axis and OD as the horizontal axis 595 The value is the vertical coordinate, and the growth curve is drawn. The result is as follows Figure 1 As shown, the growth curve shows that the logarithmic growth phase of Pediococcus pentosaceus is 2h-4h, and the stable phase is reached at 10h, indicating that the Pediococcus pentosaceus ZJPY017 of the present invention has good activity.

[0042] Study on the probiotic characteristics of Pediococcus pentosaceus ZJPY017

[0043] The Pediococcus pentosaceus ZJPY017 was inoculated into MRS liquid culture medium and cultured in a constant temperature incubator at 40°C for 12 hours to obtain the culture fluid of the Pediococcus pentosaceus ZJPY017, and the following acid resistance and bile salt resistance experiments were performed.

[0044] (1) Acid resistance test

[0045] Take 100 μL of the culture solution of Pediococcus pentosaceus ZJPY017 and culture it in PBS solutions with pH values ​​of 2, 3, 4 and 7, respectively, at a temperature of 40°C. The viable rate was determined by the plate count method at 1 h, 3 h and 4 h of culture. Viable rate (%) = (a / b) × 100%, where a represents the number of viable bacteria in the culture solution of Pediococcus pentosaceus ZJPY017 at 1 h, 3 h and 4 h, respectively, and b is the initial number of viable bacteria in 100 μL of the culture solution of Pediococcus pentosaceus ZJPY017, that is, the number of viable bacteria before inoculation into PBS. This experiment was set up in 3 parallels, and the data are mean ± SD. The results are shown in Table 1.

[0046] Table 1 Acid resistance results of Pediococcus pentosaceus ZJPY017

[0047] Live bacteria rate (%) 1h 3h 4h pH2 25.3±3.4 12.1±4.6 0 pH3 98.6±6.9 81.3±8.2 69.5±5.1 pH4 134.8±4.6 126.5±4.2 112.4±6.4 pH7 152.7±5.8 149.5±3.6 123.5±5.3

[0048] (2) Bile salt tolerance test

[0049] The culture fluid of Pediococcus pentosaceus ZJPY017 was inoculated at a volume inoculation rate of 2% into MRS liquid culture medium containing 0% and 0.3% ox bile salts, respectively, and cultured at 40°C for 4 hours. The viable bacterial rate was determined by the plate count method. The liquid culture medium without inoculation of bacterial fluid was used as a blank control, and the viable bacterial rate of the strain in bile salts was calculated according to the following formula.

[0050] Viable bacteria rate (%) = [(viable bacteria count in medium containing 0.3% bile salts - viable bacteria count in blank control) / (viable bacteria count in medium containing 0% bile salts - viable bacteria count in blank control)] × 100%.

[0051] This experiment was set up in 3 parallels, and the data are mean ± SD. The results showed that the viable bacterial rate of Pediococcus pentosaceus ZJPY017 in 0.3% bile salt for 4 hours was as high as 95.4 ± 2.6%.

[0052] The above results show that the Pediococcus pentosaceus ZJPY017 of the present invention has strong acid resistance and bile salt resistance.

[0053] Example 3

[0054] A Pediococcus pentosaceus bacterial liquid is composed of the live bacterial liquid of Pediococcus pentosaceus ZJPY017 obtained in Example 1, and the specific preparation method is as follows:

[0055] The Pediococcus pentosaceus ZJPY017 obtained in Example 1 was inoculated into a liquid culture medium and fermented at 45°C. The fermentation culture was counted and the bacterial concentration was adjusted to 10 with sterile water. 5 CFU / mL to obtain a live bacterial liquid; the liquid culture medium consists of 20g / L protein powder, 30g / L glucose, 2.1g / L dipotassium hydrogen phosphate, 0.2g / L magnesium chloride, 0.3g / L zinc sulfate, and 0.2g / L calcium carbonate, and the pH value of the liquid culture medium is 5.5.

[0056] Example 4

[0057] A Pediococcus pentosaceus bacterial liquid is composed of the inactivated bacterial liquid of Pediococcus pentosaceus ZJPY017 obtained in Example 1, and the specific preparation method is as follows:

[0058] The Pediococcus pentosaceus ZJPY017 obtained in Example 1 was inoculated into a liquid culture medium and fermented at 45°C. The fermentation culture was counted and the bacterial concentration was adjusted to 10 with sterile water. 5 CFU / mL to obtain a live bacterial liquid; the liquid culture medium consists of 20g / L protein powder, 30g / L glucose, 2.1g / L dipotassium hydrogen phosphate, 0.2g / L magnesium chloride, 0.3g / L zinc sulfate, and 0.2g / L calcium carbonate, and the pH value of the liquid culture medium is 5.5.

[0059] The live bacterial solution was sterilized at 140°C for 20 minutes to obtain an inactivated bacterial solution.

[0060] Example 5

[0061] The difference from Example 4 is that the bacterial concentration is adjusted to 3×10 5 CFU / mL, and the rest are the same as Example 4.

[0062] Example 6

[0063] A Pediococcus pentosaceus bacterial solution, comprising 1 mL of the live bacterial solution obtained in Example 3 and 1 mL of the inactivated bacterial solution obtained in Example 5, wherein the bacterial concentration of the live bacterial solution obtained in Example 3 is 10 5 CFU / mL, the bacterial concentration of the inactivated bacterial solution obtained in Example 5 was 3×10 5 CFU / mL.

[0064] Example 7

[0065] Verification of lead excretion effect in lead-poisoned rats

[0066] 70 SD rats were randomly divided into a normal group (abbreviated as: NC), a model group (abbreviated as: Pb), a Pediococcus pentosaceus group obtained in Example 1 (abbreviated as: Pb+S1), a live bacterial solution of Pediococcus pentosaceus group in Example 3 (abbreviated as: Pb+S3), an inactivated bacterial solution of Pediococcus pentosaceus group in Example 4 (abbreviated as: Pb+S4), an inactivated bacterial solution of Pediococcus pentosaceus group in Example 5 (abbreviated as: Pb+S5) and a bacterial solution of Pediococcus pentosaceus group in Example 6 (abbreviated as: Pb+S6), 10 rats in each group, and the experiment was started after one week of adaptive feeding, and the experimental period was 8 weeks. The feeding methods of each group were:

[0067] NC: Lead-free normal drinking water was provided for rats to drink freely during 8 weeks. Starting from the second week, normal saline was gavaged once a day at a dose of 0.1 mL per 100 g body weight.

[0068] Pb: During the first week of adaptive feeding, lead-free drinking water was provided for rats to drink freely. From the second week, lead-free drinking water was replaced with lead water, which was prepared by dissolving lead acetate trihydrate in drinking water to a concentration of 600 mg / L, and mice were provided with free drinking. From the second week, normal saline was gavaged once a day, with a gavage dose of 0.1 mL per 100 g body weight.

[0069] Pb+S1: During the adaptive feeding, lead-free ordinary drinking water was provided for rats to drink freely. From the second week, lead-free ordinary drinking water was replaced with lead water, which was prepared by dissolving lead acetate trihydrate in drinking water to a concentration of 600 mg / L, and mice were provided with free drinking. In addition, from the second week, the Pediococcus pentosaceus obtained in Example 1 was gavaged once a day, and the gavage dose was 0.1 mL per 100 g body weight. The bacterial concentration of the Pediococcus pentosaceus obtained in Example 1 by gavage was 10 5 CFU / mL, bacterial concentration is 10 5 The preparation method of Pediococcus pentosaceus with a CFU / mL is as follows: the Pediococcus pentosaceus obtained in Example 1 is inoculated into MRS liquid culture medium, anaerobically cultured at 40°C for 30 hours, centrifuged to obtain the bacterial cells, and the bacterial cells are adjusted to a bacterial concentration of 10 with sterile water.5 CFU / mL.

[0070] Pb+S3: During one week of adaptive feeding, lead-free ordinary drinking water was provided for rats to drink freely. From the second week, lead-free ordinary drinking water was replaced with lead water, which was prepared by dissolving lead acetate trihydrate in drinking water to a concentration of 600 mg / L, and mice were provided with free drinking. In addition, from the second week, the live bacterial solution of Pediococcus pentosaceus obtained in Example 3 was gavaged once a day, and the gavage dose was 0.1 mL per 100 g body weight.

[0071] Pb+S4: Adaptive feeding: Within one week, lead-free ordinary drinking water was provided for rats to drink freely. From the second week, lead-free ordinary drinking water was replaced with lead water, which was prepared by dissolving lead acetate trihydrate in drinking water to a concentration of 600 mg / L, and mice were provided with free drinking. In addition, from the second week, the inactivated bacterial solution of Pediococcus pentosaceus of Example 4 was gavaged once a day, and the gavage dose was 0.1 mL per 100 g body weight.

[0072] Pb+S5: Adaptive feeding: Within one week, lead-free ordinary drinking water was provided for rats to drink freely. From the second week, lead-free ordinary drinking water was replaced with lead water, which was prepared by dissolving lead acetate trihydrate in drinking water to a concentration of 600 mg / L, and mice were provided with free drinking. In addition, from the second week, the inactivated bacterial solution of Pediococcus pentosaceus of Example 5 was gavaged once a day, and the gavage dose was 0.1 mL per 100 g body weight.

[0073] Pb+S6: Adaptive feeding: Within one week, lead-free ordinary drinking water was provided for rats to drink freely. From the second week, lead-free ordinary drinking water was replaced with lead water, which was prepared by dissolving lead acetate trihydrate in drinking water to a concentration of 600 mg / L, and mice were provided with free drinking. In addition, from the second week, the bacterial solution of Pediococcus pentosaceus of Example 6 was gavaged once a day, and the gavage dose was 0.1 mL per 100 g body weight.

[0074] The feces of each rat were collected separately at regular intervals every week. After the 8-week experiment, the rats were weighed after fasting and depriving of water for 18 hours, and blood was collected from the eyeballs after being stunned with ether. 3mL of whole blood was taken for blood lead detection, which was placed in a sodium heparin tube, inverted and mixed for testing. The method for determining the lead content in feces and blood is as follows: the feces and blood samples were transferred to a conical flask soaked in 20% nitric acid overnight, 10mL of concentrated nitric acid was added, and after being placed overnight, they were placed on an adjustable electric hot plate for digestion until the digestion solution was colorless and transparent or slightly yellowish. After cooling, the volume was fixed, and the lead content in the sample was determined using a flame atomic absorption spectrophotometer, and the final result was displayed as an average value.

[0075] The results of the lead content in feces are as follows: Figure 2 As shown by Figure 2It can be seen that the normal group contained almost no lead in the feces during the whole cycle. In other groups, when fed with lead water, the lead ion content in the feces increased significantly. Compared with the model group, each group of Pediococcus pentosaceus or Pediococcus pentosaceus bacterial liquid of the present invention can increase the lead content in the feces, and the live Pediococcus pentosaceus bacterial group and the inactivated bacterial liquid group have better lead removal effects than the Pediococcus pentosaceus group.

[0076] The results of the lead content in the blood are shown in Table 2. It can be seen from Table 2 that the normal group contains almost no lead in the blood during the entire cycle. Compared with the model group, each group of the Pediococcus pentosaceus ZJPY017 or the Pediococcus pentosaceus ZJPY017 bacterial solution of the present invention can reduce the lead content in the blood, and the live bacteria group and the inactivated bacterial solution group of the Pediococcus pentosaceus ZJPY017 have better lead removal effects than the Pediococcus pentosaceus ZJPY017 group.

[0077] Table 2 Results of lead content determination in blood

[0078] Group Blood lead content (μg / mL) NC 0 Pb 6.2 Pb+S1 5.4 Pb+S3 5.1 Pb+S4 4.6 Pb+S5 4.1 Pb+S6 3.6

[0079] Example 8

[0080] Improve sleep effect verification

[0081] (1) Prolonging the sleep time of mice induced by sodium pentobarbital

[0082] 70 SPF-grade ICR healthy male mice, aged 2-3 months and weighing 20-22 g, were selected. After one week of adaptive feeding, the mice were randomly divided into 7 groups, each with 10 mice, including a blank control group (abbreviated as NC), a positive group, a Pediococcus pentosaceus group obtained in Example 1 (abbreviated as Pb+S1, the bacterial concentration of the Pediococcus pentosaceus group obtained in Example 1 was 10 5 CFU / mL, the preparation method is as follows: the Pediococcus pentosaceus obtained in Example 1 is inoculated into MRS liquid culture medium, anaerobically cultured at 40°C for 30 hours, centrifuged to obtain the bacterial cells, and the bacterial cells are adjusted to a bacterial concentration of 10 with sterile water. 5CFU / mL), Example 3 Pentosaceae live bacterial liquid group (abbreviated as Pb+S3), Example 4 Pentosaceae inactivated bacterial liquid group (abbreviated as Pb+S4), Example 5 Pentosaceae inactivated bacterial liquid group (abbreviated as Pb+S5) and Example 6 Pentosaceae bacterial liquid group (abbreviated as Pb+S6), except for the NC group and the positive group, each group of mice was gavaged with the corresponding group of bacterial agents at an amount of 1 mL per 100 g body weight, once a day, for 15 consecutive days. The NC group of mice was gavaged with physiological saline at an amount of 1 mL per 100 g body weight, once a day, for 15 consecutive days. The positive group of mice was given rotundine tablets for consumption at an amount of 14 mg per 1 kg body weight, once a day, for 15 consecutive days. 15 minutes after the last administration, each group of mice was intraperitoneally injected with a sodium pentobarbital solution with a dose of 38 mg / kg determined by preliminary experiments, and the injection volume was 0.2 mL / 20 g. The disappearance of the righting reflex for more than 1 minute was used as the sleep judgment standard, and the extended pentobarbital sodium sleep time of each group of mice was recorded, and the final results were displayed as average values, as shown in Table 3. As can be seen from Table 3, the Pediococcus pentosaceus ZJPY017 obtained in Example 1 of the present invention, the live bacterial solution of Pediococcus pentosaceus ZJPY017 obtained in Example 3, the inactivated bacterial solution of Pediococcus pentosaceus ZJPY017 obtained in Example 4, the inactivated bacterial solution of Pediococcus pentosaceus ZJPY017 obtained in Example 5, and the Pediococcus pentosaceus ZJPY017 bacterial solution obtained in Example 6 can all significantly improve sleep time.

[0083] Table 3 Results of prolonged pentobarbital sodium sleep time in mice in each group

[0084] Group Sleep time(s) NC 1863.5 Positive group 3350.6 Pb+S1 3142.8 Pb+S3 3226.2 Pb+S4 3287.6 Pb+S5 3310.2 Pb+S6 3348.5

[0085] (2) Sodium barbiturate sleep latency test

[0086] 70 SPF-grade ICR healthy male mice, aged 2-3 months and weighing 20-22 g, were selected. After one week of adaptive feeding, the mice were randomly divided into 7 groups, each with 10 mice, including a blank control group (abbreviated as NC), a positive group, a Pediococcus pentosaceus group obtained in Example 1 (abbreviated as Pb+S1, the bacterial concentration of the Pediococcus pentosaceus group obtained in Example 1 was 10 5 CFU / mL, the preparation method is as follows: the Pediococcus pentosaceus obtained in Example 1 is inoculated into MRS liquid culture medium, anaerobically cultured at 40°C for 30 hours, centrifuged to obtain the bacterial cells, and the bacterial cells are adjusted to a bacterial concentration of 10 with sterile water. 5CFU / mL), Example 3 Pentosaceae live bacterial liquid group (abbreviated as Pb+S3), Example 4 Pentosaceae inactivated bacterial liquid group (abbreviated as Pb+S4), Example 5 Pentosaceae inactivated bacterial liquid group (abbreviated as Pb+S5) and Example 6 Pentosaceae bacterial liquid group (abbreviated as Pb+S6), except for the NC group and the positive group, each group of mice was gavaged with the corresponding group of bacterial agents at a volume of 1 mL per 100 g body weight, once a day, for 15 consecutive days. The NC group of mice was gavaged with physiological saline at a volume of 1 mL per 100 g body weight, once a day, for 15 consecutive days. The positive group of mice was given rotundine tablets at a volume of 14 mg per 1 kg body weight, once a day, for 15 consecutive days. 15 minutes after the last administration, each group of mice was intraperitoneally injected with sodium barbital at a dose of 230 mg / kg determined by preliminary experiments, and the injection volume was 0.2 mL / 20 g. The disappearance of righting reflex for more than 1 minute was used as the index of falling asleep, and the sleep latency of each group of mice was recorded. The final results were displayed as average values, and the results are shown in Table 4. As can be seen from Table 4, the Pediococcus pentosaceus ZJPY017 obtained in Example 1 of the present invention, the live bacterial solution of Pediococcus pentosaceus ZJPY017 obtained in Example 3, the inactivated bacterial solution of Pediococcus pentosaceus ZJPY017 obtained in Example 4, the inactivated bacterial solution of Pediococcus pentosaceus ZJPY017 obtained in Example 5, and the bacterial solution of Pediococcus pentosaceus ZJPY017 obtained in Example 6 can all significantly shorten the sleep latency.

[0087] Table 4 Results of shortening the sleep latency of mice in each group with sodium pentobarbital

[0088] Group Sleep latency(s) NC 2238.6 Positive group 1256.4 Pb+S1 1312.4 Pb+S3 1289.5 Pb+S4 1269.7 Pb+S5 1259.3 Pb+S6 1248.2

[0089] Example 9

[0090] Emotional regulation effect verification

[0091] A chronic unpredictable mild stimulation (CUMS) model was established to simulate the disease state of patients with depression. The specific modeling method is:

[0092] 70 7-week-old male C57BL / 6 mice were selected as experimental mice, and 60 of them were randomly selected to establish the CUMS mouse model. The method of establishing the CUMS model is: 8 stimuli, including empty cage for 17 hours, tilted cage at 45° for 17 hours, wet cage for 17 hours, food deprivation for 14 hours, night light for 12 hours, hot water swimming for 5 minutes, water deprivation for 16 hours, and tail clamping for 5 minutes, were randomly arranged in 28 days, 2 times a day, and the same stimulus did not appear continuously, so that the mice could not anticipate the occurrence of the stimulus. The results showed that the mice in the model group showed behavioral and physiological characteristics similar to depression, indicating that the model was successfully established in 28 days. The remaining 10 mice were used as the normal control group (abbreviated as NC), which were raised normally and not stimulated.

[0093] Starting from the 5th week, the 60 CUMS model mice that were successfully modeled were randomly divided into 6 groups, namely the model group (abbreviated as M), the Pediococcus pentosaceus group obtained in Example 1 (abbreviated as Pb+S1, the bacterial concentration of the Pediococcus pentosaceus group obtained in Example 1 was 10 5 CFU / mL, the preparation method is as follows: the Pediococcus pentosaceus obtained in Example 1 is inoculated into MRS liquid culture medium, anaerobically cultured at 40°C for 30 hours, centrifuged to obtain the bacterial cells, and the bacterial cells are adjusted to a bacterial concentration of 10 with sterile water. 5 CFU / mL), Example 3 Pentosaceae live bacterial liquid group (abbreviated as Pb+S3), Example 4 Pentosaceae inactivated bacterial liquid group (abbreviated as Pb+S4), Example 5 Pentosaceae inactivated bacterial liquid group (abbreviated as Pb+S5) and Example 6 Pentosaceae bacterial liquid group (abbreviated as Pb+S6), 10 mice in each group. The control group mice and the model group mice were fed normally without other treatments; the mice in the Pb+S1, Pb+S3, Pb+S4, Pb+S5 and Pb+S6 groups were gavaged with the corresponding group of bacterial agents once a day, and the gavage dose was 1 mL per 100 g body weight for 15 consecutive days. Then the following tests were performed:

[0094] (1) Open Field Test (OFT)

[0095] The experiment was carried out in a quiet environment. A total of 70 boxes with exactly the same specifications were prepared, and 70 mice were tested at the same time. Different animals of different groups were placed in the center of the bottom surface of different boxes, and video and timing were performed at the same time. Video recording was stopped after 6 minutes of observation, and the total moving distances of different mice were calculated according to the video recording results. The final value was reflected in the form of an average. The calculation results are shown in Table 5. Compared with the normal control group mice, the total moving distance of the model group CUMS mice was significantly shorter. However, the total moving distances of the mice in the Pb+S1, Pb+S3, Pb+S4, Pb+S5 and Pb+S6 groups were significantly improved, indicating that the pentosaceous Pediococcus ZJPY017 of the present invention can significantly improve the depression and depressive tendency of CUMS model mice.

[0096] Table 5 Open field test results

[0097] Group Total moving distance (m) NC 35.4 M 18.9 Pb+S1 25.8 Pb+S3 27.3 Pb+S4 28.6 Pb+S5 30.2 Pb+S6 33.7

[0098] (2) Tail suspension test (TST)

[0099] The tail of the mouse was fixed to the top of the experimental box using medical tape, and the behavior of the mouse within 5 minutes was recorded with a camera, and analyzed using Tail Suspension ScanTM (Clever Sys Inc, VA, USA) analysis software, and the time when the mouse limbs and trunk were completely immobile within 2-5 minutes was counted. The final value was embodied in the form of an average, and the calculation results are shown in Table 6. Compared with the normal control group, the model group mice showed a longer immobility time in the tail suspension test, while the immobility time of the mice in the Pb+S1, Pb+S3, Pb+S4, Pb+S5 and Pb+S6 groups was significantly shortened relative to the model group mice group, indicating that the present invention Pediococcus pentosaceus ZJPY017 can significantly improve the depression and depressive tendency of CUMS model mice.

[0100] Table 6 Tail suspension test results

[0101] Group Immobility time(s) NC 50 M 90 Pb+S1 79 Pb+S3 65 Pb+S4 62 Pb+S5 59 Pb+S6 54

[0102] Example 10

[0103] Verification of the effect of increasing bone density

[0104] The osteoporosis rat model was established by removing the ovaries. Specifically, 70 SD female (8-week-old) SPF rats in stable health were randomly divided into a blank control group (abbreviated as NC, also known as the sham operation group, 10 rats) and an ovariectomized group (60 rats) according to their body weight. The 60 rats in the ovariectomized group had their bilateral ovaries removed and then sutured. The blank control group (sham operation group) underwent corresponding operations except for the removal of bilateral ovaries, and disinfection was performed after the operation. On the 6th day of modeling, the ovariectomized rats were randomly divided into 6 groups according to their body weight, specifically:

[0105] Model group (10 rats): 1 mL of sterile water was gavaged per 100 g body weight, once a day;

[0106] The Pediococcus pentosaceus group obtained in Example 1 (abbreviated as Pb+S1, the bacterial concentration of the Pediococcus pentosaceus group obtained in Example 1 is 10 5 CFU / mL, the preparation method is as follows: the Pediococcus pentosaceus obtained in Example 1 is inoculated into MRS liquid culture medium, anaerobically cultured at 40°C for 30 hours, centrifuged to obtain the bacterial cells, and the bacterial cells are adjusted to a bacterial concentration of 10 with sterile water. 5 CFU / mL): 1 mL was administered intragastrically for every 100 g of body weight, and the intragastrical concentration was 10 5 CFU / mL of Pediococcus pentosaceus obtained in Example 1, once a day;

[0107] Example 3 Pediococcus pentosaceus live bacterial liquid group (abbreviated as Pb+S3): 1 mL of the Pediococcus pentosaceus live bacterial liquid obtained in Example 3 was gavaged per 100 g body weight, once a day;

[0108] Example 4 Pediococcus pentosaceus inactivated bacterial solution group (abbreviated as Pb+S4): 1 mL of the Pediococcus pentosaceus inactivated bacterial solution obtained in Example 4 was intragastrically administered per 100 g body weight, once a day;

[0109] Example 5 Pediococcus pentosaceus inactivated bacterial solution group (abbreviated as Pb+S5): 1 mL of the Pediococcus pentosaceus inactivated bacterial solution obtained in Example 5 was intragastrically administered per 100 g body weight, once a day;

[0110] Example 6 Pediococcus pentosaceus bacterial solution group (abbreviated as Pb+S6): 1 mL of the Pediococcus pentosaceus bacterial solution obtained in Example 6 was gavaged per 100 g body weight, once a day.

[0111] Each group of mice was gavaged continuously for 6 weeks. After the end, the rats were anesthetized with 1% sodium pentobarbital and fixed on a small animal in vivo tomography imaging device with a resolution of 20 μm. After the scanning, image reconstruction was performed and the bone density of each group of rats was analyzed using Inveon Research Workplace 2.2 software. The results were expressed as average values, as shown in Table 7. This shows that the Pediococcus pentosaceus ZJPY017 of the present invention has the effect of significantly improving bone density.

[0112] Table 7 Bone density measurement results

[0113] Group <![CDATA[Bone density (μg / cm 3 )]]> NC 0.359 Model Group 0.274 Pb+S1 0.308 Pb+S3 0.312 Pb+S4 0.323 Pb+S5 0.336 Pb+S6 0.348

[0114] Embodiment 11

[0115] Promoting height growth effect verification

[0116] 60 newly born Kunming rats with a body length difference of no more than 0.5 cm and a weight difference of no more than 1 g were purchased and randomly divided into 6 groups: a blank control group, a group of Pediococcus pentosaceus obtained in Example 1 (abbreviated as Pb+S1, the bacterial concentration of the group of Pediococcus pentosaceus obtained in Example 1 was 10 5 CFU / mL, the preparation method is as follows: the Pediococcus pentosaceus obtained in Example 1 is inoculated into MRS liquid culture medium, anaerobically cultured at 40°C for 30 hours, centrifuged to obtain the bacterial cells, and the bacterial cells are adjusted to a bacterial concentration of 10 with sterile water. 5CFU / mL), Example 3 Pentosaceae live bacterial liquid group (abbreviated as Pb+S3), Example 4 Pentosaceae inactivated bacterial liquid group (abbreviated as Pb+S4), Example 5 Pentosaceae inactivated bacterial liquid group (abbreviated as Pb+S5) and Example 6 Pentosaceae bacterial liquid group (abbreviated as Pb+S6)), 10 mice in each group. The 6 groups of mice were fed with the same amount of milk at the same time every day. Except for the blank control group, the remaining groups were gavaged with 1mL of the corresponding group of bacterial agents per 10g body weight, once a day, and the blank control group was gavaged with sterile water per 10g body weight, once a day, for 30 consecutive days. During the entire experiment, the body length of the offspring was measured at 0d, 10d, 20d and 30d, and the results were expressed as averages, as shown in Table 8. The results show that the Pentosaceae ZJPY017 of the present invention has a significant effect on promoting height growth.

[0117] Table 8 Measurement results of body length changes of offspring mice

[0118] Group 0d(cm) 10d(cm) 20d(cm) 30d(cm) NC 2.52 4.22 5.67 6.16 Pb+S1 2.53 4.62 6.23 7.45 Pb+S3 2.50 4.71 6.87 7.79 Pb+S4 2.51 4.82 6.98 7.96 Pb+S5 2.52 4.98 7.12 8.35 Pb+S6 2.53 5.34 7.86 8.98

[0119] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Pediococcus pentosaceus, characterized in that The Pediococcus pentosaceus is Pediococcus pentosaceus ZJPY017, which is classified and named as Pediococcus pentosaceus and is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration with a deposit number of CGMCC No.1.60177.

2. A bacterial liquid of Pediococcus pentosaceus, characterized in that: The bacterial solution of Pediococcus pentosaceus comprises at least one of a live bacterial solution and an inactivated bacterial solution of Pediococcus pentosaceus according to claim 1.

3. The Pediococcus pentosaceus bacterial liquid according to claim 2, characterized in that The preparation method of the live bacterial liquid comprises the following steps: inoculating the Pediococcus pentosaceus described in claim 1 into a liquid culture medium, fermenting and culturing at 40° C.-50° C., counting the fermentation culture liquid, and adjusting the bacterial concentration to 10 with sterile water. 4 CFU / mL-10 6 CFU / mL, and obtain live bacterial solution.

4. The Pediococcus pentosaceus bacterial liquid according to claim 2, characterized in that The preparation method of the inactivated bacterial liquid comprises the following steps: inactivating the active bacterial liquid to obtain the inactivated bacterial liquid.

5. The Pediococcus pentosaceus bacterial liquid according to claim 2, characterized in that When the Pediococcus pentosaceus bacterial solution comprises a live bacterial solution and an inactivated bacterial solution of the Pediococcus pentosaceus according to claim 1, the ratio of the number of bacteria in the live bacterial solution to the number of bacteria in the inactivated bacterial solution is 1:2-4.

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