Lactobacillus casei C7-2 and application thereof in preparation of composition for conditioning / improving thyroiditis and brain function activity and protecting neurons

Abstract

The invention discloses a lactobacillus casei C7-2 and application thereof in preparation of a composition for conditioning / improving thyroiditis and brain function activity and protecting neurons. The lactobacillus casei C7-2 has the Latin literature name of Lacticaseibacillus casei, the preservation number of the lactobacillus casei C7-2 is GDMCC 63609, the preservation time of the lactobacillus casei C7-2 is June 30, 2023, and the preservation unit of the lactobacillus casei C7-2 is Guangdong Province Microbial Culture Collection Center. Compared with the prior art, the lactobacillus casei C7-2 has the advantages that the motor function and cognitive function of mice are obviously improved, and the Abeta42 level of hippocampus and cerebral cortex can be effectively reduced. In addition, the composition also can reduce the level of inflammatory factors to a certain extent, and condition / improve further deterioration of neurodegeneration and cognitive impairment. In addition, the lactobacillus casei C7-2 disclosed by the invention also has a relatively good conditioning / improving effect on thyroiditis.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a Lactobacillus casei C7-2 and an application thereof in preparing a composition for regulating / improving thyroiditis, brain function activity and protecting neurons. Background Art

[0002] Lactobacillus casei, Latin name: Lacticaseibacillus casei It is a Gram-positive, non-motile, non-spore-forming, short-rod-shaped, pleomorphic bacillus. It heterofermentes lactose and has numerous beneficial effects, including regulating intestinal flora balance, promoting digestion and absorption, enhancing immunity, alleviating lactose intolerance, and improving cardiovascular health. In the food industry, it is commonly used as a starter and auxiliary starter for dairy products. It is also manufactured into a variety of products in the health supplement industry, with a wide range of applications.

[0003] CN116948916A discloses a Lactobacillus casei strain and its applications. This strain has excellent safety and does not exhibit hemolysis on Columbia blood agar plates. It is sensitive to a variety of common antibiotics and can tolerate artificial gastric and intestinal fluids well. This strain has a strong adhesion ability to human colon cancer cells. When co-cultured with human colon cancer cells HCT-116, its fermentation supernatant exhibits significant cancer cell growth inhibition and apoptosis-promoting activity. Furthermore, this strain has a strong inhibitory effect on a range of pathogenic bacteria, including Escherichia coli and Pseudomonas aeruginosa. It has a particularly strong ability to produce acetic acid and lactic acid, has a high cholesterol degradation level, and exhibits a strong antioxidant effect.

[0004] CN114874952A discloses a Lactobacillus casei HJD strain and its applications. This Lactobacillus casei HJD strain exhibits broad and strong antibacterial activity against intestinal indicator bacteria such as Salmonella choleraesuis and Escherichia coli. It exhibits good tolerance in a low pH environment and in the presence of 0.30% bile salts, exhibits strong adhesion to Caco-2 cells, and carries no plasmid-encoded, transferable antibiotic resistance genes. In safety testing, the HJD strain did not produce α- or β-hemolysis on blood agar plates. In oral toxicity testing in mice, no adverse reactions were observed after gavage. Furthermore, mice gavaged with the HJD strain showed significant weight gain compared to the control group. In summary, Lactobacillus casei HJD is a potential candidate probiotic strain with the potential to enhance animal growth and immunity. Summary of the Invention

[0005] The present invention provides a Lactobacillus casei C7-2 with the functions of conditioning / improving thyroiditis, brain function activity and protecting neurons. Lacticaseibacillus casei; Deposit number GDMCC 63609, deposit date June 30, 2023, deposit unit: Guangdong Microbiological Culture Collection Center (GDMCC), address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0006] Special note, Lactobacillus casei, Latin name Lactibacillus casei , was renamed Lactobacillus casei in 2022, the Latin name Lacticaseibacillus casei .

[0007] The present invention also discloses the application of Lactobacillus casei C7-2 in regulating / improving thyroiditis, specifically, the application of Lactobacillus casei C7-2 in preparing a composition for regulating / improving thyroiditis.

[0008] The present invention also discloses the use of Lactobacillus casei C7-2 in regulating / improving brain function activity. Specifically, Lactobacillus casei C7-2 is used to prepare a composition for regulating / improving Alzheimer's disease, or Lactobacillus casei C7-2 is used to prepare a composition for promoting brain health. The present invention also discloses the use of Lactobacillus casei C7-2 in protecting neurons. Specifically, Lactobacillus casei C7-2 is used to prepare a composition for regulating / improving Parkinson's disease.

[0009] More specifically, the preparation method of the composition for regulating / improving thyroiditis, regulating / improving Alzheimer's disease, or regulating / improving Parkinson's disease is as follows: dissolving chitosan in an acetic acid aqueous solution; dissolving gelatin in water; mixing the two evenly, adding Lactobacillus casei C7-2 and cinnamaldehyde, and adjusting the pH to 5-6; stirring and mixing at 30-40° C. for 6-12 hours, and then cooling to room temperature to obtain a microcoagulation system; heating again to 30-45° C., adding a cross-linking agent, and stirring and mixing for 1-3 hours; cooling to room temperature, filtering, washing, and freeze-drying to obtain the composition; the bacterial content of Lactobacillus casei C7-2 is 1×10 9 -3×10 10 CFU / g.

[0010] Further preferably, the preparation method of the composition for conditioning / improving thyroiditis or conditioning / improving Alzheimer's disease or promoting brain health or conditioning / improving Parkinson's disease is as follows: dissolving 3-8 g of chitosan in 300-1000 mL of 1-5 wt% acetic acid aqueous solution; dissolving 5-15 g of gelatin in 300-1000 mL of water; mixing the two evenly, adding 5-15 g of Lactobacillus casei C7-2 and 0.1-0.3 g of cinnamaldehyde, and adjusting the pH value to 5-6 with sodium hydroxide aqueous solution; stirring and mixing at 30-40 ° C for 6-12 hours, and then cooling to room temperature to obtain a microcoagulation system; heating again to 30-45 ° C, adding 0.5-3 g of a cross-linking agent, and stirring and mixing for 1-3 hours; cooling to room temperature, filtering, washing, and freeze-drying to obtain; the bacterial content of Lactobacillus casei C7-2 is 1×10 9 -3×10 10 CFU / g.

[0011] Preferably, the cross-linking agent is at least one of transglutaminase, casein, lectin, and tannic acid.

[0012] Most preferably, the preparation method of the composition for regulating / improving thyroiditis, regulating / improving Alzheimer's disease, promoting brain health, or regulating / improving Parkinson's disease is as follows: dissolving 3-8 g of chitosan in 300-1000 mL of 1-3 wt% acetic acid aqueous solution; dissolving 5-15 g of gelatin in 300-1000 mL of water; mixing the two evenly, adding 5-15 g of Lactobacillus casei C7-2 and 0.1-0.3 g of cinnamaldehyde, and adjusting the pH value to 5-6 with sodium hydroxide aqueous solution; stirring and mixing at 30-40° C. for 6-12 hours, and then cooling to room temperature to obtain a microcoagulation system; heating again to 30-45° C., adding 0.25 g-1.5 g of casein, and stirring and mixing for 0.5-1.5 hours; then adding 1-3 mL 10-30wt% tannic acid aqueous solution, continue stirring at 30-45℃ for 0.5-1.5h; cool to room temperature, filter, wash, and freeze-dry to obtain; the bacterial content of Lactobacillus casei C7-2 is 1×10 9 -3×10 10 CFU / g.

[0013] Beneficial effects of the present invention: Compared to the prior art Lactobacillus casei, the present invention's Lactobacillus casei C7-2 significantly improves motor and cognitive function in mice and effectively reduces Aβ42 levels in the hippocampus and cerebral cortex. Furthermore, the present invention's Lactobacillus casei C7-2 can, to a certain extent, reduce levels of inflammatory factors (IL-6, IL-1β, and TNF-ɑ), modulating / ameliorating further deterioration of neurodegeneration and cognitive impairment. Furthermore, the present invention's Lactobacillus casei C7-2 has a significant modulating / ameliorating effect on thyroiditis.

[0014] The Lactobacillus casei C7-2 of the present invention has good application prospects in regulating / improving thyroiditis, brain function activity and protecting neurons. DETAILED DESCRIPTION

[0015] The source of Lactobacillus casei C7-2 of the present invention: Collection location: Pickled vegetables sold at Wushan Market, Tianhe District, Guangzhou City, Guangdong Province Separation and screening method: The collected pickles were diluted with 0.85wt% sterile NaCl aqueous solution for 10 4 times and mix thoroughly; then, draw 0.2 mL of the dilution and apply it on MRS agar at a concentration of 1% (v / v); select litmus as an indicator and incubate at 30°C for 48 hours; select the colonies surrounded by red circles and re-streak them on the MRS agar plate to obtain a pure culture, namely, the casei C7-2 of the present invention. The 16S rRNA sequence identification results of the casei C7-2 of the present invention show that it is Lacticaseibacillus casei The complete genome of Lactobacillus casei has been uploaded to the GENBANK database, with the authorized accession number being JWZV00000000.

[0016] Culture method: Lactobacillus casei C7-2 was inoculated into MRS liquid culture medium at a culture temperature of 37°C.

[0017] MRS liquid medium: 10 g peptone, 5 g beef extract, 4 g yeast extract powder, 20 g glucose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 2 g diammonium hydrogen citrate, 5 g anhydrous sodium acetate, 0.054 g MnSO4, 0.2 g MgSO4, and 1000 mL distilled water. Stir well, adjust the pH to 6.3, and sterilize at 121°C for 20 min.

[0018] The taxonomic characteristics of the Lactobacillus casei C7-2 of the present invention are: Gram-positive bacteria, short rod-shaped, without spores and without flagella.

[0019] Table 1 Biochemical characteristics test of Lactobacillus casei C7-2 Item Result Catalase test - Sugar alcohol fermentation test + Fructose + Glucose + Lactose + Mannitol + Sorbitol + Sucrose + Maltose + Acid and gas production using glucose + Starch hydrolysis + V-P test + <![CDATA[H2S试验]]> - Indole test + Gelatin liquefaction test + Litmus milk test + Arginine hydrolysis - Growth at 16℃ + Growth at 70℃ - Growth at pH 4.5 + Growth at pH 9.0 + Growth at 4% sodium chloride + Growth at 6.5% sodium chloride + Growth at 10% sodium chloride - Growth at 15% sodium chloride - Note: - represents negative, + represents positive. Example 1

[0020] Effects of Lactobacillus casei C7-2 on the behavior of Alzheimer's disease mice (1) Establishment of Alzheimer's disease mouse model The mice used in the animal experiment were purchased from the Guangdong Medical Laboratory Animal Center. Forty 8-week-old male C57BL / 6 mice weighing 20-25 g were randomly divided into four groups (blank control group, model control group, live bacteria group, and inactivated bacteria group). Among them, the model control group, live bacteria group, and inactivated bacteria group were injected with streptozotocin into the bilateral ventricles of the rats (coordinates: 0.8 mm behind the bregma, 2.2 mm left and right on the sagittal suture, and 4.5 mm vertically) on the 1st day and the 3rd day, with 1.5 mg / kg of streptozotocin injected into each side. The Alzheimer's disease (AD) animal model with impaired cerebral energy metabolism and oxidative stress injury was prepared, and the impaired spatial learning and memory ability of the animals and the oxidative stress injury in the hippocampus of the brain were used as the basis for judging the success of the modeling. The blank control group was injected with artificial cerebrospinal fluid [NaCl (147 mmol / L) + KCl (2.9 mmol / L) + MgCl2 (21.6 mmol / L) + CaCl2 (21.7 mmol / L) + glucose (2.2 mmol / L)] into the bilateral ventricles of the rats, with an injection dose of 1.5 mg / kg. After the modeling was completed, the live bacteria group was given 1 x 10 9 CFU of Lactobacillus casei C7-2 live bacteria, with a gavage dose of 0.1 mL / 10 g, and the inactivated bacteria group was given an equal amount of inactivated bacteria suspension daily. The blank control group and the model control group were given an equal volume of normal saline, and the gavage was continued for 4 weeks.

[0021] (2) Morris water maze (MWM) behavior test The Morris water maze test lasted for 7 days and was conducted in a quiet environment. On the 1st day, it was the training adaptation stage, and the mice were given platform practice during this stage. On the 2nd-6th day, the swimming time of the mice from entering the water to finding the platform was used as the observation index, which was called escape latency. If the mouse did not find the platform within 60 s, it was recorded as 60 s. On the 7th morning, the platform was removed, and the time the mouse spent in the fourth quadrant where the platform was located and the number of times it crossed the platform location were measured.

[0022] Table 2 Comparison of escape latency (s) of mice in each group in the positioning navigation experiment Days Blank control group Model control group Live bacteria group Inactivated bacteria group 2 40.1±3.2 61.4±2.4 55.9±3.6 56.1±2.9 3 39.5±2.8 58.3±3.3 51.8±2.9 51.2±1.6 4 38.2±2.9 56.1±2.8 43.6±1.9 43.4±2.1 5 35.7±3.1 55.6±3.7 40.8±2.4 39.2±2.3 6 32.6±2.7 54.4±4.2 38.6±3.1 36.9±4.2 Table 3 Comparison of platform quadrant swimming time of mice in each group in the positioning navigation experiment Group Blank control group Model control group Live bacteria group Inactivated bacteria group Cross-platform times 4.81±0.76 1.46±0.24 3.56±0.31 3.71±0.28 Target quadrant residence time (s) 25.85±7.30 12.84±6.16 25.57±6.19 26.37±8.17 The Morris Water Maze (MWM) behavioral test is a standard experimental method for assessing cognitive function, particularly spatial memory and learning abilities, in animal models of Alzheimer's disease (AD) and humans. In AD research, the MWM can reveal cognitive deficits in model animals during the course of the disease, which is important for understanding the pathological mechanisms of AD, discovering new therapeutic targets, and evaluating the efficacy of potential therapeutic / conditioning / improvement drugs. By observing how efficiently animals find hidden platforms in the maze, researchers can quantify their spatial memory abilities, indirectly reflecting the functional status of brain regions such as the hippocampus, which are the first areas affected by AD. Therefore, the MWM is widely used in AD research to declare (indicate) the presence and extent of cognitive impairment, providing a strong experimental basis for the diagnosis and treatment / conditioning / improvement of the disease.

[0023] The live bacteria group and the inactivated bacteria group of Lactobacillus casei C7-2 of the present invention can regulate / improve the motor function and cognitive function of mice.

[0024] Example 2 Effect of Lactobacillus casei C7-2 on Aβ42 Concentration in the Brains of Alzheimer's Disease Mice After the experimental period of intragastric administration of the bacterial solution, the mice were immediately sacrificed by cervical dislocation. The hippocampus and cerebral cortex of the mice were isolated and stored at -80°C until further use. Aβ42 levels in the samples were measured using an Aβ42 enzyme-linked immunosorbent assay (ELISA) kit.

[0025] Table 4 Aβ42 levels in the hippocampus and cerebral cortex of mice in different groups Group Blank control group Model control group Live bacteria group Inactivated bacteria group Hippocampus (ng / L) 40.5±0.3 78.6±0.2 45.7±0.4 46.1±0.3 Cerebral cortex (ng / L) 42.3±0.2 64.5±0.3 36.1±0.2 36.7±0.3 The hippocampus and cerebral cortex are key brain areas closely related to Alzheimer's disease, among which the abnormal deposition of Aβ42 is one of the main pathological characteristics of the disease and is closely related to cognitive decline and disease progression.

[0026] It can be seen from the above table that both the live bacteria group and the inactivated bacteria group of the present invention can effectively reduce the Aβ42 levels in the hippocampus and cerebral cortex.

[0027] Example 3 Effect of Lactobacillus casei C7-2 on neuronal inflammatory response in Alzheimer's disease mice (pg / mL) The blood (from Example 2) was coagulated at room temperature for 2 h, centrifuged at 3000 rpm for 5 min at 4°C to separate the serum, and stored in a refrigerator at -2°C. The levels of inflammatory factors in the serum of mice in different groups were determined according to the ELISA kit instructions.

[0028] Table 5 Levels of inflammatory factors in the serum of mice in different groups Group Blank control group Model control group Live bacteria group Inactivated bacteria group IL-6 (pg / mL) 27.84±5.48 82.17±8.38 44.92±7.29 46.18±5.64 IL-1β (pg / mL) 15.73±2.84 29.55±3.62 21.72±4.28 22.32±3.16 TNF-ɑ (pg / mL) 53.72±9.24 114.92±10.11 74.24±10.36 75.39±9.82 Neuronal inflammatory response plays a crucial role in the pathogenesis of Alzheimer's disease. It promotes the pathological process of the disease by releasing inflammatory mediators, damaging neurons, affecting amyloid protein metabolism, promoting tau protein phosphorylation, exacerbating oxidative stress, and destroying the blood-brain barrier, leading to the gradual deterioration of neurodegeneration and cognitive dysfunction.

[0029] As can be seen from the above table, the live bacteria group and the inactivated bacteria group of Lactobacillus casei C7-2 of the present invention can reduce the levels of inflammatory factors (IL-6, IL-1β, TNF-ɑ) to a certain extent, and regulate / improve the further deterioration of neurodegeneration and cognitive dysfunction.

[0030] Example 4 Effects of Lactobacillus casei C7-2 on Hormone Levels in Hypothyroidism Model Mice 6-8 week old SPF mice (16-18 g, 24 males) were randomly divided into the following 4 groups: blank control group, model control group, live bacteria group and inactivated bacteria group. The blank control group received an intramuscular injection of 50 μL of Ad-β-gal (diluted to 1×10 9 pfu / mL), and the other groups were given 50 μL of Ad-TSHR-289 (diluted to 1×10 9 pfu / mL), each mouse was immunized 3 times, with an interval of 2 weeks between each immunization. Starting from the 5th week after model establishment, the live bacteria group was given 1×10 9 Live Lactobacillus casei C7-2 bacteria (CFU) were gavaged orally at a dose of 0.1 mL / 10 g. The inactivated bacteria group received an equal volume of a resuspension of the inactivated bacteria, while the blank and model control groups received an equal volume of normal saline. All groups were gavaged continuously for 30 days. After administration, blood was collected from the eyeballs and the animals were sacrificed. After stratification, serum was collected by centrifugation and stored at 2°C. Thyrotropin (TSH), T3, and T4 levels were measured according to the ELISA kit instructions.

[0031] Table 6 Levels of triiodothyronine T3, thyroxine T4 and thyroid stimulating hormone TSH in serum of mice in different groups Group Blank control group Model control group Live bacteria group Inactivated bacteria group Triiodothyronine T3 (ng / mL) 1.53±0.17 0.91±0.24 1.38±0.14 1.41±0.12 Thyroxine T4 (ng / mL) 58.2±1.9 21.5±1.1 57.4±1.4 58.2±1.5 Thyrotropin TSH (IU / mL) 2.1±0.22 14.8±0.24 6.3±0.32 6.1±0.47 During different stages of thyroiditis, levels of triiodothyronine (T3), thyroxine (T4), and thyroid-stimulating hormone (TSH) vary. In the thyrotoxic stage, such as in the early stages of subacute thyroiditis, destruction of thyroid follicles leads to the release of large amounts of T3 and T4 into the bloodstream, causing elevated levels. This negative feedback loop lowers TSH. In the hypothyroid stage, such as in the late stages of Hashimoto's thyroiditis, damage to thyroid tissue leads to decreased synthesis and secretion of T3 and T4, resulting in decreased T3 and T4 and elevated TSH. During the normal function stage, all three levels remain within normal ranges.

[0032] These indicators play an important role in the diagnosis of thyroiditis. By measuring them, the functional status of patients with thyroiditis can be assessed. Abnormal combinations of T3, T4, and TSH can indicate conditions such as hyperthyroidism and hypothyroidism. The "dissociation phenomenon" of early subacute thyroiditis, combined with symptoms, can aid in diagnosis. In Hashimoto's thyroiditis, measuring these indicators and autoantibodies can aid in diagnosis and predict disease progression.

[0033] As can be seen from the above table, both the live bacteria group and the inactivated bacteria group of Lactobacillus casei C7-2 of the present invention have a good conditioning / improving effect on thyroiditis.

[0034] Application Example 1 A preparation method of a Lactobacillus casei bacterial agent for conditioning / improving thyroiditis and Alzheimer's disease is as follows: 5 g of chitosan was dissolved in 500 mL of a 2 wt% aqueous acetic acid solution; 10 g of gelatin was dissolved in 500 mL of deionized water; the two were mixed evenly, 10 g of Lactobacillus casei C7-2 and 0.2 g of cinnamaldehyde were added, and the pH value was adjusted to 5.5 by dissolving with 0.1 mol / L sodium hydroxide water; the mixture was mixed at 200 rpm and 35°C for 8 h, and then cooled to room temperature to obtain a microcoagulation system; the temperature was raised to 40°C again, 1 g of transglutaminase was added, and the mixture was stirred at 200 rpm for 2 h; the mixture was cooled to room temperature, filtered, washed with deionized water, and freeze-dried to obtain a Lactobacillus casei agent for conditioning / improving thyroiditis and Alzheimer's disease.

[0035] The bacterial content of Lactobacillus casei C7-2 is 10 10 CFU / g.

[0036] Application Example 2 A preparation method of a Lactobacillus casei bacterial agent for conditioning / improving thyroiditis and Alzheimer's disease is as follows: 5 g of chitosan was dissolved in 500 mL of a 2 wt% aqueous acetic acid solution; 10 g of gelatin was dissolved in 500 mL of deionized water; the two were mixed evenly, 10 g of Lactobacillus casei C7-2 and 0.2 g of cinnamaldehyde were added, and the pH value was adjusted to 5.5 by dissolving with 0.1 mol / L sodium hydroxide water; the mixture was mixed at 200 rpm and 35°C for 8 h, and then cooled to room temperature to obtain a microcoagulation system; the temperature was raised to 40°C again, 1 g of casein was added, and the mixture was stirred at 200 rpm for 2 h; the mixture was cooled to room temperature, filtered, washed with deionized water, and freeze-dried to obtain a Lactobacillus casei agent for conditioning / improving thyroiditis and Alzheimer's disease.

[0037] The bacterial content of Lactobacillus casei C7-2 is 10 10 CFU / g.

[0038] Application Example 3 A preparation method of a Lactobacillus casei bacterial agent for conditioning / improving thyroiditis and Alzheimer's disease is as follows: 5 g of chitosan was dissolved in 500 mL of a 2 wt% aqueous acetic acid solution; 10 g of gelatin was dissolved in 500 mL of deionized water; the two were mixed evenly, 10 g of Lactobacillus casei C7-2 and 0.2 g of cinnamaldehyde were added, and the pH value was adjusted to 5.5 by dissolving with 0.1 mol / L sodium hydroxide water; the mixture was mixed at 200 rpm and 35°C for 8 h, and then cooled to room temperature to obtain a micro-coagulation system; the temperature was raised to 40°C again, 1 g of lectin was added, and the mixture was stirred at 200 rpm for 2 h; the mixture was cooled to room temperature, filtered, washed with deionized water, and freeze-dried to obtain a Lactobacillus casei agent for conditioning / improving thyroiditis and Alzheimer's disease.

[0039] The bacterial content of Lactobacillus casei C7-2 is 10 10 CFU / g.

[0040] Application Example 4 A preparation method of a Lactobacillus casei bacterial agent for conditioning / improving thyroiditis and Alzheimer's disease is as follows: 5 g of chitosan was dissolved in 500 mL of a 2 wt% aqueous acetic acid solution; 10 g of gelatin was dissolved in 500 mL of deionized water; the two were mixed evenly, 10 g of Lactobacillus casei C7-2 and 0.2 g of cinnamaldehyde were added, and the pH value was adjusted to 5.5 by dissolving with 0.1 mol / L sodium hydroxide solution; the mixture was mixed at 200 rpm and 35°C for 8 h, and then cooled to room temperature to obtain a microcoagulation system; the temperature was raised to 40°C again, and then 4 mL of a 25 wt% aqueous tannic acid solution was added dropwise, and the mixture was stirred at 200 rpm and 40°C for 2 h; the mixture was cooled to room temperature, filtered, washed with deionized water, and freeze-dried to obtain a Lactobacillus casei agent for conditioning / improving thyroiditis and Alzheimer's disease.

[0041] The bacterial content of Lactobacillus casei C7-2 is 10 10 CFU / g.

[0042] Application Example 5 A preparation method of a Lactobacillus casei bacterial agent for conditioning / improving thyroiditis and Alzheimer's disease is as follows: 5 g of chitosan was dissolved in 500 mL of a 2 wt% aqueous acetic acid solution; 10 g of gelatin was dissolved in 500 mL of deionized water; the two were mixed evenly, 10 g of Lactobacillus casei C7-2 and 0.2 g of cinnamaldehyde were added, and the pH value was adjusted to 5.5 with 0.1 mol / L sodium hydroxide solution; the mixture was mixed at 200 rpm and 35°C for 8 h, and then cooled to room temperature to obtain a microcoagulation system; the temperature was raised to 40°C again, 0.5 g of casein was added, and the mixture was stirred at 200 rpm for 1 h; then 2 mL of a 25 wt% aqueous tannic acid solution was added dropwise, and stirring was continued at 200 rpm and 40°C for 1 h; the mixture was cooled to room temperature, filtered, washed with deionized water, and freeze-dried to obtain a Lactobacillus casei agent for conditioning / improving thyroiditis and Alzheimer's disease.

[0043] The bacterial content of Lactobacillus casei C7-2 is 10 10 CFU / g.

[0044] Application Example 6 A preparation method of a Lactobacillus casei bacterial agent for conditioning / improving thyroiditis and Alzheimer's disease is as follows: 5 g of chitosan was dissolved in 500 mL of a 2 wt% aqueous acetic acid solution; 10 g of gelatin was dissolved in 500 mL of deionized water; the two were mixed evenly, 10 g of Lactobacillus casei C7-2 and 0.2 g of cinnamaldehyde were added, and the pH value was adjusted to 5.5 with 0.1 mol / L sodium hydroxide solution; the mixture was mixed at 200 rpm and 35°C for 8 h, and then cooled to room temperature to obtain a microcoagulation system; the temperature was raised to 40°C again, 0.5 g of transglutaminase was added, and the mixture was stirred at 200 rpm for 1 h; then 2 mL of a 25 wt% aqueous tannic acid solution was added dropwise, and stirring was continued at 200 rpm and 40°C for 1 h; the mixture was cooled to room temperature, filtered, washed with deionized water, and freeze-dried to obtain a Lactobacillus casei agent for conditioning / improving thyroiditis and Alzheimer's disease.

[0045] The bacterial content of Lactobacillus casei C7-2 is 10 10 CFU / g.

[0046] Comparative Example 1 A preparation method of a Lactobacillus casei bacterial agent for conditioning / improving thyroiditis and Alzheimer's disease is as follows: 5 g of chitosan was dissolved in 500 mL of a 2 wt% aqueous acetic acid solution; 10 g of gelatin was dissolved in 500 mL of deionized water; the two were mixed evenly, 10 g of Lactobacillus casei C7-2 and 0.2 g of cinnamaldehyde were added, and the pH value was adjusted to 5.5 by dissolving with 0.1 mol / L sodium hydroxide solution; the mixture was mixed at 200 rpm and 35°C for 8 h, and then cooled to room temperature to obtain a micro-coagulation system; the temperature was raised to 40°C again, and then 4 mL of a 25 wt% aqueous glutaraldehyde solution was added dropwise, and the mixture was stirred at 200 rpm and 40°C for 2 h; the mixture was cooled to room temperature, filtered, washed with deionized water, and freeze-dried to obtain a Lactobacillus casei agent for conditioning / improving thyroiditis and Alzheimer's disease.

[0047] The bacterial content of Lactobacillus casei C7-2 is 10 10 CFU / g.

[0048] Comparative Example 2 The preparation method of a Lactobacillus casei agent for regulating / improving thyroiditis and Alzheimer's disease is basically the same as that of Comparative Example 1, except that the dropwise addition of 4 mL of a 25 wt % glutaraldehyde aqueous solution is replaced by the addition of 200 mL of a 0.5 wt % genipin aqueous solution.

[0049] Some raw material parameters and sources in the application examples are as follows: Chitosan has a molecular weight of about 100,000 and a deacetylation degree of 90%.

[0050] Transglutaminase, with an optimum pH of 6-7 and an optimum temperature of 50-55°C, was obtained from Taixing Dongsheng Biotechnology Co., Ltd.

[0051] Other raw materials not mentioned are commercially available medical grade.

[0052] The relevant tests were conducted on the Lactobacillus casei bacterial agent of the application example and the comparative example, as follows: Storage stability The freshly prepared Lactobacillus casei inoculum from the application examples and comparative examples was tested for viable cell counts in accordance with the national standard "GB4789.35-2016 National Food Safety Standard - Microbiological Examination of Food - Lactic Acid Bacteria," and the average value of multiple tests was calculated. The Lactobacillus casei inoculum from the application examples and comparative examples was stored in sealed glass bottles (wrapped in aluminum foil) at 4°C for 90 days. The viable cell count was then tested again using the above method, and the average value of multiple tests was calculated.

[0053] It should be noted that the Lactobacillus casei agent of the present application is in the form of microcapsules, and the Lactobacillus casei is wrapped and not easy to dissolve. Before detection, swelling and decomposition need to be carried out, specifically: take 1 g of Lactobacillus casei agent, add 50 mL of sterile normal saline, adjust the pH to 5.5, swell for 20 min; add 0.5 g of chitosanase (enzyme activity 100,000 U / g), and after enzymolysis at 100 rpm and 50°C for 30 min, it is used as the sample to be detected.

[0054] Survival rate calculation formula: Survival rate (%) = (dried active bacteria concentration / dried active bacteria concentration) x 100%.

[0055] Table 7 4℃ storage stability

[0056] As can be seen from Table 7, using glutamine transaminase, casein, lectin and tannic acid as the crosslinking agent of microcapsules is obviously more conducive to the long-term stable storage of the Lactobacillus casei agent compared with the glutaraldehyde and genipin of the prior art. The possible reason is that the crosslinking effects of different crosslinking agents are not the same, although they all play a crosslinking role, but the influence on the effective ingredients wrapped / coated / encapsulated by the microcapsules is not consistent. According to the experimental results, glutamine transaminase, casein, lectin and tannic acid as the crosslinking agent may be more gentle for the Lactobacillus casei C7-2 of the present application, which helps to maintain its activity; and glutaraldehyde and genipin may be more likely to cause damage or death of the bacteria.

[0057] The application example 5 adopts secondary crosslinking of casein and tannic acid, which can further reinforce the structure of the agent compared with the single crosslinking of application examples 1-4. This more compact structure can better maintain the internal environment required for the survival of Lactobacillus casei, reduce the influence of external factors (such as temperature, humidity, etc.) on Lactobacillus casei, thereby improving the storage stability. Moreover, the synergistic effect of casein and tannic acid may form a more complex and stable network structure, which not only can better fix Lactobacillus casei, but also can enhance the mechanical properties and chemical stability of the whole agent, and reduce the mortality of Lactobacillus casei during storage.

[0058] In vitro simulated gastric digestion survival test The human stomach environment has high acidity (pH is usually between 1.5-3.5), and contains digestive enzymes such as pepsin. Probiotics can only exert their probiotic function after passing through the stomach and reaching the intestine. Through the simulation of gastric conditions test, the survival of probiotic microcapsules under the action of gastric acid and pepsin can be understood.

[0059] Artificial gastric juice was prepared by weighing 8.5 g of sodium chloride and dissolving it in 900 mL of deionized water. Stirring thoroughly until completely dissolved, gradually add 10 wt % concentrated hydrochloric acid dropwise while stirring continuously to adjust the pH to 3.0. Add 1 g of pepsin powder and continue stirring until the pepsin powder is completely dissolved. Transfer the mixture to a volumetric flask and dilute to 1 L with deionized water.

[0060] 1 g of each of the Lactobacillus casei inoculum from the application example and the comparative example was dispersed in 9 mL of artificial gastric fluid, stored at 37°C, and removed after 2 hours to obtain gastric treatment fluid. The viable cell count of the Lactobacillus casei inoculum was tested in accordance with the national standard "GB4789.35-2016 National Food Safety Standard - Microbiological Examination of Food - Lactic Acid Bacteria", and the average value of multiple tests was calculated.

[0061] It should be noted that swelling and decomposition are required before testing. Specifically, take 1 mL of gastric treatment fluid, add 50 mL of sterile saline, adjust the pH to 5.5, and swell for 20 minutes; add 0.5 g of chitosanase (enzyme activity 100,000 U / g), and enzymatically hydrolyze at 100 rpm and 50°C for 30 minutes to use as the sample to be tested.

[0062] Table 8 In vitro simulated gastric digestion survival test

[0063] Note: 8.65×10 9 CFU / g=9.94 Log CFU / g Compared with glutaraldehyde and genipin commonly used in the microcapsule cross-linking process in the prior art, the cross-linked structure formed by the cross-linking agents (transglutaminase, casein, lectin, tannic acid) in Application Examples 1-5 may be tighter and have good stability, and can better protect Lactobacillus casei from damage by factors such as gastric acid and pepsin in simulated artificial gastric juice.

[0064] Obviously, those skilled in the art may make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if such changes and modifications fall within the scope of the claims and their equivalents, the present invention is intended to include such changes and modifications.

Claims

1. Lactobacillus casei C7-2, Latin name: Lacticaseibacillus casei , deposit number GDMCC63609, deposit time June 30, 2023, and the depository unit is Guangdong Provincial Microbiological Culture Collection Center.

2. Lactobacillus casei C7-2 as claimed in claim 1 is used in conditioning / improving thyroiditis.

3. The use according to claim 2, characterized in that: The Lactobacillus casei C7-2 described in claim 1 is used to prepare a composition for regulating / improving thyroiditis.

4. Lactobacillus casei C7-2 as claimed in claim 1 is used in conditioning / improving brain function activity.

5. The use according to claim 4, characterized in that: The Lactobacillus casei C7-2 described in claim 1 is used to prepare a composition for regulating / improving Alzheimer's disease.

6. The use according to claim 4, characterized in that: The Lactobacillus casei C7-2 described in claim 1 is used to prepare a composition for promoting brain health.

7. Lactobacillus casei C7-2 is used in protecting neurons as claimed in claim 1.

8. The use according to claim 6, characterized in that: The Lactobacillus casei C7-2 described in claim 6 is used to prepare a composition for regulating / improving Parkinson's disease.

9. The use according to claim 3, 5, 6 or 8, characterized in that The preparation method of the composition for regulating / improving thyroiditis, regulating / improving Alzheimer's disease, promoting brain health, or regulating / improving Parkinson's disease comprises the following steps: dissolving chitosan in an acetic acid aqueous solution; dissolving gelatin in water; mixing the chitosan and gelatin uniformly, adding the Lactobacillus casei C7-2 and cinnamaldehyde described in claim 1, and adjusting the pH to 5-6; stirring and mixing at 30-40° C. for 6-12 hours, and then cooling to room temperature to obtain a microcoagulation system; heating the mixture again to 30-45° C., adding a cross-linking agent, and stirring and mixing for 1-3 hours; cooling the mixture to room temperature, filtering, washing, and freeze-drying to obtain the microcoagulation system. The bacterial content of Lactobacillus casei C7-2 was 1×10 9 -3×10 10 CFU / g; The cross-linking agent is at least one of transglutaminase, casein, lectin, and tannic acid.

Images