Preparation method and application of lactic acid bacteria fermented Chinese chestnut protein postbiotics for improving systemic chronic inflammation

Abstract

The invention belongs to the technical field of preparation of postbiotics, and discloses a preparation method and application of lactic acid bacteria fermented Chinese chestnut protein postbiotics for improving systemic chronic inflammations, Chinese chestnuts are subjected to shelling, grinding and degreasing treatment, and degreased Chinese chestnut powder is obtained; resuspending the degreased Chinese chestnut powder in distilled water, adjusting the pH value, and stirring; performing solid-liquid separation and magnetic stirring; carrying out solid-liquid separation, resuspending and adjusting the pH value; dialyzing to obtain Chinese chestnut protein powder; mixing with glucose powder, and redissolving in distilled water; and mixing with lactic acid bacteria and carrying out aerobic fermentation to obtain the Chinese chestnut protein metaplasm product. The bioactive peptides contained in the Chinese chestnut protein metaplasm prepared by the method have potential anti-inflammatory activity, can effectively inhibit proinflammatory medium secretion of macrophages and intestinal epithelial cells, and improve glucose and lipid metabolism disorder and intestinal barrier function damage related to chronic inflammation of a body. The bioactive peptide contained in the postbiotic product disclosed by the invention has an anti-inflammatory effect.

Description

Technical Field

[0001] The invention belongs to the technical field of postbiotic preparation, and in particular to a preparation method and application of a lactic acid bacteria fermented chestnut protein postbiotic for improving systemic chronic inflammation. Background Art

[0002] Obesity associated with systemic chronic inflammation is a major factor in inducing chronic metabolic syndromes such as hypertension, cardiovascular disease, and type 2 diabetes. On the one hand, the endogenous release of free fatty acids in adipocytes activates macrophages and increases the level of systemic proinflammatory factors. On the other hand, the imbalance of intestinal microecology mediated by a high-fat diet has a negative impact on the diversity of intestinal microbiota and intestinal integrity. Endotoxins such as lipopolysaccharide (LPS) from Gram-negative bacteria enter the submucosal layer, causing a subclinical increase in the level of proinflammatory mediators and inducing systemic inflammation. Currently, chronic inflammatory-related diseases are a major challenge facing the global public health system and have imposed a serious economic burden on the sick population.

[0003] Currently, many bioactive compounds in foods have anti-inflammatory activity and are used as potential methods to prevent and improve chronic inflammation. Plant protein contains no cholesterol and almost no saturated fat, which can effectively limit calorie intake and reduce the incidence of chronic metabolic syndrome. It is reported that chestnut protein is rich in essential amino acids and acidic amino acids (aspartic acid and glutamic acid), thus having high potential anti-inflammatory and antioxidant activity. Lactic acid bacteria fermentation can utilize the proteolytic system of the strain to degrade protein, increase the content of bioactive peptides in the fermentation product and enhance the protein function. Therefore, lactic acid bacteria fermented chestnut protein has broad application prospects in the consumer market for chronic inflammatory-related diseases.

[0004] Probiotic preparations are currently used to improve chronic low-grade inflammation in the body, see CN118086154B, CN116478874B and CN112190600B, and studies have shown that the introduction of live bacteria has the risk of disrupting the intestinal microecology of patients with chronic inflammation and aggravating inflammatory responses. On the other hand, studies have also confirmed the important role of plant protein in reducing chronic metabolic syndrome. Therefore, the present invention intends to make full use of the advantages of lactic acid bacteria and plant protein in improving chronic inflammatory-related diseases, overcome the shortcomings of the prior art, and provide a preparation method and application of lactic acid bacteria fermented chestnut protein postbiotics for improving systemic chronic inflammation. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a preparation method and application of a lactic acid bacteria fermented chestnut protein postbiotic for improving systemic chronic inflammation.

[0006] The technical solution adopted by the present invention to solve the technical problem is:

[0007] A method for preparing lactic acid bacteria fermented chestnut protein postbiotics for improving systemic chronic inflammation comprises the following steps:

[0008] Shelling, grinding and defatting the chestnuts to obtain defatted chestnut powder;

[0009] The defatted chestnut powder was resuspended in distilled water at a ratio of defatted chestnut powder to distilled water of 1:15 g:mL, and the pH was adjusted to 9.0 with 0.2 M NaOH, followed by magnetic stirring at room temperature for 2 h;

[0010] The mixture was separated into solid and liquid, and the supernatant was retained and the pH value was adjusted to 4.5 with 1 M HCl and magnetically stirred for 1 h;

[0011] The mixture was separated into solid and liquid, the precipitate was collected and resuspended in distilled water, and the pH was adjusted to 7.0 with 0.2 M NaOH;

[0012] The mixture is placed in a dialysis bag and dialyzed for 36-48 hours, and then the dialysate is vacuum freeze-dried to obtain a lyophilized product, which is chestnut protein powder;

[0013] Mixing the chestnut protein powder and glucose powder and redissolving them in distilled water to obtain a chestnut protein fermentation substrate;

[0014] The chestnut protein fermentation substrate is mixed with lactic acid bacteria for aerobic fermentation to obtain a chestnut protein postbiotic product.

[0015] Furthermore, the ratio of the chestnut protein powder: glucose powder: distilled water (g:g;mL) is 5:2:100.

[0016] Furthermore, the lactic acid bacteria is any one of Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei, and Pediococcus acidilactici, and the effective viable count of the lactic acid bacteria is (1-10)×10 7 CFU / g chestnut protein powder.

[0017] Furthermore, the aerobic fermentation conditions are a temperature of 37° C. and a time of 24 hours.

[0018] The chestnut protein postbiotics are prepared by the preparation method described above.

[0019] The use of the chestnut protein postbiotics as described above in the preparation of chronic inflammation drugs.

[0020] Furthermore, the chestnut protein postbiotics can inhibit the secretion of pro-inflammatory mediators by macrophages and intestinal epithelial cells, and improve the body's chronic inflammation-related glucose and lipid metabolism disorders and impaired intestinal barrier function.

[0021] The use of the chestnut protein postbiotics as described above in the preparation of drugs for inhibiting the secretion of pro-inflammatory mediators in macrophages and intestinal epithelial cells.

[0022] The use of the chestnut protein postbiotics as described above in the preparation of drugs for improving chronic inflammation-related glucose and lipid metabolism disorders and / or impaired intestinal barrier function.

[0023] The advantages and positive effects achieved by the present invention are:

[0024] 1. The bioactive peptides contained in the chestnut protein postbiotics prepared by the method of the present invention have potential anti-inflammatory activity, can effectively inhibit the secretion of pro-inflammatory mediators by macrophages and intestinal epithelial cells, and improve the body's chronic inflammation-related glucose and lipid metabolism disorders and impaired intestinal barrier function. The bioactive peptides contained in the postbiotic products of the present invention have anti-inflammatory effects.

[0025] 2. The chestnut protein postbiotics of the present invention can significantly reduce the level of serum pro-inflammatory cytokines, inhibit the activation of adipose tissue macrophages, maintain blood sugar and insulin levels, and maintain intestinal barrier function through dietary therapy.

[0026] The chestnut protein postbiotic can inhibit the secretion of pro-inflammatory mediators by macrophages and intestinal epithelial cells, and improve the body's chronic inflammation-related glucose and lipid metabolism disorders and impaired intestinal barrier function.

[0027] 3. The method of the present invention first selects lactic acid bacteria that can effectively metabolize chestnut protein based on the fermentation characteristics of chestnut. The preferred lactic acid bacteria is Lactobacillus rhamnosus. The postbiotic product of chestnut protein fermented by lactic acid bacteria is rich in active polypeptides and essential amino acids, and has strong intestinal absorbability; it can effectively inhibit the inflammatory response of macrophages and intestinal epithelial cells; it can effectively alleviate the inflammatory response of inflammatory mice. The postbiotic products in the present invention can be widely used to improve the increased levels of systemic inflammatory factors and inflammatory intestinal barrier damage related to aging, obesity, diabetes, irritable bowel syndrome, etc., and have high market application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a graph showing the organic acid content in chestnut protein and its postbiotic products in the present invention;

[0029] Figure 2 This is a graph showing the free amino acid content in chestnut protein and its postbiotic products in the present invention;

[0030] Figure 3 This is a graph showing the release of inflammatory factors in the macrophage model of the present invention;

[0031] Figure 4 This is a diagram of inflammatory damage in the intestinal epithelial cell model of the present invention;

[0032] Figure 5 is a distribution diagram of tight junction proteins in intestinal epithelial cells in the present invention;

[0033] Figure 6 This is a graph showing the effect of the chestnut protein postbiotic product of the present invention on the weight change of mice;

[0034] Figure 7 This is a graph showing the effect of the chestnut protein postbiotic product of the present invention on the content of serum pro-inflammatory mediators in mice with low-grade inflammation;

[0035] Figure 8 This is a diagram showing the effect of the chestnut protein postbiotic product of the present invention on the glucose and lipid metabolism homeostasis in mice with low-grade inflammation;

[0036] Fig. 9 This is a graph showing the effects of the chestnut protein postbiotic product of the present invention on the intestinal function of mice with low-grade inflammation. DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0038] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0039] A method for preparing lactic acid bacteria fermented chestnut protein postbiotics for improving systemic chronic inflammation comprises the following steps:

[0040] Shelling, grinding and defatting the chestnuts to obtain defatted chestnut powder;

[0041] The defatted chestnut powder was resuspended in distilled water at a ratio of defatted chestnut powder to distilled water of 1:15 g:mL, and the pH was adjusted to 9.0 with 0.2 M NaOH, followed by magnetic stirring at room temperature for 2 h;

[0042] The mixture was separated into solid and liquid, and the supernatant was retained and the pH value was adjusted to 4.5 with 1 M HCl and magnetically stirred for 1 h;

[0043] The mixture was separated into solid and liquid, the precipitate was collected and resuspended in distilled water, and the pH was adjusted to 7.0 with 0.2 M NaOH;

[0044] The mixture is placed in a dialysis bag and dialyzed for 36-48 hours, and then the dialysate is vacuum freeze-dried to obtain a lyophilized product, which is chestnut protein powder;

[0045] Mixing the chestnut protein powder and glucose powder and redissolving them in distilled water to obtain a chestnut protein fermentation substrate;

[0046] The chestnut protein fermentation substrate is mixed with lactic acid bacteria for aerobic fermentation to obtain a chestnut protein postbiotic product.

[0047] Preferably, the ratio of the chestnut protein powder: glucose powder: distilled water (g:g; mL) is 5:2:100.

[0048] Preferably, the lactic acid bacteria is any one of Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei, and Pediococcus acidilactici, and the effective viable count of the lactic acid bacteria is (1-10)×10 7 CFU / g chestnut protein powder.

[0049] Preferably, the aerobic fermentation is carried out at a temperature of 37° C. and for 24 hours.

[0050] The chestnut protein postbiotics are prepared by the preparation method described above.

[0051] The use of the chestnut protein postbiotics as described above in the preparation of chronic inflammation drugs.

[0052] Preferably, the chestnut protein postbiotic can inhibit the secretion of pro-inflammatory mediators by macrophages and intestinal epithelial cells, and improve the body's chronic inflammation-related glucose and lipid metabolism disorders and impaired intestinal barrier function.

[0053] The use of the chestnut protein postbiotics as described above in the preparation of drugs for inhibiting the secretion of pro-inflammatory mediators in macrophages and intestinal epithelial cells.

[0054] The use of the chestnut protein postbiotics as described above in the preparation of drugs for improving chronic inflammation-related glucose and lipid metabolism disorders and / or impaired intestinal barrier function.

[0055] Specifically, the relevant preparation and detection are as follows:

[0056] Example 1

[0057] A method for preparing lactic acid bacteria fermented chestnut protein postbiotics for improving systemic chronic inflammation comprises the following steps:

[0058] (1) First, the chestnuts were ground and defatted: the chestnuts were shelled, peeled, sliced, and placed in a -80°C refrigerator for overnight freezing, followed by dehydration and freeze drying for 24 hours. The dried chestnut slices were ground in a blender and then further ground in a mortar. Dehydrated chestnut powder was weighed and mixed with n-hexane at a solid-liquid ratio of 1:9 (w / v, g / mL), ultrasonically extracted for 1 hour, centrifuged at 2000g, the supernatant was discarded, and the above operation was repeated once for the remaining residue. The collected residue was placed in a fume hood to dry naturally to obtain defatted chestnut powder.

[0059] (2) Weigh the defatted chestnut powder obtained by the method described in (1) and mix it with distilled water at a solid-liquid ratio of 1:15 (w / v, g / mL), adjust the pH to 9.0 with 0.2M NaOH, and stir magnetically at room temperature for 2 hours. Pass the stirred mixture through gauze, retain the supernatant and centrifuge at 3000rpm for 20 minutes, adjust the pH value of the mixed supernatant to 4.5 with 1M HCl, stir magnetically for 1 hour, and then centrifuge at 3000rpm for 20 minutes. Collect the precipitated protein, resuspend it with distilled water, and neutralize it to pH=7.0. Then centrifuge to remove insoluble protein, put the supernatant into a dialysis bag, dialyze for 36-48 hours, and vacuum freeze-dry the collected liquid to obtain chestnut protein powder.

[0060] (3) Weigh 5 g of chestnut protein powder obtained by the method described in (2) and 2 g of glucose powder (purchased from Beijing Solebow Technology Co., Ltd., product number: G8150), mix them and dissolve them in 100 mL of distilled water. The resulting chestnut protein-rich aqueous solution is labeled as CNP.

[0061] (4) The bacterial suspension of Lactobacillus rhamnosus was prepared as follows: Lactobacillus rhamnosus was cultured in MRS medium (purchased from Beijing Solebaugh Technology Co., Ltd., product number: M8540) to the third stable stage, centrifuged at 4000 g for 10 min, and then the precipitate was washed with distilled water, and then resuspended with distilled water to obtain a bacterial suspension. The bacterial suspension was adjusted to a viable cell count of 5×10 6 CFU, 5×10 7 CFU, 5×10 9 CFU and 5×10 8 CFU.

[0062] The products obtained from (3) and (4) were mixed in a conical flask, the mouth of the conical flask was sealed with a sterile filter membrane, and fermented in a fermentation box at 37°C for 24 h. The chestnut protein postbiotic products obtained by fermentation with different bacterial amounts were labeled as CNPL1 (5×10 6 CFU / g chestnut protein), CNPL2 (5×10 7 CFU / g chestnut protein), CNPL3 (5×10 9 CFU / g chestnut protein) and CNPL (5×10 8 CFU / g chestnut protein).

[0063] Example 2

[0064] Determination of organic acid and polypeptide content in chestnut protein and its postbiotic products:

[0065] The change in sample acidity during the fermentation process in Example 1 was evaluated using a fully automatic pH meter. 1 g of the sample was redissolved in 10 mL of distilled water and the pH value was measured after homogenization. The results were as follows: Figure 1 As shown. The changes in polypeptide content during the fermentation process were determined by high performance liquid chromatography. The TSK Gel2000 SWXL column was used for analysis at a flow rate of 0.5 mL / min. The mobile phase was a H2O solution containing 0.1% trifluoroacetic acid and 45% acetonitrile. 1 g of the sample was redissolved in 10 mL of distilled water, homogenized and centrifuged at 10000 g for 15 min, the supernatant was ultrafiltered, and the ultrafiltrated solution was dissolved in the mobile phase at 2 mg / mL for determination. GLY-GLY-TYR-ARG with a molecular weight of 451 Da, bacitracin zinc with a molecular weight of 1450 Da, aprotinin with a molecular weight of 6500 Da, and cytochrome C with a molecular weight of 12384 Da were used as standards to calibrate the molecular weight distribution of the polypeptide to be tested. The results are shown in Table 1.

[0066] Depend on Figure 1 It can be seen that the pH value of the chestnut protein solution group did not change significantly. After inoculation with lactic acid bacteria, the pH values ​​of all groups decreased as the fermentation progressed. 6 The pH value of CNPL1 group with a CFU / g decreased most slowly. 9 The pH value of CNPL3 group with a CFU / g decreased most rapidly. 7-8 The pH value of CNPL2 and CNPL groups with CFU / g changed moderately. It can be concluded from Table 1 that the content of polypeptides and potential small molecule functional peptides (<0.45 kDa) in CNPL and CNPL2 is significantly higher than that in CNPL1 and CNPL3, and higher than that in unfermented chestnut protein (CNP). Combined with the results of the determination of the organic acid and polypeptide content in the postbiotic product of chestnut protein in Example 2, it can be seen that too little bacterial amount is not enough to complete the conversion of protein into functional small peptides within 24 hours, while too much bacterial amount accelerates the fermentation process and produces more organic acids, thereby inhibiting the protein metabolism activity of lactic acid bacteria. Therefore, (1-10)×10 7 CFU / g chestnut protein powder is the best inoculation amount, especially 1×10 7 CFU / g chestnut protein powder is the best inoculation amount.

[0067] At the same time, through Figure 1 It can also be seen from Table 1 that the aqueous solution rich in chestnut protein in the method of the present invention has a CNP and (1-10)×10 7 CFU / g chestnut protein powder has a synergistic effect, which can synergistically improve the relevant properties of postbiotic products obtained by fermentation, especially the chestnut protein-rich aqueous solution CNP and 1×10 7There is a particularly significant synergistic effect between CFU / g chestnut protein powder, which can significantly synergistically improve the relevant properties of postbiotic products obtained by fermentation.

[0068] Table 1 Molecular weight distribution of free polypeptides in chestnut protein and its postbiotic products

[0069] Group >12kDa 1.45-6.5 kDa 0.45-1.45 kDa <0.45kDa CNP 342353 654855 2124543 9834720 CNPL1 370997 802763 2600258 49751570 CNPL2 660453 1282036 3162730 99751572 CNPL3 406020 879316 2875829 34150333 CNPL 691900 1452129 3627068 113367253

[0070] Example 3

[0071] Identification of active peptide sequences and free amino acids in chestnut protein and its postbiotic products:

[0072] UPLC-MS / MS analysis was performed using Orbitrap Fusion-Lumos. A C18 column (3 μm, 75 μm×15 cm) was used for analysis at a flow rate of 300 μL / min. Mobile phase A was H2O containing 0.1% formic acid, and mobile phase B was 80% acetonitrile / H2O containing 0.1% acetic acid. The elution conditions were as follows: 0-4 min, 5%-10% B; 4-40 min, 10%-28% B; 40-47 min, 28%-38% B; 47-48 min, 38%-100% B; 48-60 min, 100% B. A critical value of -101 gP>15 was used to eliminate biased peptide identifications, and the sequences were manually inspected and compared with the protein coding information of the Uniprot database to determine the source protein.

[0073] According to the UPLC-MS / MS results (Table 2), 1585 and 1172 peptide sequences were identified in chestnut protein (CNP) and chestnut protein postbiotic products (CNPL), respectively. PeptideRanker scores and BIOPEP protein database were used to predict key peptides with potential anti-inflammatory effects in chestnut protein hydrolysates. Relative peak area was also used as a screening criterion. According to the screening results, 570 (total abundance of 7.59E+010) and 485 (total abundance of 1.07E+011) peptides were screened out in CNP and CNPL, respectively, with peak area relative abundance>0.1% and PeptideRanker score>0.2 as screening criteria. According to the BIOPEP search results, the content of anti-inflammatory peptides in CNP was 4.67E+09, and the content of anti-inflammatory peptides in CNPL was 6.84E+09. The key active sequences of these peptides were IPP, VPP, PY, GGW, ANP, NLQ, YW, and HY.

[0074] The free amino acid content in chestnut protein (CNP) and chestnut protein postbiotic product (CNPL) was determined by reverse phase high performance liquid chromatography. A Waters AccQ.Tag amino acid analysis column (4μm, 3.9mm×150mm) was used for analysis at a flow rate of 1mL / min. The mobile phase A was 10% AccQ·Tag Ultra concentrated solvent A, the mobile phase B was 100% acetonitrile, and the mobile phase C was HPLC grade water. A nonlinear separation gradient was used for elution. Amino acids were detected by excitation at 395nm and emission at 250nm.

[0075] According to the results of reversed-phase high performance liquid chromatography ( Figure 2 ), the main free amino acids in CNP and CNPL are serine (Ser), glycine (Gly), proline (Pro) and cysteine ​​(Cys). The contents of leucine and total essential amino acids in CNPL are significantly higher than those in CNP, indicating that fermentation treatment promotes the release of leucine, the first limiting amino acid of chestnut protein, and the release of essential amino acids except tryptophan, and chestnut protein postbiotic products have a higher amino acid nutritional level.

[0076] Table 2 Anti-inflammatory bioactive peptides from chestnut protein and its postbiotic products

[0077]

[0078]

[0079] Example 4

[0080] Chestnut protein and its postbiotic products inhibit the secretion of pro-inflammatory mediators in macrophages:

[0081] The inflammatory macrophage model was established using the RAW264.7 cell line. The cells were cultured at 3×10 5 Each well was inoculated into a 24-well cell culture plate and cultured for 24 hours, then different concentrations of CNP and CNPL (0.5, 1, 2 mg / mL) were added to treat the cells for 2 hours, and finally treated with 100 ng / mL of LPS for 24 hours to stimulate cell inflammatory response. The cell culture supernatant was collected and the secretion of macrophage proinflammatory mediators (NO, TNF-α, IL-6) was determined using a kit. The NO kit was purchased from Nanjing Jiancheng Biotechnology Co., Ltd. with a product number of A013-2-1; the TNF-α and IL-6 kits were purchased from Jiangsu Enzyme Biotechnology Co., Ltd. with product numbers of MB6426A and MB50054A, respectively.

[0082] The results of the inflammatory macrophage model showed that ( Figure 3), the release of proinflammatory cytokines NO, TNF-α and IL-6 in macrophages significantly increased after LPS treatment, which was about 7.5 times, 2 times and 1.4 times higher than that in the control group, respectively. 0.5, 1, and 2 mg / mL CNP\CNPL can inhibit LPS-induced macrophage inflammatory response to varying degrees. 0.5 mg / mL of CNP and CNPL can reduce the secretion of NO by 47% and 90%, the secretion of TNF-α by 8% and 21%, and the secretion of IL-6 by 29% and 31%, respectively; 1 mg / mL of CNP and CNPL can reduce the secretion of NO by 14% and 60%, the secretion of TNF-α by 10% and 14%, and the secretion of IL-6 by 29% and 18%, respectively; 2 mg / mL of CNP and CNPL can reduce the secretion of NO by 28% and 45%, the secretion of TNF-α by 15% and 2%, and the secretion of IL-6 by 11% and 2%, respectively; it can be seen that 0.5 mg / mL of CNPL can most significantly reduce the secretion of TNF-α, IL-6, and NO, indicating that chestnut protein postbiotic products can most significantly alleviate the LPS-induced macrophage inflammatory response.

[0083] Example 5

[0084] Chestnut protein and its postbiotic products alleviate inflammatory damage to intestinal epithelial cells:

[0085] The inflammatory intestinal epithelial cell model was established using the Caco-2 cell line. The cells were cultured at a rate of 2×10 5 Cells were inoculated into 12-well Transwell cell culture plates. The culture medium was changed every other day for 7 days after cell inoculation, and then changed every day until the 21st day. Cell resistance was measured using Millicell-ERS, and the resistance value was greater than 500Ω×cm 2 The cell wells were considered as successful Caco-2 monolayer cell models and were further used in the following experiments. Different concentrations of CNP and CNPL (0.1, 0.2 mg / mL) were added to the above cell wells to treat the cells for 2 hours, and finally treated with 100 μg / mL of LPS for 24 hours to stimulate cellular inflammatory response. The cell culture supernatant was removed, the cells were collected, and total RNA was extracted from the cells using TRIzol reagent. After determining the RNA concentration and purity, it was reverse transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit. qRT-PCR was used to detect the mRNA expression levels of genes related to impaired intestinal barrier function. β-actin was used as an internal reference, and the 2-ΔΔCt method was used for relative quantitative analysis. The primer information of the relevant genes is as follows. Figure 3As shown. At the same time, the collected cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature and permeated with 0.1% TritonX-100 for 10 minutes for further immunofluorescence experiments. Phalloidin-AlexaFlour 448 fluorescent antibody was used to directly stain cells for 50 minutes to observe the distribution of intestinal tight junction protein F-actin; rabbit anti-mouse ZO-1 and Claudin-1 primary antibodies and goat anti-rabbit IgG secondary antibodies were used to incubate cells in sequence to observe the distribution of intestinal tight junction proteins ZO-1 and Claudin-1.

[0086] The results of the inflammatory intestinal epithelial cell model showed that the mRNA expression levels of ZO-1, Occludin and Claudin-1 in small intestinal epithelial cells were significantly reduced after LPS treatment, while different concentrations of chestnut protein (CNP) and chestnut protein postbiotic products (CNPL) increased the expression of related factors. Among them, the effect of chestnut protein postbiotic products (CNPL) was the most significant. Compared with the LPS group, 0.1 mg / mL CNPL increased the mRNA expression levels of ZO-1, Occludin and Claudin-1 by 7.5 times, 3.6 times and 0.2 times, respectively; 0.2 mg / mL CNPL increased the mRNA expression levels of ZO-1, Occludin and Claudin-1 by 7.1 times, 3.3 times and 0.8 times, respectively ( Figure 4 ). In addition, LPS treatment destroyed the tight junctions between intestinal epithelial cells, causing cell aggregation and vacuoles, and the fluorescence intensity of three proteins (F-actin, ZO-1 and Claudin-1) was weakened. 0.2 mg / L chestnut protein (CNP) and chestnut protein postbiotic product (CNPL) intervention treatment can improve the above phenomena. CNPL can most significantly reduce the aggregation behavior of cells and increase the fluorescence intensity of F-actin and ZO-1 ( Figure 5 ). Taken together, the above results indicate that chestnut protein postbiotic products can most significantly alleviate LPS-induced intestinal epithelial cell inflammatory response.

[0087] Table 3 Primer information of related genes

[0088]

[0089] Example 6

[0090] Animal Experimentation:

[0091] The experimental animals were specific pathogen-free C57BL / 6J mice (8 weeks old, female) with a body weight of 15-17 g. The mice were placed in a polycarbonate box under the controlled conditions of temperature 22±2°C, humidity 65%±5%, and light / dark photoperiod of 12 h. After one week of adaptive feeding, all mice were randomly divided into 4 groups, the high-dose chestnut protein postbiotics (100 mg / kg / day CNPL by oral gavage) intervention group was marked as FCPH, the low-dose chestnut protein postbiotics (50 mg / kg / day CNPL by oral gavage) intervention group was marked as FCPL, the chronic inflammation model group (HFL) and the blank control group (NC); all groups were provided with normal diet from 0 to 4 weeks, and from 4 to 16 weeks, except for the blank control group, the intervention group and the model group were provided with 400 μg / Kg LPS drinking water and a high-fat diet containing 60% fat by total mass, and the body weight changes were recorded. At the same time, the intervention group was continuously gavaged with high-dose chestnut protein postbiotics and low-dose chestnut protein postbiotics, and the model group was gavaged with an equal amount of sterile water; the mice were killed by cervical dislocation at the 16th week, and serum, adipose tissue, and intestinal tissue were collected for further analysis.

[0092] The changes in mouse body weight Figure 6 As shown in the figure, the body weight of mice in the chronic inflammation model group (HFL) was significantly lower than that of mice in the blank control group (NC) (21.77±1.925g). The FCPL (24.23±3.317g) and FCPH (24.48±1.185g) groups were gavaged with chestnut protein postbiotics, which effectively improved the weight loss of mice, indicating that high and low doses of chestnut protein postbiotics have an improvement effect on weight loss caused by inflammation.

[0093] Example 7

[0094] Example 6 Effect of chestnut protein postbiotic products on the secretion of inflammatory factors in the body:

[0095] After the 16th week of intervention in Example 6, serum was collected and the levels of inflammatory factors and endotoxin LPS in the mouse serum were measured using Elisa kits. TNF-α, IL-1β, IL-6 and LPS kits were purchased from Jiangsu Enzyme Biotechnology Co., Ltd. with product numbers MB-2868A, MB-2776A, MB-2899A and MB-3418A, respectively. The measurement results are shown in the figure. Figure 7 shown.

[0096] The results showed that the TNF-α content in the HFL group mice (104.8±8.106pg / mL) was significantly higher than that in the NC group mice (119.2±9.828pg / mL) (P<0.05). Intragastric administration of different concentrations of fermented chestnut protein could inhibit the TNF-α level in mice (FCPL: 100.5±7.411pg / mL, FCPH: 103.9±4.582pg / mL), and had a significant effect (P<0.01, P<0.01). In addition, compared with the NC group (82.57±2.828pg / mL), the plasma IL-1β of the HFL group (88.77±2.006pg / mL) mice was significantly increased (P<0.05). Both low-dose (82.98±4.170pg / mL) and high-dose (84.68±4.630pg / mL) chestnut protein postbiotics could inhibit the abnormal increase of IL-1β induced by HFD+LPS, but only the low-dose intervention group had a significant inhibitory effect (P<0.05). At the same time, compared with the NC group (123.3±4.536pg / mL), the IL-6 level in the HFL group (137.2±7.490pg / mL) mice was also significantly increased (P<0.01), while the IL-6 content in the FCPL group (122.0±4.513pg / mL) and FCPH group (123.9±4.001pg / mL) mice was significantly lower than that in the HFL group (P<0.001, P<0.001). In addition, the serum endotoxin LPS of mice was also measured in this study. The results showed that the LPS level in the HFL group (132.3±11.88EU / L) was significantly higher than that in the NC group (163.2±9.790EU / L) (P<0.001), while chestnut protein postbiotics (FCPL: 143.5±6.510EU / L, FCPH: 139.9±8.413EU / L) could significantly inhibit the endotoxin level in mice (P<0.01, P<0.001). In summary, chestnut protein postbiotics (FCP) can effectively improve the increased secretion of inflammatory factors induced by high-fat diet and LPS.

[0097] Example 8

[0098] Effects of chestnut protein postbiotic products on glucose and lipid metabolism disorders associated with chronic inflammation in Example 6:

[0099] Animal experiment refers to the relevant operation of Example 6, and a glucose tolerance test (GTT) is performed in the 15th week to determine the occurrence of insulin resistance. Before the test, mice fasted for 12-14 hours (food was removed but water was retained). After fasting, the tail tip of the mouse was cut off. A drop of blood was squeezed out from the incision and placed in a blood glucose meter to measure the initial glucose level. Then 200 μL 2g / kg BW oral glucose solution was given to each mouse immediately by gavage. Then the same scheme as above was used to measure and record the mouse glucose level at 15, 30, 60 and 120 minutes; the total cholesterol, triglyceride, low density / high density lipoprotein and transaminase (alanine and aspartate transaminase) levels in mouse serum were measured using Nanjing Jiancheng kit. Among them, the catalog numbers of lipid indicators TC, TG, LDL-C and HDL-C are A111-1-1, A110-1-1, A113-1-1 and A112-1-1 respectively; the catalog numbers of transaminase ALT and AST kits are C009-2-1 and C010-2-1 respectively.

[0100] Glucose tolerance test results Figure 8 As shown in AB, the GTT results evaluated by the area under the curve (AUC) showed that the AUC value of the HFL group (1335±70.41mmol / L×120min) was higher than that of the NC group (1047±64.12mmol / L×120min), and it was statistically significant (P<0.0001). Compared with the HFL group, the glucose AUC of the mice in the FCP group was significantly reduced (P<0.0001, P<0.001); in addition, it was observed that the glucose AUC value of the mice in the FCPL group (1135±58.14mmol / L×120min) was significantly lower than that in the FCPH group (1217±59.08mmol / L×120min) (P<0.01). It can be seen that chestnut protein postbiotics can regulate glucose metabolism in mice induced by HFD and LPS, and the effect of the low-dose chestnut protein supplementation group was significantly better than that of the high-dose chestnut protein supplementation group.

[0101] Depend on Figure 8The CF results showed that compared with mice fed a normal diet, the high-fat diet supplemented with LPS significantly increased the levels of total cholesterol, triglycerides, and low-density lipoprotein in the serum of mice. Compared with the NC group (2.582±0.3624mmol / L), the total cholesterol level of mice in the HFL group (3.323±0.1766mmol / L) was significantly increased, while intragastric administration of different doses of chestnut protein postbiotics (FCPL: 3.295±0.3169mmol / L, FCPH: 2.503±0.6627mmol / L) could inhibit the abnormal increase in serum cholesterol level, and the inhibitory effect of the FCPH group was significant (P<0.01). In addition, compared with the HFL group (1.741±0.1674mmol / L), the serum triglyceride content of mice gavaged with different doses of chestnut protein postbiotics (FCPL: 1.469±0.1087mmol / L, FCPH: 1.474±0.2770mmol / L) was reduced, especially in the FCPL group (P<0.01). At the same time, the low-density lipoprotein content of mice in the HFL group (0.7193±0.1068mmol / L) was significantly higher than that in the NC group (0.4001±0.1144mmol / L), while the low-density lipoprotein content of mice in the chestnut protein postbiotic intervention group (FCPL: 0.3948±0.0697mmol / L, FCPH: 0.3771±0.1067mmol / L) was significantly lower than that in the HFL model group (P<0.0001, P<0.0001). The results of high-density lipoprotein HDL-C determination showed that oral administration of FCP could significantly increase the content of HDL-C in mice (P<0.0001, P<0.001) and regulate blood lipid levels. Figure 7The results of GI measurement showed that compared with the NC group (ALT: 5.572±0.7804U / L, AST: 16.70±5.050U / L), the ALT and AST in the serum of the HFL group (ALT: 8.233±2.106U / L, AST: 24.60±2.337U / L) were significantly increased (P<0.01), while oral administration of low-dose (ALT: 5.831±1.676U / L, AST: 11.73±2.101U / L) and high-dose (ALT: 3.306±1.389U / L, AST: 17.54±4.360U / L) fermented chestnut protein could significantly inhibit the abnormal changes of transaminase levels in mice (P<0.0001), and significantly reduce the liver index (P<0.01). It can be seen that long-term intake of high-fat diet and LPS leads to liver damage in mice, which in turn induces low-grade inflammation; and different doses of fermented chestnut protein can significantly inhibit abnormal changes in serum aminotransferase levels. In summary, chestnut protein postbiotic products can effectively improve the glucose and lipid metabolism disorders induced by high-fat diet and LPS, alleviate liver damage, and then inhibit systemic inflammatory response.

[0102] Example 9

[0103] Example 6 Effects of chestnut protein postbiotic products on intestinal barrier function damage associated with chronic inflammation

[0104] After the 16-week intervention in Example 6, the colon and small intestinal tissues of the mice were collected to observe the colon morphology and to measure the inflammatory factors (TNF-α and IL-1β) in the intestinal tissues. The TNF-α and IL-1β kits were purchased from ELISA reagent company, with product numbers MM-0132M1 and MM-0040M1, respectively. Figure 8 AB showed that compared with the NC group (6.833±0.1966cm), the colon length of the HFL group (6.833±0.1966cm) was significantly shortened and the intestinal morphology was damaged. However, supplementation with different doses of chestnut protein postbiotics could alleviate the colon damage to varying degrees (FCPL: 7.300±0.3950cm, FCPH: 7.500±0.4435cm).

[0105] Colon tissue Fig. 9The results of CE inflammatory factor determination showed that the content of TNF-α in the colon tissue of mice in the HFL group (55.80±8.290ng / L) was increased compared with that in the NC group (50.77±0.7115ng / L). Chestnut protein postbiotics could inhibit the abnormal increase of inflammatory factors, but had no significant effect (FCPL: 53.50±3.264ng / L, FCPH: 52.46±3.015ng / L). In addition, this study measured the inflammatory conditions in the small intestinal tissue. The results showed that the TNF-α content in the HFL group (65.52±5.366ng / L) was significantly increased compared with the NC group (60.08±2.551ng / L) (P<0.05). Supplementation of low and high doses of chestnut protein postbiotics (FCP) can inhibit the release of proinflammatory cytokines (TNF-α and IL-1β) in colon and small intestinal tissues (FCPL: 56.32±5.131ng / L, FCPH: 61.09±5.653ng / L), and the inhibitory effect of the low-dose group was more significant (P<0.05). At the same time, the results of IL-1β determination also showed that the small intestine of mice in the HFL group (5.974±0.3635ng / L) was significantly inflamed, and its content was significantly higher than that in the NC group (7.679±1.331ng / L) (P<0.01). Different concentrations of chestnut protein postbiotics (FCPL: 6.703±0.4497ng / L, FCPH: 7.542±0.6255ng / L) could inhibit the abnormal release of IL-1β, but there was no significant difference. In summary, our results show that supplementation with different doses of chestnut protein postbiotics (FCP) can maintain the integrity of intestinal morphology, improve intestinal function, inhibit the release of proinflammatory cytokines, and relieve systemic inflammation.

[0106] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for preparing a lactic acid bacteria fermented chestnut protein postbiotic for improving systemic chronic inflammation, characterized in that: The steps include: Shelling, grinding and defatting the chestnuts to obtain defatted chestnut powder; The defatted chestnut powder was resuspended in distilled water at a ratio of 1:15 g:mL of defatted chestnut powder: distilled water, and the pH was adjusted to 9.0 with 0.2 M NaOH, followed by magnetic stirring at room temperature for 2 h; The mixture was separated into solid and liquid, and the supernatant was retained and the pH value was adjusted to 4.5 with 1 M HCl and magnetically stirred for 1 h; The mixture was separated into solid and liquid, the precipitate was collected and resuspended in distilled water, and the pH was adjusted to 7.0 with 0.2 M NaOH; The mixture is placed in a dialysis bag and dialyzed for 36-48 hours, and then the dialysate is vacuum freeze-dried to obtain a lyophilized product, which is chestnut protein powder; Mixing the chestnut protein powder and glucose powder and redissolving them in distilled water to obtain a chestnut protein fermentation substrate; The chestnut protein fermentation substrate is mixed with lactic acid bacteria for aerobic fermentation to obtain a chestnut protein postbiotic product.

2. The preparation method according to claim 1, characterized in that: The ratio of the chestnut protein powder: glucose powder: distilled water (g:g;mL) is 5:2:

100.

3. The preparation method according to claim 1, characterized in that: The lactic acid bacteria is any one of Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei, and Pediococcus acidilactici, and the effective viable count of the lactic acid bacteria is (1-10)×10 7 CFU / g chestnut protein powder.

4. The preparation method according to any one of claims 1 to 3, characterized in that: The aerobic fermentation conditions are a temperature of 37° C. and a time of 24 hours.

5. Chestnut protein postbiotics prepared by the preparation method according to any one of claims 1 to 4.

6. Use of the chestnut protein postbiotic as claimed in claim 5 in the preparation of a chronic inflammation drug.

7. The use according to claim 6, characterized in that: The chestnut protein postbiotic can inhibit the secretion of pro-inflammatory mediators by macrophages and intestinal epithelial cells, and improve the body's chronic inflammation-related glucose and lipid metabolism disorders and impaired intestinal barrier function.

8. Use of the chestnut protein postbiotic as claimed in claim 5 in the preparation of a drug for inhibiting the secretion of pro-inflammatory mediators by macrophages and intestinal epithelial cells.

9. Use of the chestnut protein postbiotic as claimed in claim 5 in the preparation of a drug for improving chronic inflammation-related glucose and lipid metabolism disorders and / or impaired intestinal barrier function.

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