Application of fermented functional feed of Jiang-flavor liquor lees in improving uterine immune homeostasis of female animals in intestinal mucositis environment

Abstract

This invention discloses the application of a soy sauce-flavored fermented distillers' grains-fermented functional feed in improving uterine immune homeostasis in female animals under conditions of enteritis. This invention belongs to the field of biotechnology, specifically relating to the application of a soy sauce-flavored fermented distillers' grains-fermented functional feed in improving uterine immune homeostasis in female animals under conditions of enteritis. This invention provides applications of the soy sauce-flavored fermented distillers' grains-fermented functional feed in improving systemic inflammation and / or stress in subjects; in the preparation of drugs for preventing and / or treating and / or improving systemic inflammation and stress in subjects; and in maintaining uterine immune homeostasis in subjects. The soy sauce-flavored fermented distillers' grains-fermented functional feed has promising application prospects for uterine repair and maintaining uterine immune homeostasis in the context of enteritis.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the application of a soy sauce-flavored fermented functional feed to improve uterine immune homeostasis in female animals under conditions of enteritis. Background Technology

[0002] In the physiological processes of female animals, there is a close relationship between gut health and reproductive system function. Recent studies have shown that when animals are in a state of enteritis, chemical stimulation, or continuous stress, the damage to the intestinal barrier and immune imbalance are not limited to the gut itself, but may also affect the immune homeostasis of distal organs through inflammatory factors, immune mediators, and microbial-related molecules, especially having an adverse effect on the local immune environment of reproductive organs such as the uterus.

[0003] In cases of intestinal inflammation, the uterus may experience disordered immune signaling, enhanced inflammation recognition, and imbalances in repair pathways, thereby affecting the normal immune homeostasis and functional maintenance of the endometrium. This can potentially impact the reproductive or egg production performance of female animals. However, current feed or nutritional intervention technologies primarily aim to improve gut health or overall production performance, and systematic research and patent protection for the application of directly improving uterine immune homeostasis in the context of intestinal mucosal inflammation remain relatively limited.

[0004] The lees produced during the production of Maotai-flavor liquor are a representative agricultural byproduct, rich in protein, amino acids, polyphenols, and various fermentation derivatives, possessing the potential for development and utilization as a feed resource. Appropriate microbial fermentation treatment can further improve its nutritional composition and bioavailability. However, existing technologies have not explicitly disclosed the use of fermented Maotai-flavor liquor lees as functional feed for regulating uterine immune homeostasis in female animals under conditions of intestinal mucositis. Therefore, it is necessary to provide a new application scheme to address the problem of impaired uterine immune homeostasis in female animals under intestinal inflammation or stress conditions. Summary of the Invention

[0005] The main problem this invention aims to solve is how to improve systemic inflammation and stress environment in subjects.

[0006] To address the aforementioned problems, this invention provides the following applications of fermented functional feed made from soy sauce-flavored distiller's grains: 1. Application of fermented functional feed with soy sauce aroma in improving systemic inflammation and stress in subjects.

[0007] 2. Application of fermented functional feed with soy sauce aroma in the preparation of drugs for the prevention and / or treatment or / or improvement of systemic inflammation and stress environment in subjects.

[0008] Furthermore, the systemic inflammation is intestinal mucositis; the stress environment is an intestinal mucositis environment.

[0009] The aforementioned intestinal mucositis can significantly disrupt the local immune homeostasis of the uterus without causing obvious morphological damage.

[0010] The positive manifestations of enteromucositis were as follows: although the overall morphology of the subject's uterine tissue was highly consistent with that of the negative control group, significant changes were observed at the molecular level, including downregulation of mTOR signaling in the uterine tissue, enhanced TLR4-mediated inflammation recognition, imbalance of chemokine and anti-inflammatory factor expression, and abnormal activation of apoptosis-proliferation-related pathways.

[0011] This invention also provides the application of a soy sauce-flavored fermented functional feed in maintaining uterine immune homeostasis in subjects.

[0012] This invention also provides the application of fermented functional feed with soy sauce aroma in the preparation of drugs to improve or maintain uterine immune homeostasis in subjects.

[0013] Furthermore, maintaining the subject's uterine immune homeostasis involves enhancing inflammation recognition, reducing systemic inflammatory stress, restoring barrier function from a stress state to homeostasis, and maintaining immune regulatory balance.

[0014] The maintenance of uterine immune homeostasis can be manifested in the following ways: 1) The number of vacuoles in the cytoplasm of uterine glandular epithelial cells in the treatment group treated with the soy sauce-flavored fermented lees functional feed slightly increased, and the secretion and functional activity of uterine glands were mildly promoted without inducing inflammation or abnormal proliferation; 2) The soy sauce-flavored fermented lees functional feed can promote the reconstruction of uterine repair-related signaling pathways more effectively than dry baijiu lees feed in the context of enteritis, which is conducive to maintaining the integrity and functional stability of uterine epithelium; 3) The ability to inhibit the recognition of local uterine inflammation and excessive recruitment of immune cells helps to reduce secondary uterine immune activation caused by enteritis; 4) The soy sauce-flavored fermented lees functional feed caused the high level of IL-10 induced by enteritis to show a downward trend, reflecting the reduction of systemic inflammatory pressure, and the transition of the uterus from a "high-load anti-inflammatory compensation state" to a "balanced immune homeostasis state".

[0015] The immune regulation is manifested in the following way: through the synergistic effect of multiple targets, it corrects the abnormal anti-apoptotic state and inflammatory signal disorder of the endometrium, thereby restoring the physiological apoptosis-proliferation and immune balance.

[0016] In this article, the genes related to immune homeostasis, inflammation recognition, barrier function and immune regulation include mTOR, TLR4, MUC2, ZO-1, CXCL2 and IL-10.

[0017] In this study, proteins associated with apoptosis, inflammation, and immune regulation include Bcl-2, Caspase-3, NF-κB, IFN-γ, and STAT3.

[0018] Among them, the imbalance of immune regulation was manifested by downregulation of NF-κB and IFN-γ, and STAT3 was overactivated in the positive control group of intestinal mucositis.

[0019] The above applications are not for disease diagnosis. These applications are not intended to directly obtain disease diagnoses or health status results in living humans or animals.

[0020] The above applications are for non-disease treatment purposes. These applications are not intended to restore or restore health or reduce suffering in living human or animal bodies.

[0021] The samples to be tested in the above applications can be samples from non-living human or animal bodies.

[0022] In this article, the subjects include human or non-human animals, or cells, blood, tissues, body fluids or secretions derived from human or non-human mammals, such as humans, monkeys, mice, rats, rabbits, donkeys, cattle, horses, pigs or dogs.

[0023] In this article, the functional feed made from fermented lees of soy sauce-flavored liquor is obtained by microbial fermentation of lees of soy sauce-flavored liquor. The microorganisms include a compound microbial agent composed of one or more of yeast, Bacillus subtilis and Aspergillus niger.

[0024] Furthermore, in the compound microbial agent of the soy sauce-flavored fermented functional feed, the ratio of yeast, Bacillus subtilis and Aspergillus niger is 2:1:1.

[0025] In one specific embodiment, the preparation method of the soy sauce-flavored fermented functional feed can be as follows: 1) Select the lees produced during the production of fresh sauce-flavored baijiu as raw materials.

[0026] First, the lees are dried with hot air at 60-70℃ to control their moisture content below 12%. The dried lees are then crushed and passed through a 40-60 mesh sieve to obtain the lees fermentation substrate.

[0027] 2) Yeast, Bacillus subtilis, and Aspergillus niger were used as a compound microbial inoculant, mixed in a 2:1:1 ratio. The total inoculant dosage was controlled at 1×10⁻⁶ per gram of fermentation substrate. 8 ~1×10 9CFU. Adjust the moisture content of the fermentation substrate to 45%–55%, inoculate with compound microbial agents and mix thoroughly, then place in a fermentation pan for solid-state fermentation.

[0028] Fermentation temperature was controlled at 30–34 °C, and initial pH was controlled between 5.5 and 6.5. Oxygen supply was ensured during fermentation by intermittent turning of the compost pile every 12 hours, with a fermentation time of 48–96 hours. Fermentation was terminated when the pH of the fermentation system stabilized at 4.2–4.8 and the fermentation characteristics became stable.

[0029] 3) After fermentation, the material is dried at a low temperature below 60 ℃, then crushed and passed through a 40-mesh sieve to obtain the finished product of fermented functional feed with soy sauce aroma.

[0030] In this article, the soy sauce-flavored fermented functional feed can be a product of Guizhou Maotai Distillery (Group) Circular Economy Industry Investment and Development Co., Ltd., and the feed production license number of the product is: Qian Si Zheng (2023) 02093.

[0031] In this article, the amount of the soy sauce-flavored fermented functional feed added to animal diets can be 0.8% to 2.0%.

[0032] Compared with the prior art, the present invention has at least the following beneficial effects: 1) Clearly distinguish between enteritis and non-enteritis states, and elucidate the systemic impact of enteritis on uterine immune homeostasis; 2) Without causing structural damage to the uterus, fermented functional feed with soy sauce aroma can gently regulate the uterine function. 3) Compared with dry liquor lees feed, sauce-flavored liquor lees fermented functional feed is more effective in restoring suppressed uterine mTOR signals under enteritis conditions; 4) Soy sauce-flavored fermented functional feed has a significant advantage in inhibiting excessive uterine inflammation recognition and chemotactic signals; 5) By regulating the expression of barrier-related molecules, the uterine immune response is transformed from "stress compensation" to "homeostasis maintenance"; 6) At the level of immune regulation, functional feeds fermented with soy sauce-flavored distillers' grains can more precisely reshape the balance of uterine immune factors; 7) By regulating apoptosis-proliferation and inflammation signaling pathways through multiple targets, it systematically improves enteritis-related uterine immune imbalance. Attached Figure Description

[0033] Figure 1 Uterine morphology and structure of mice fed with different distillers' grains feed after H&E staining.

[0034] Figure 2This represents the mRNA expression level of a protein related to mouse uterine tissue.

[0035] Figure 3 This represents the expression level of a key protein in the mouse uterus. Detailed Implementation

[0036] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0038] Unless otherwise specified, the quantitative experiments in the following examples are all repeated three times, and the results are averaged.

[0039] The yeast used in the following examples was purchased from the China General Microbiological Culture Collection Center, accession number CGMCC No. 2.1882.

[0040] The Bacillus subtilis used in the following examples was purchased from the China General Microbiological Culture Collection Center, accession number CGMCC No. 28017.

[0041] The Aspergillus niger used in the following examples was purchased from the China General Microbiological Culture Collection Center, accession number CGMCC No. 41129.

[0042] The following examples use statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA test was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0043] Example 1: Application of fermented functional feed with soy sauce aroma and distillers' grains in maintaining uterine immune homeostasis under conditions of enteritis. 1. Preparation method of functional feed made from fermented distiller's grains with soy sauce aroma The lees produced during the production of fresh soy sauce-flavored baijiu are selected as raw materials. First, the lees are dried with hot air at 60-70℃ to control their moisture content below 12%. The dried lees are then crushed and passed through a 40-60 mesh sieve to obtain the lees fermentation substrate.

[0044] Yeast, Bacillus subtilis, and Aspergillus niger are used as a composite microbial inoculum, and the three strains are mixed in a ratio of 2:1:1 before use. The total inoculation amount of the composite inoculum is controlled at 1×10 8 ~1×10 9 CFU per gram of fermentation substrate.

[0045] The moisture content of the distiller's grains fermentation substrate is adjusted to 45% - 55%. After inoculating the composite microbial inoculum and thoroughly mixing, it is placed in a fermentation tray for solid-state fermentation.

[0046] The fermentation temperature is controlled at 30 - 34 °C, and the initial pH is controlled between 5.5 - 6.5. During the fermentation process, oxygen supply is ensured by intermittent turning of the pile, turning the pile once every 12 hours, and the fermentation time is 48 - 96 hours. Fermentation ends when the pH of the fermentation system stabilizes at 4.2 - 4.8 and the fermentation characteristics tend to be stable.

[0047] After fermentation, the material is subjected to low-temperature drying treatment below 60 °C, dried and then crushed and passed through a 40-mesh sieve to obtain the finished product of the functional feed for fermented sauce-flavored distiller's grains. The relevant products are already on the market (Production License Number: Qian Si Zheng (2023) 02093).

[0048] 2. Determination of polar metabolite and free amino acid components in feed before and after fermentation of sauce distiller's grains (1) Determination method In the determination of polar metabolites, freeze-drying and pulverization, methanol / water solvent extraction are used, and then multiple metabolites, including amino acids, organic acids, and water-soluble vitamins, are analyzed by liquid chromatography-mass spectrometry (LC / MS). According to the results of polar metabolites, the content of free functional amino acids is further determined by targeted quantitative metabolomics.

[0049] (2) Test results Table 1. Targeted metabolomics determination of free amino acids in dry baijiu distiller's grains feed and fermented distiller's grains feed

[0050] The content of free functional amino acids is determined by targeted quantitative metabolomics. The results are shown in Table 1: The distiller's grains feed contains rich functional amino acids; compared with the dry baijiu distiller's grains feed, the contents of 14 functional amino acids in the metabolites of the fermented distiller's grains feed have increased significantly.

[0051] 3. Construction of an intestinal mucositis environment (verification model) An intestinal mucositis environment was constructed by intraperitoneal injection of 5-fluorouracil (5-FU). 5-FU was dissolved in sterile PBS solution (concentration 0.01 mol / L, pH 7.4), and female mice (Beijing Vital River Laboratory Animal Technology Co., Ltd., catalog number: SCXK (Beijing) 2021-0006) were intraperitoneally injected at a dose of 40 mg / kg body weight for 5 consecutive days. The control group was injected with an equal volume of PBS. This method was used to form a systemic inflammatory and stress environment in mice to induce disturbances in uterine immune homeostasis and to verify the practicality of this study. The intestinal mucositis model was only used as a background condition and not as an evaluation endpoint for the technical effects of this invention.

[0052] 4. Effects of fermented functional feed from Jiangxiang-type distiller's grains on uterine immune homeostasis in an intestinal mucositis environment 1) Experimental animals and grouping Female C57BL / 6J mice aged 5 - 6 weeks (Beijing Vital River Laboratory Animal Technology Co., Ltd., catalog number: SCXK (Beijing) 2021-0006) were selected. After adaptive feeding, they were randomly divided into the following 4 groups, with no less than 15 mice in each group: Negative control group: basal diet + PBS injection; Model control group: basal diet + 5-FU injection; Dry Jiangxiang-type distiller's grains control group: basal diet + 1% unfermented Jiangxiang-type distiller's grains + 5-FU injection; Experimental group: basal diet + 1% fermented functional feed from Jiangxiang-type distiller's grains + 5-FU injection.

[0053] 2) Collection of uterine tissue The mice were sacrificed on the 3rd day after the 5-FU injection. Under sterile conditions, the abdominal cavity was quickly opened to separate the intact uterine tissue, and the surrounding fat and connective tissue were removed. After rinsing with sterile PBS, the samples were processed.

[0054] Part of the uterine tissue was placed in 4% paraformaldehyde fixative for histological analysis, and the rest was immediately frozen in liquid nitrogen and transferred to be stored at 80 °C for RNA and protein detection.

[0055] 3) Morphological observation of uterine tissue Approximately 2 cm of uterine tissue from each group of mice was fixed with 4% paraformaldehyde, dehydrated through gradients, embedded in paraffin, and sectioned with a thickness of 4 - 5 μm. After the sections were stained with hematoxylin - eosin (H&E), they were scanned using a digital slide scanning system, and the endometrial structure, gland morphology, stromal state, and inflammatory cell infiltration were observed under the same magnification.

[0056] The morphological structure of uterine tissue from each group of mice after H&E staining is as follows: Figure 1 As shown: By comprehensively comparing the uterine tissue sections of each group, the overall morphology of the uterus in the positive control group of intestinal mucositis was highly consistent with that in the negative control group of intestinal mucositis. No obvious endometrial thinning, glandular destruction or large-scale infiltration of inflammatory cells was observed, indicating that the intestinal mucositis model established in this study did not cause direct morphological damage to the uterus.

[0057] Based on this, the uterine tissue structure in the 1% dry distillers' grains feed group was similar to that in the negative control group, suggesting that at this level, unfermented distillers' grains had a limited impact on uterine tissue morphology. In the 1% soy sauce-flavored distillers' grains fermented functional feed group, a slight increase in the number of intracytoplasmic vacuoles in some uterine glandular epithelial cells was observed, suggesting a slight increase in glandular secretory activity; however, endometrial thickness, matrix state, and inflammatory infiltration did not show significant changes.

[0058] The above results indicate that, in the context of enteritis, fermented functional feed made from fermented mash of soy sauce-flavored liquor may have a mild regulatory effect on the functional state of uterine glands through the active components produced during fermentation, without causing structural damage to the uterus.

[0059] 4) Detection of gene expression related to uterine immune homeostasis (qRT-PCR) Uterine tissue samples of approximately 2 cm were collected from each group of mice, quickly washed, and cryopreserved in liquid nitrogen. Total RNA was extracted from the uterine tissue, reverse transcribed into cDNA, and the mRNA expression levels of genes related to immune homeostasis, inflammation recognition, barrier function, and immune regulation, including mTOR, TLR4, MUC2, ZO-1, CXCL2, and IL-10, were detected using qRT-PCR. Six biological replicates were set up for each group.

[0060] The relative mRNA expression levels of relevant genes in the uterine tissues of mice in each group are as follows: Figure 2 As shown: In the negative control group of enteritis, uterine tissue mTOR expression remained at a high level; while in the positive control group of enteritis, mTOR expression was significantly reduced, suggesting that intestinal inflammation can inhibit local metabolic and repair-related signals in the uterus. Adding 1% dry distillers' grains feed did not significantly restore mTOR expression; however, adding 1% fermented functional feed of soy sauce-flavored distillers' grains showed a significant upward trend in uterine mTOR expression compared to both the positive control group of enteritis and the dry distillers' grains group, indicating that it is more beneficial for maintaining uterine repair capacity under the background of enteritis.

[0061] Regarding the expression of the inflammation recognition receptor TLR4, both the negative and positive control groups of enteritis showed high TLR4 expression levels, while the TLR4 expression in the fermented functional feed group of soy sauce-flavored liquor lees was significantly reduced. The dry liquor lees feed did not show a significant regulatory effect on TLR4 expression, suggesting that fermentation treatment significantly enhanced the ability of liquor lees feed to inhibit excessive uterine inflammation recognition signals.

[0062] Regarding barrier-related genes, the expression of MUC2 and ZO-1 was significantly increased in the enteritis-positive control group and the dry liquor lees feed group, reflecting that the uterine epithelium was in a compensatory defense state; while the expression of MUC2 and ZO-1 was relatively decreased in the sauce-flavored liquor lees fermentation functional feed group and approached the non-enteritis level, indicating that it helps to reduce systemic inflammatory pressure and restore the uterine barrier function from a stress state to homeostasis.

[0063] Regarding chemokines and anti-inflammatory factors, the functional feed fermented with soy sauce-flavored distillers' grains restored CXCL2 expression to a state close to that of non-enteritis, while also causing IL-10 to show a downward trend on the basis of high expression induced by enteritis, reflecting that it can more effectively regulate the balance of uterine immune factors.

[0064] 5) Detection of expression of proteins related to uterine immunity and repair (Western Blot) Total protein was extracted from the uterine tissue of mice in each group, and the expression levels of proteins related to apoptosis, inflammation and immune regulation in the uterus were detected by Western blotting, including Bcl-2, Caspase-3, NF-κB, IFN-γ and STAT3.

[0065] The expression bands and grayscale analysis results of key proteins in the uterine tissues of mice in each group are as follows: Figure 3 As shown: Compared with the negative control group of enteritis, the positive control group of enteritis showed downregulation of NF-κB and IFN-γ, suggesting that the endometrium was in a state of immune regulation imbalance.

[0066] In the dry baijiu lees feed group, the above-mentioned abnormal changes were not significantly improved; while in the sauce-flavored baijiu lees fermentation functional feed group, Caspase-3 expression was significantly increased and Bcl-2 expression was inhibited, indicating that it helps to restore the physiological apoptosis-proliferation balance of uterine cells.

[0067] Regarding inflammation and immune signals, the expression of NF-κB and IFN-γ decreased in the positive control group of enteritis, suggesting impaired local immune surveillance in the uterus; while the expression of NF-κB and IFN-γ showed an upward trend in the functional feed group fermented with soy sauce-flavored lees, indicating that it helps to restore the normal immune defense and repair signals of the uterus.

[0068] Furthermore, STAT3 was overactivated in the positive control group of enteritis, while the functional feed containing fermented baijiu (distillers' grains) significantly reduced STAT3 expression levels. The regulatory effect of dry baijiu feed on STAT3 was relatively limited, indicating that fermented baijiu has a significant advantage in inhibiting abnormal inflammation and proliferative signals related to enteritis. Under enteritis conditions, compared with the model control group, the experimental group supplemented with the functional feed containing fermented baijiu showed an overall regulatory trend in the expression of immune homeostasis-related genes and proteins in uterine tissue, suggesting that this feed can improve the uterine immune microenvironment in the context of intestinal inflammation.

[0069] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application of fermented functional feed with soy sauce aroma in improving systemic inflammation and / or stress in subjects.

2. Application of fermented functional feed with soy sauce aroma in the preparation of drugs for the prevention and / or treatment and / or improvement of systemic inflammation and stress environment in subjects.

3. The application according to claim 1 or 2, characterized in that, The systemic inflammation is intestinal mucositis; the stressful environment is an intestinal mucositis environment.

4. Application of fermented functional feed with soy sauce aroma in maintaining uterine immune homeostasis in subjects.

5. Application of fermented functional feed with soy sauce aroma in the preparation of drugs to improve or maintain uterine immune homeostasis in subjects.

6. The application according to claim 4 or 5, characterized in that, Maintaining uterine immune homeostasis in the subject involves enhancing inflammation recognition, reducing systemic inflammatory stress, restoring barrier function from a stress state to homeostasis, and balancing immune regulation.

7. The application according to any one of claims 1-6, characterized in that: The soy sauce-flavored fermented functional feed is made from soy sauce-flavored liquor lees through microbial fermentation. The microorganisms include a compound microbial agent composed of one or more of yeast, Bacillus subtilis, and Aspergillus niger.

8. The application according to claim 7, characterized in that: In the compound microbial agent of the soy sauce-flavored fermented functional feed, the ratio of yeast, Bacillus subtilis and Aspergillus niger is 2:1:

1.

9. The application according to any one of claims 1-6, characterized in that: The amount of the soy sauce-flavored fermented functional feed added to the animal diet is 0.8% to 2.0%.

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