Application of lactobacillus intestinalis and metabolites thereof in preparation of products for relieving high temperature stress

Abstract

The application discloses a lactobacillus intestinalis (Lactobacillus intestinalis) Lactobacillus intestinalis ) and metabolites thereof in the preparation of products to alleviate high temperature stress. The application obtains lactobacillus intestinalis and metabolites thereof by culture, detects the open field behavior of rats, collects rat blood samples, determines relevant indexes and performs statistical analysis, and the results show that supplementing live lactobacillus intestinalis or metabolites can significantly reduce the anxiety behavior of mice caused by high temperature stress, alleviate the reproductive damage of mice under high temperature, reduce the stress level of mice under high temperature, improve the energy metabolism level of mice, regulate the immunosuppressive inflammation of mice, and enhance the antioxidant capacity of mice, thereby confirming the alleviating effect of lactobacillus intestinalis and metabolites thereof on high temperature stress, and providing technical support for preventing and alleviating the health hazards of high temperature stress on animals and humans.

Description

Technical Field

[0001] This invention relates to an enteric lactobacillus, specifically to the application of enteric lactobacillus and its metabolites in the preparation of products to alleviate high-temperature stress. Background Technology

[0002] Climate change has become a major international issue affecting the stability of Earth's ecosystems and human health. The continued rise in global temperatures not only exacerbates biodiversity loss and reduces agricultural productivity, but also significantly threatens animal and human health through pathways such as heat stress, metabolic disorders, and immune imbalances. Therefore, developing novel intervention strategies and safe and effective protective measures to enhance the body's adaptability to high-temperature environments has become a crucial scientific and applied problem that urgently needs to be addressed.

[0003] Hainan Island exhibits typical tropical marine monsoon climate characteristics, with local animals chronically exposed to high temperatures and humidity. With the intensification of global warming, the frequency and duration of extreme heat events on Hainan Island have shown a significant upward trend. Wild brown rats, widely distributed in Hainan, may have evolved a series of physiological and behavioral adaptation mechanisms to cope with heat stress under long-term natural selection pressure. Therefore, we hypothesize that gut microbiota may play a crucial regulatory role in the adaptation of wild brown rats in Hainan to high-temperature environments. However, systematic research in this area remains lacking.

[0004] Currently, intervention strategies for human or animal adaptation to high-temperature environments mainly focus on three aspects. (1) Antioxidant and anti-stress drugs, such as vitamin C, vitamin E, polyphenolic compounds, melatonin, etc., mainly reduce the adverse effects of high temperature on the body by scavenging reactive oxygen species and alleviating oxidative stress damage. (2) Endocrine or neuromodulatory drugs, CN120837510A reduces cortisol secretion and alleviates oxidative damage by regulating the HPA axis through dexmedetomidine hydrochloride and gastrodin. (3) Functional additives and feed modifiers, CN107189947A discloses a method for preparing a Cordyceps feed additive. The feed additive obtained from the fermentation product retains the active functional components in Cordyceps to the maximum extent, and also contains highly active heat-resistant proteases, as well as functional active peptides and amino acids obtained by hydrolysis by the enzyme. Existing anti-high temperature related drugs or patents mostly focus on passively alleviating heat stress damage, while products that improve the body's intrinsic adaptability to high-temperature environments from the perspective of ecological, microbial, and host synergistic adaptation are still relatively lacking.

[0005] In recent years, the functional study of animal gut microbiota has become a hot topic in the life sciences. Numerous studies have shown that the gut microbiota has a profound impact on host physiological functions; its composition and metabolic functions can be significantly regulated by external environmental factors, and it participates extensively in key life processes such as host immune regulation, energy metabolism, behavioral regulation, and maintenance of reproductive function. However, research on the role and mechanisms of gut microbiota in host responses to high-temperature environmental stress, especially in wild animals, remains very limited.

[0006] In the past, regarding enterobacteria ( Lactobacillus intestinalis Research on this topic remains relatively limited, primarily focusing on areas related to immunity and inflammation. For example, in 2025, Sui et al. discovered that *Lactobacillus enterica* can inhibit CD8 in C57BL / 6N mice. + Abnormal infiltration of T cells and macrophages effectively alleviates chronic pancreatitis. A 2023 study showed that aldehyde dehydrogenase from *Lactobacillus enterica* co-metabolic with the host's ALDH1A2, promoting increased retinoic acid biosynthesis and thus mediating an anti-colitis effect through retinoic acid receptor α. Currently, there are no literature reports on the role of *Lactobacillus enterica* in assisting the host to cope with high-temperature environmental stress, especially whether *Lactobacillus enterica* can improve the host's adaptability under high-temperature conditions by improving host reproductive function, inhibiting anxiety responses, and alleviating inflammation, stress, and injury. Summary of the Invention

[0007] To address the aforementioned problems, this invention discloses an enterobacterial lactobacillus (... Lactobacillus intestinalis In the preparation of products to alleviate high-temperature stress, *Lactobacillus enterica*, as a potential probiotic, has significant advantages and can be used as a pharmaceutical, microbial agent, or feed additive.

[0008] This invention is achieved through the following technical solution: This invention first provides the application of Enterobacter ilex and its metabolites in the preparation of products that improve high temperature stress.

[0009] In some embodiments, the product for improving high-temperature stress is a product having any one or more of the functions in A1)-A5): A1) Inhibit anxiety behavior in animals under high temperatures; specifically, improve open field behavior in animals; A2) Alleviate reproductive damage in animals under high temperature; specifically, increase sperm density and increase the concentration levels of gonadotropin-releasing hormone, follicle-stimulating hormone, luteinizing hormone and testosterone along the hypothalamic-pituitary-gonadal axis (HPG). A3) Inhibit the stress level of animals under high temperature; specifically, reduce the concentration levels of adrenocorticotropic hormone-releasing hormone, adrenocorticotropic hormone, and corticosterone along the hypothalamic-pituitary-adrenal (HPA) axis; A4) Improve the energy metabolism level of animals; specifically, increase the concentration levels of leptin, triiodothyronine, and thyroxine (T4); A5) Alleviate inflammation and oxidative stress levels in animals under high temperature conditions; specifically, reduce the concentration levels of lipopolysaccharide, immunoglobulin A, and tumor necrosis factor α, and increase the concentration level of superoxide dismutase.

[0010] In some embodiments, the product is food, health product, topical skin agent, pharmaceutical, microbial agent, or feed additive.

[0011] In some embodiments, the product is a pharmaceutical product, which further includes pharmaceutically acceptable excipients, such as stabilizers, wetting agents, emulsifiers, binders, and isoosmotic agents.

[0012] In some embodiments, the dosage form of the drug is a tablet, granule, powder, capsule, solution, suspension, or lyophilized preparation.

[0013] In some embodiments, when the product is a topical skin agent, it is selected from the following forms: face cream, lotion, eye cream, face mask, serum.

[0014] Beneficial effects This invention, in a high-temperature stress experiment on rats, found that *Lactobacillus enterica* and its metabolites significantly improved anxiety-like behavior, alleviated reproductive damage caused by high temperatures, suppressed stress levels, increased energy metabolism, and reduced inflammation and oxidative stress in rats under high-temperature conditions. These results suggest that *Lactobacillus enterica* has potential application value in the prevention and treatment of diseases caused by high temperatures in animals. Attached Figure Description

[0015] Figure 1 The effects of *Lactobacillus enterica* and its metabolites on anxiety-like behavior in rats (open field behavior test); (A) time spent in the central region; (B) frequency of entering the central region; (C) latency to enter the central region; (D) distance traveled in the central region; (E) thermograms of activity in the open field for the five groups of rats; PBS represents rats receiving saline under high temperature; Li represents rats receiving live *Lactobacillus enterica* under high temperature; Heated represents rats receiving inactive *Lactobacillus enterica* under high temperature; CM represents rats receiving blank MRS medium containing *Lactobacillus enterica* under high temperature; L.iCM represents rats receiving the supernatant of MRS medium containing *Lactobacillus enterica* under high temperature (containing all metabolites produced by *Lactobacillus enterica* during culture). Figure 2The effects of *Lactobacillus enterica* and its metabolites on immunity, inflammation, stress, reproduction, metabolic status, and oxidative stress; (A) sperm density; (B) gonadotropin-releasing hormone (GnRH); (C) follicle-stimulating hormone (FSH); (D) luteinizing hormone (LH); (E) testosterone (T); (F) corticotropin-releasing hormone (CRH); (G) adrenocorticotropic hormone (ACTH); (H) corticosterone (CORT); (I) triiodothyronine (T3); (J) thyroxine (T4); (K) lipopolysaccharide (LPS); (L) tumor necrosis factor-α (TNF-α); (M) immunoglobulin A (IgA); (N) superoxide dismutase (SOD); (O) leptin. Detailed Implementation

[0016] 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.

[0017] 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, reagents, instruments, etc., used in the following examples are commercially available.

[0018] Enterobacteriaceae ( Lactobacillus intestinalis ATCC 49335 was purchased from Mingzhou Biotechnology Co., Ltd., Ningbo, China.

[0019] In one embodiment of the present invention, the basic feed used for rats is a product of Beijing Keao Biotechnology Co., Ltd.

[0020] MRS medium (i.e., blank medium group; Qingdao Haibo Biotechnology, catalog number HB0384-1) contains the following components: 10.0 g / L peptone, 8.0 g / L beef extract, 4.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L diammonium citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.04 g / L manganese sulfate, and 1.0 g / L Tween-80. Peptone, beef extract, and yeast extract provide nitrogen, vitamins, and growth factors; glucose is a fermentable sugar; dipotassium hydrogen phosphate is an acid-base buffer; and diammonium citrate, magnesium sulfate, manganese sulfate, Tween-80, and sodium acetate provide growth factors for culturing various lactobacilli. These components also inhibit certain contaminating bacteria and neutralize cytotoxic substances, providing a favorable growth environment for lactobacilli.

[0021] Growth curve determination of Lactobacillus enterica: 10 mL of Lactobacillus enterica culture was inoculated into a 90 mL Erlenmeyer flask containing MRS liquid medium and incubated at 37°C. Uninoculated MRS liquid medium was used as a blank control. Starting from 0 h, the OD600 of the culture was measured every 1 h, and samples were continuously taken until 48 h. Each sample was performed in triplicate. A smooth S-shaped growth curve was obtained by plotting the OD600 value on the ordinate and the incubation time on the abscissa.

[0022] Lactobacillus Count and Metabolite Preparation: Lactobacillus culturing for 16 hours was counted using the dilution plating method. Specifically, the original culture of Lactobacillus culturing for 16 hours was serially diluted 10-fold. ‑6 ~10 ‑8 Inoculation was performed, with three replicates for each dilution gradient. After thoroughly vortexing the diluted bacterial suspension, 100 μL of the suspension was quickly inoculated onto a prepared MRS plate and spread evenly with a spreader. After inoculation, the plate was left to stand for 10–30 min until the suspension was completely absorbed. The plate was then inverted and incubated at 37°C for 24–48 h. Colony counts were then performed (plates with 30–300 colonies were selected). After culturing *Lactobacillus enterica* in MRS liquid medium for 16 h, the suspension was evenly transferred to sterile centrifuge tubes and centrifuged at 3000 rpm for 10 min at 4°C. After centrifugation, the supernatant was pipetted into a sterile centrifuge tube to obtain the *Lactobacillus enterica* culture supernatant. Sterile PBS solution was added to the *Lactobacillus enterica* precipitate after removing the supernatant. The cells were thoroughly washed with a vortex mixer and resuspended by centrifugation twice to prepare a concentration of 1 × 10⁻⁶. 9 A suspension of CFU / mL (PBS as solvent).

[0023] The oral dose of enterobacterium lactis is 1×10 9 CFU / mL / day, orally for 15 consecutive days, then orally once every 3 days for a total of 15 days); High-temperature inactivation of enterobacteria: 1×10 9 CFU / mL Lactobacillus enterica was inactivated in a 100℃ water bath for 2 hours to prepare high-temperature inactivated Lactobacillus enterica; 1 mL / time / day for the first 15 days, and 1 mL / time / 3 days for the next 15 days. Enterobacterial lactobacillus culture medium supernatant: Enterobacterial lactobacillus content in the supernatant is 1×10⁻⁶. 9 CFU / mL; 1 mL / time / day, orally for 30 consecutive days.

[0024] Example 1 1. Enterobacterial colonization experiment 1.1 Experimental Procedure Twenty-four male SPF-grade rats weighing 160-200 g were purchased from Guangzhou Ruige Biotechnology Co., Ltd. and randomly divided into four groups (n=8 per group). Each group was housed individually in polycarbonate ventilated cages and acclimatized for 30 days under laboratory conditions of 32℃, a photoperiod of 12L:12D, a light intensity of 100-200 lux, and a humidity of 55 ± 5%. Before acclimatization, all four groups of rats were pretreated with antibiotics (50 μg / mL streptomycin, 100 μg / mL neomycin, and 100 U / mL penicillin; 1 mL / time / day) for 7 days to release space for the intestinal flora already colonized in the recipient rats, thus providing a site for the growth of the newly transplanted flora. During the subsequent 30-day high-temperature acclimatization, the PBS group received oral PBS suspension for 30 days (1 mL / time / day for the first 15 days, and 1 mL / time / 3 days for the next 15 days), and the Li group received oral live Lactobacillus (1×10⁻⁶) for 30 days. 9 CFU / mL, 1 mL / time / day for the first 15 days, 1 mL / time / 3 days for the next 15 days; Heated group orally administered high-temperature inactivated Lactobacillus enterica for 30 days (inactivated in a 100℃ water bath for 2 hours, 1×10⁻⁶ CFU / mL). 9 CFU / mL, 1 mL / day for the first 15 days, 1 mL / day for the next 15 days (3 days). During the experiment, rats had free access to maintenance feed (Beijing Keao Biotechnology Co., Ltd.) and purified water. After the *Lactobacillus enterica* colonization experiment, the rats' open field behavior (central area 45 cm × 45 cm) was tested within 5 minutes in a 75 cm × 75 cm × 50 cm acrylic plate. Rats were anesthetized with isoflurane between 8:00 and 10:00 and euthanized by decapitation. Fresh blood was immediately collected, centrifuged at 3000 rpm for 20 min, and the supernatant was used to detect serum reproductive hormones using ELISA. The left epididymal tail was harvested, and the sperm suspension was diluted 10-fold with 0.9% sterile saline. The rat sperm density was detected under a microscope using a hemocytometer.

[0025] 1.2 Experimental Results 1.2.1 Open field behavior One-way ANOVA results showed that, compared with the Heated group, the Li group had a higher frequency of entering the central region ( P = 0.020; Figure 1 B) and distance of activity in the central area ( P = 0.026; Figure 1 D) all increased significantly, and the latency period to enter the central region was significantly shortened. P = 0.045; Figure 1 (C) indicates that live lactobacillus can improve anxiety-like behavior in rats induced by heat stress. Furthermore, the time it took for rats to enter the central region did not differ significantly among the three groups, but the Li group showed an increasing trend compared to the other two groups (C). Figure 1A). Three sets of activity levels as follows: Figure 1 As shown in Figure E, the heatmap visually demonstrates that the time, distance, and frequency of entering the central region were higher in the Li group than in the PBS and Heated groups. Although the PBS group did not show significant differences from the other two groups, it exhibited an anxiety trend compared to the Li group, but was less anxious than the Heated group. These results indicate that live *Lactobacillus enterica* significantly improves anxiety in rats under high temperature, while heat-inactivated *Lactobacillus enterica* (inactivation of bacterial nucleic acid, retention of cell wall and unreleased intracellular molecules) has no effect on improving anxiety-like behavior in rats, and may even enhance it. Therefore, *Lactobacillus enterica* may improve anxiety-like behavior by producing metabolites.

[0026] 1.2.2 Sperm density One-way ANOVA (LSD) results showed that sperm density in the Li group was significantly higher than that in the PBS and Heated groups, indicating that live *Lactobacillus enterica* could improve sperm count reduction caused by high-temperature stress. Although the cell wall and intracellular unreleased molecules were preserved after the *Lactobacillus enterica* was killed by high temperature, these substances did not improve reproduction. Therefore, combining the results in 1.2.3 (see below), it is further confirmed that *Lactobacillus enterica* probiotics require an active state to exert their effect, and are ineffective in an inactive state. Therefore, we hypothesize that *Lactobacillus enterica* needs metabolites or secreted factors to alleviate reproductive damage in rats under high temperature.

[0027] 1.2.3 Serum Reproduction-Related Hormone Levels One-way ANOVA (LSD) results showed that gonadotropin-releasing hormone (GnRH) was present. Figure 2 B) Follicle-stimulating hormone (FSH); Figure 2 C) Luteinizing hormone (LH) Figure 2 D) Testosterone (T; Figure 2 E) The highest levels of gonadotropins were observed in the Li group and the lowest in the PBS group. Furthermore, the concentrations of gonadotropins in the Heated group were either lower or equal to those in the Li group, and vice versa. Specifically, GnRH: Li group > Heated group > PBS group; FSH and LH: Li group = Heated group > PBS group; T: Li group > PBS group = Heated group. P All values ​​were less than 0.001. These results indicate that live *Lactobacillus enterica* can improve heat-induced reproductive damage by upregulating the hypothalamic-pituitary-gonadal (HPG) axis, while heat-inactivated *Lactobacillus enterica* has a weaker effect on improving reproduction compared to live *Lactobacillus enterica*. Therefore, *Lactobacillus enterica* needs to rely on metabolites or secreted factors to alleviate reproductive damage in rats under high temperatures.

[0028] 1.2.4 Serum stress-related hormone levels One-way ANOVA (LSD) results showed that corticotropin-releasing hormone (CRH) was significantly elevated. Figure 2 F), Adrenocorticotropic hormone (ACTH); Figure 2 G) and corticosterone (CORT); Figure 2 H) was lowest in the Li group and highest in the PBS group, with the concentrations of stress-related hormones in the Heated group falling between those in the Li and PBS groups. Specifically, the order of stress-related hormone concentrations was PBS group > Heated group > Li group. P All values ​​were less than 0.01. These results indicate that live *Lactobacillus enterica* inhibits thermo-induced stress activation by suppressing the hypothalamic-pituitary-adrenal (HPA) axis, while thermo-inactivated *Lactobacillus enterica* has a weaker inhibitory effect on stress compared to live *Lactobacillus enterica*. Therefore, *Lactobacillus enterica* needs to rely on metabolites or secreted factors to alleviate the stress level in rats under high temperatures.

[0029] 1.2.5 Serum levels of hormones related to energy metabolism One-way ANOVA (LSD) results showed that triiodothyronine (T3; Figure 2 I) Thyroxine (T4; Figure 2 J) and leptin; Figure 2 O) was highest in the Li group and lowest in the PBS group, with the concentration of energy metabolism-related hormones in the Heated group falling between that of the Li and PBS groups. Specifically, the concentrations of all three energy metabolism-related hormones were: Li group > PBS group > Heated group. P All values ​​were less than 0.05. These results indicate that live *Lactobacillus enterica* significantly enhances the metabolic level of rats under high temperatures, while heat-inactivated *Lactobacillus enterica* has a weaker effect on enhancing metabolism compared to live *Lactobacillus enterica*. Therefore, *Lactobacillus enterica* needs to rely on metabolites or secreted factors to enhance the metabolic level of rats under high temperatures.

[0030] 1.2.6 Serum inflammation and oxidative stress levels One-way ANOVA (LSD) results showed that lipopolysaccharide (LPS)... Figure 2 K), tumor necrosis factor α (TNF-α); Figure 2 L) and immunoglobulin A (IgA); Figure 2 The levels of endotoxins (M) were lowest in the Li group and highest in the PBS group. Furthermore, the concentrations of endotoxins, pro-inflammatory factors, and immune-related proteins in the Heated group fell between those in the Li and PBS groups. Specifically, the levels of the three stress-related hormones were: PBS group > Heated group > Li group. Meanwhile, superoxide dismutase (SOD) levels were... Figure 2 SOD levels were highest in the Li group and lowest in the PBS group, with the Heated group falling in between. Specifically, the SOD levels were: Li group > PBS group > Heated group. PAll values ​​were less than 0.05. These results indicate that live *Lactobacillus enterica* significantly inhibited endotoxin LPS and pro-inflammatory factors in heat-stressed rats under high temperature, and significantly improved immune function and antioxidant capacity. In contrast, heat-inactivated *Lactobacillus enterica* showed weaker inhibitory and antioxidant capacity compared to live *Lactobacillus enterica*. Therefore, *Lactobacillus enterica* needs to rely on metabolites or secreted factors to inhibit inflammation levels in rats under high temperature and enhance their immune and antioxidant capacity under high-temperature conditions.

[0031] In summary, based on the results in sections 1.2.2-1.2.6 above, this patent demonstrates that active *Lactobacillus enterica* can improve reproductive, metabolic, immune, and antioxidant disorders in rats induced by heat stress, and possesses the ability to inhibit stress and inflammation. The anti-heat stress effect of inactivated *Lactobacillus enterica* is less than that of active *Lactobacillus enterica*. Therefore, *Lactobacillus enterica* may help animals resist or alleviate reproductive, metabolic, immune, and oxidative stress damage caused by heat stress by producing metabolites or secreting factors. Next, this patent focuses on verifying whether *Lactobacillus enterica* metabolites have the same effect (see *Lactobacillus enterica* metabolite oral administration experiment).

[0032] 2. Oral administration of enterobacterial metabolites 2.1 Experimental Procedure Sixteen male SPF-grade rats weighing 160-200 g were purchased from Guangzhou Ruige Biotechnology Co., Ltd. and randomly divided into two groups (n=8 per group). They were housed individually in polycarbonate ventilated cages and acclimatized for 30 days under laboratory conditions of 32℃, a photoperiod of 12L:12D, a light intensity of 100-200 lux, and a humidity of 55 ± 5%. During the 30-day high-temperature acclimatization period, the CM group was orally administered MRS culture medium (blank medium group, 1 mL / time / day) for 30 days, and the L.iCM group was orally administered 1×10⁻⁶ mcg of MRS culture medium for 30 days. 9 CFU / mL *Lactobacillus enterica* culture supernatant (conditioned medium group, 1 mL / time / day). During the experiment, rats had free access to maintenance diet (Beijing Keao Biotechnology Co., Ltd.) and purified water. After the oral administration of *Lactobacillus enterica* metabolites, rats' open field behavior was assessed over 5 minutes in a 75 cm × 75 cm × 50 cm acrylic plate. Rats were anesthetized with isoflurane between 8:00 and 10:00 and euthanized by decapitation. Fresh blood was immediately collected, and the supernatant was collected after centrifugation at 3000 rpm for 20 min for serum reproductive hormone detection. The left epididymal tail was harvested, and the sperm suspension was diluted 10-fold with 0.9% sterile saline. Rat sperm density was measured under a microscope using a hemocytometer.

[0033] 2.2 Experimental Results 2.2.1 Open field behavior tThe test results showed that, compared with the oral culture medium control group (CM), the latency time for entering the central region of the oral Lactobacillus metabolite group (L.iCM) was significantly shorter. P = 0.047; Figure 1 C) is significantly shortened, and the time to enter the central region ( Figure 1 A) Frequency ( Figure 1 B) and activity distance ( Figure 1 D) all show an upward trend (but not significantly, all) P >0.05), indicating that the metabolites of *Lactobacillus enterica* have a role in improving anxiety-like behaviors induced by heat stress. The activity levels of the two groups were as follows: Figure 1 As shown in E, the heatmap visually demonstrates that the L.iCM group enters the central region at a higher time, distance, and frequency than the CM group.

[0034] 2.2.2 Sperm density t The test results showed that, compared with the CM group, the sperm density in the L.iCM group was significantly increased ( P = 0.027; Figure 2 (A) indicates that enterobacterial lactobacillus metabolites play an important role in improving sperm density.

[0035] 2.2.3 Serum levels of reproductive-related hormones t The test results showed that, compared with the CM group, the serum GnRH (increased blood glucose) in the L.iCM group was significantly lower. P <0.001; Figure 2 B), FSH P <0.001; Figure 2 C), LH ( P <0.001; Figure 2 D) and T ( P <0.001; Figure 2 E) were significantly upregulated, indicating that Enterobacterial lactobacillus metabolites can improve reproductive function damage in rats caused by high temperature by upregulating the HPG axis, which is consistent with the effect of live Enterobacterial lactobacillus, further demonstrating that Enterobacterial lactobacillus can improve the decline in reproductive performance caused by high temperature through its metabolites.

[0036] 2.2.4 Serum stress-related hormone levels t The test results showed that, compared with the CM group, the serum CRH (C6H2O) in the L.iCM group was significantly lower. P <0.001; Figure 2 F), ACTH P <0.001; Figure 2 G) and CORT ( P <0.001; Figure 2Both H) were significantly downregulated, and these results are consistent with the effects of live *Lactobacillus enterica*, meaning that the reduction was consistent with that of live *Lactobacillus enterica* compared to their respective control groups. These results indicate that *Lactobacillus enterica* can alleviate stress levels by inhibiting the HPA axis through its metabolites.

[0037] 2.2.5 Serum levels of hormones related to energy metabolism t The test results showed that, compared with the CM group, the serum T3 in the L.iCM group was lower. P <0.001; Figure 2 I), T4 ( P <0.001; Figure 2 J) and Leptin P <0.001; Figure 2 All levels of O (oxidation) increased significantly, consistent with the effects of live *Lactobacillus enterica*, meaning the increase was similar to that of the control groups. These results indicate that *Lactobacillus enterica* can increase the levels of energy metabolism-related hormones through its metabolites.

[0038] 2.2.6 Serum inflammation and oxidative stress levels t The test results showed that, compared with the CM group, the serum LPS (increased by serum iodine) in the L.iCM group was significantly lower. P <0.001; Figure 2 K), TNF-α ( P <0.001; Figure 2 L) and IgA ( P <0.001; Figure 2 Serum SOD (M) levels were significantly decreased in the L.iCM group compared to the CM group. P <0.001; Figure 2 (N) increased significantly. These results are consistent with the effects of live *Lactobacillus enterica*, meaning that the decrease or increase was consistent with that of the respective control groups. These results indicate that *Lactobacillus enterica* metabolites can significantly inhibit the increase in endotoxin LPS and pro-inflammatory factor levels in rats induced by heat stress, and can enhance the immune and antioxidant stress capabilities of rats under heat stress. These results suggest that *Lactobacillus enterica* reduces the level of inflammation in rats induced by heat stress through its metabolites, and enhances the immune and antioxidant capabilities of rats under high-temperature environments.

[0039] In summary, *Lactobacillus enterica* and its metabolites can improve anxiety behavior, reproductive function impairment, metabolic levels, and oxidative stress levels induced by heat stress, while also inhibiting heat-induced stress and inflammation, indicating that *Lactobacillus enterica* has the effect of improving heat stress. This invention has excellent application prospects in the preparation of products for improving heat stress.

[0040] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways without departing from its spirit and scope, and without requiring unnecessary experiments, under equivalent parameters, concentrations, and conditions. Although specific embodiments have been given, 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. Enterobacteriaceae ( Lactobacillus intestinalis Application of ) and its metabolites in the preparation of products to alleviate high temperature stress.

2. The application according to claim 1, characterized in that, The aforementioned product for improving high-temperature stress is a product having any one or more functions from A1) to A5): A1) Inhibit anxiety behavior in animals under high temperatures; A2) Alleviating reproductive damage in animals under high temperatures; A3) Inhibits stress levels in animals under high temperatures; A4) Improve the energy metabolism level of animals; A5) Relieves inflammation and oxidative stress levels in animals under high temperatures.

3. The application according to claim 2, characterized in that, The application of suppressing anxiety behavior in animals under high temperatures aims to improve open field behavior in animals.

4. The application according to claim 2, characterized in that, The application of alleviating reproductive damage in animals under high temperature is to increase sperm density or increase the concentration levels of gonadotropin-releasing hormone, follicle-stimulating hormone, luteinizing hormone, and testosterone along the hypothalamic-pituitary-gonadal axis.

5. The application according to claim 2, characterized in that, The application of inhibiting stress levels in animals under high temperatures involves reducing the concentrations of adrenocorticotropic hormone-releasing hormone, adrenocorticotropic hormone, and corticosterone along the hypothalamic-pituitary-adrenal axis.

6. The application according to claim 2, characterized in that, The application of improving animal energy metabolism involves increasing the concentrations of leptin, triiodothyronine, and thyroxine (T4).

7. The application according to claim 1, characterized in that, The application of reducing inflammation and oxidative stress in animals under high temperature involves lowering the concentration levels of lipopolysaccharide, immunoglobulin A, and tumor necrosis factor α, while increasing the concentration level of superoxide dismutase.

8. The application according to claim 1, characterized in that, The products mentioned are food, health products, topical skin agents, fungicides, pharmaceuticals, or feed additives.

9. The application according to claim 8, characterized in that, The product is a pharmaceutical product, which also includes pharmaceutically acceptable excipients, which are one or more of stabilizers, wetting agents, emulsifiers, binders, and isotonic agents.

10. The application according to claim 9, characterized in that, The dosage form of the drug is tablet, granule, powder, capsule, solution, suspension, or lyophilized preparation.

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