A heat stress-resistant probiotic preparation and its application

Abstract

This invention belongs to the field of probiotic functional technology. This invention discloses a heat stress-resistant probiotic, which was deposited on May 12, 2022, at the China Culture Collection Center for Microbial Cultures, located at No. 3, Courtyard 1, Beichen West Road, Beijing, with accession number CGMCC NO. 24883, and classified as *Enterococcus faecalis*. This invention primarily addresses the problem of intestinal damage caused by heat stress in animals.

Description

Technical Field

[0001] This invention belongs to the field of microbial engineering, specifically relating to the field of probiotic functional technology. Background Technology

[0002] Heat stress refers to the sum of non-specific physiological responses of an organism to heat exposure under high-temperature conditions. When livestock and poultry are excessively exposed to high temperatures, various physiological and pathological changes occur. The inability of livestock and poultry to effectively dissipate heat leads to symptoms such as open-mouth breathing, lethargy, decreased feed intake, and slowed weight gain; in severe cases, it can cause death, thus challenging animal welfare. During heat stress, animal body temperature rises, metabolism increases, and more free radicals are produced. The physiological balance between the production and elimination of free radicals within the body or cells is disrupted, leading to the accumulation of reactive oxygen species that damage cells and harm the animal's body, ultimately resulting in decreased livestock and poultry production performance and impacting economic benefits. Numerous studies have shown that antioxidants can reduce or alleviate cell damage and even apoptosis through free radical scavenging. However, most commercially available chemically synthesized antioxidants have side effects; therefore, finding natural antioxidants with few or no side effects is of great importance.

[0003] Probiotics are live microorganisms that can improve the host's associated or surrounding microbial flora, enhance feed nutrition, strengthen the host's resistance to disease infection, or improve the quality of the living environment. They can maintain the balance of the intestinal flora and improve the body's disease resistance through biological oxygen depletion, competitive inhibition, and the production of acidic substances. Developing a heat stress-resistant probiotic will significantly improve the livestock industry's ability to cope with heat stress in animals. Summary of the Invention

[0004] This invention discloses a heat stress-resistant probiotic, which was deposited on May 12, 2022, at the China Culture Collection Center for Microbial Cultures, located at No. 3, Courtyard 1, Beichen West Road, Beijing, with accession number CGMCC NO.24883 and classified as Enterococcus faeca lis. This invention primarily addresses the problem of intestinal damage caused by heat stress in animals.

[0005] The technical solution of this invention is as follows:

[0006] 1. Prepare MRS culture medium plates, spread the serially diluted bovine rumen fluid onto the culture plates, and invert them in an anaerobic incubator at 39°C for 1-2 days until lactic acid bacteria-like colonies grow.

[0007] 2. Select the lactic acid bacteria-like colonies with superior growth, and perform streak isolation and purification until single colonies are obtained.

[0008] The beneficial effects of this invention are: it has antioxidant and anti-inflammatory effects, and can alleviate the damage to the intestinal tissue structure of animals caused by heat stress. Attached Figure Description

[0009] Figure 1 Jejunal structures of mice in each group (HE staining, 100×): A: Blank group; B: Heat-treated model group; C: Low-dose probiotic group; D: Medium-dose probiotic group; E: High-dose probiotic group. Specific Implementation

[0010] Verification of the heat stress resistance function of isolated lactic acid bacteria

[0011] 1. Healthy, disease-free 4-week-old Kunming mice were selected as experimental animals. The experiment was divided into 5 groups: Group 1 was the blank control group, which was raised at an ambient temperature of 25℃; Group 2 was the heat stress model group, which was heat-treated in a constant temperature incubator at 42℃ for 2 hours every day; and Groups 3-5 were the Enterococcus faecalis test groups.

[0012] The experimental mice had unlimited access to water and feed. Each group consisted of 6 mice. Group 1 served as a blank control group, receiving the same dose of physiological saline as the experimental groups. Group 2 was treated at 42℃ for 2 hours daily. Groups 3-5 were administered Enterococcus faecalis (concentration 1×10⁻⁶) via gavage. 7 CFU / mL, 1×10 8 CFU / mL, 1×10 9 After 1 hour, the group (CFU / mL) was subjected to heat treatment simultaneously with group 2.

[0013] All bacterial solutions used in the experiment were prepared on the same day. One tube of bacterial solution of different concentrations was prepared each day to observe the physiological status and survival of the mice, and records were kept every day.

[0014] 2. Rectal temperature detection

[0015] The rectal temperature of mice was recorded before and after heat treatment.

[0016] The rectal temperatures of mice are shown in Table 1. Before heat treatment, there was no difference between the control group and the model group (p>0.05). Compared with the model group, the high-dose group orally administered with Enterococcus faecalis showed a significant difference (p<0.05), while the low- and medium-dose groups showed no difference (p>0.05). After heat treatment, the rectal temperature of the model group was significantly higher than that of the control group (p<0.05). Compared with the model group, the rectal temperature of the low- and medium-dose groups orally administered with Enterococcus faecalis was lower (p<0.05), with the medium-dose group showing the largest decrease in temperature.

[0017]

[0018] Table 1 Rectal temperature of mice. Note: Multiple comparisons were performed using the Duncan method. Different lowercase letters superscripted in the same row indicate significant differences between groups (P<0.05), while the same lowercase letter indicates no significant differences between groups (P>0.05). Data are expressed as mean ± standard error.

[0019] 3. Detection of serum markers in mice

[0020] On the last day after heat treatment, blood was collected from mice in each group by enucleation. After the blood coagulated for 1 hour at room temperature, it was centrifuged at 3500 rpm for 10 minutes to collect serum, which was then stored at -20°C. The expression levels of IL-1, IL-10, IL-6, and TNF-α were detected using a kit.

[0021] The effects of various serum indicators are shown in Table 2. The results showed that, compared with the blank group, the serum levels of pro-inflammatory factors TNF-α, IL-1, and IL-6 in the model group were significantly increased (p<0.01), while the level of anti-inflammatory factor IL-10 was significantly decreased (p<0.01), indicating that the model group mice exhibited an inflammatory response during heat treatment. Additionally, oral administration of Enterococcus faecalis significantly increased the level of anti-inflammatory factor IL-10 and significantly decreased the levels of pro-inflammatory factors TNF-α, IL-1, and IL-6 in each dose group compared with the model group. This indicates that oral administration of Enterococcus faecalis can reduce the levels of inflammation-related blood indicators and increase the levels of anti-inflammatory factors, thus exerting an anti-inflammatory effect. Among these, the medium-dose group showed the best effect, with serum indicator levels approaching those of the blank control group.

[0022]

[0023] Table 2. Serum Inflammatory Factor Levels in Mice. Note: Multiple comparisons were performed using the Duncan method. Different lowercase letters superscripted in the same row indicate significant differences between groups (P<0.05), while the same lowercase letter indicates no significant differences between groups (P>0.05). Data are expressed as mean ± standard error.

[0024] 4. HE staining to detect jejunal tissue damage

[0025] After the animals were sacrificed, jejunal tissue was collected, washed with physiological saline to remove intestinal contents, fixed in 4% paraformaldehyde, and embedded in paraffin. The integrity of the intestinal structure of mice in each group was observed. The section results for each group are shown below. Figure 1 The results showed that the jejunum structure in the control group was intact and the villi were densely packed. Compared with the control group, the heat-treated group showed villus breakage and shortening. The jejunum structure in the group administered Enterococcus faecalis was intact compared with the model group. The low- and high-dose groups showed a small amount of villus breakage, but the structure was intact. The medium-dose group showed intact structure and densely packed villi. This indicates that administration of Enterococcus faecalis can maintain the integrity of the intestinal structure.

[0026] The experimental results show that oral administration of Enterococcus faecalis to heat stress model mice has anti-inflammatory and antioxidant effects, can alleviate intestinal tissue damage caused by heat stress, and plays an anti-heat stress role.

Claims

1. A heat stress-resistant probiotic, characterized by: Preservation number CGMCC NO.24883, classification and name: Enterococcus faecalis ( Enterococcusfaecalis ).

2. The use of the probiotics as described in claim 1 in the preparation of a drug to address intestinal damage caused by heat stress in mice.

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