Feed for improving the anti-transport stress ability of livestock and poultry and preparation method thereof

Abstract

The present application belongs to the technical field of feed additives, and particularly relates to a feed for improving the anti-transportation stress capacity of livestock and poultry and a preparation method thereof. The feed comprises a basic daily ration and an anti-transportation stress additive. The anti-transportation stress additive is prepared by collecting the fermentation products after stage-by-stage fermentation of Aspergillus oryzae CICC 40214, Bacillus licheniformis CICC 10332 and Lactobacillus rhamnosus CICC 6137, and then drying and crushing. Through the stage-by-stage fermentation process of a specific time sequence, the three strains are synergistically used, and the prepared anti-transportation stress additive can significantly reduce the cortisol level in the serum of livestock and poultry, increase the content of immunoglobulin G and interferon-gamma, effectively relieve the transportation stress of mutton sheep, pigs and laying hens, and improve the production performance of the laying hens after stress. The present application is suitable for stress prevention and control in large-scale livestock and poultry breeding.

Description

Technical Field

[0001] This invention belongs to the field of feed additive technology, specifically relating to a feed that improves the resistance of livestock and poultry to transportation stress and its preparation method. Background Technology

[0002] Stress refers to a series of non-specific, systemic adaptive protective mechanisms triggered by various stimuli from the internal and external environment. These physiological changes are collectively known as General Adaptation Syndrome (GAS), also called "asymptomatic disease." Its mechanism of action permeates almost all tissues, organs, and cells of the body, constituting a highly programmed process of bodily adaptation and regulation. Based on the physiological response process, stress can be divided into three stages: the alarm phase, the resistance phase, and the exhaustion phase. Mild stress typically only triggers the alarm and resistance phases, allowing the body to recover through self-regulation. However, if subjected to continuous, high-intensity harmful stress, the body enters the exhaustion phase, leading to endocrine disorders, homeostasis imbalance, organ damage, and even inducing disease, shock, or death.

[0003] Under large-scale, intensive livestock and poultry farming models, stress has become one of the core factors restricting the healthy development of the livestock industry and causing economic losses. After transportation, meat sheep may experience significant dehydration, spiked cortisol levels, muscle damage, abnormal body temperature, and sharp weight loss. Low-temperature transportation in winter can also lead to deterioration in the quality of livestock and poultry muscle.

[0004] Pigs, due to underdeveloped sweat glands and abundant subcutaneous fat, have extremely poor heat dissipation capabilities and are highly sensitive to transportation stress. Heat stress, jolting, and oxygen deprivation during transport can lead to abnormal breathing and body temperature, and a significant decrease in feed intake. This not only delays sexual maturity and impairs reproductive performance in young sows but also induces deterioration in pork quality. In the egg-laying hen industry, the large-scale separation of rearing and egg production makes the inter-farm transport and intra-farm relocation of young laying hens a necessity. Transportation stress can cause a sharp drop in the immunity of laying hens, a decline in egg production performance, and an increase in soft-shelled, rough-shelled, deformed, and broken eggs. It can also induce secondary respiratory and intestinal diseases, significantly prolonging the recovery period and significantly increasing breeding losses.

[0005] There are currently no reports of specialized feeds that target and improve oxidative stress and immune function in livestock and poultry, are safe and residue-free, and are suitable for livestock and poultry transportation stress scenarios. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention has developed a feed and its preparation method that enhances the resistance of livestock and poultry to transportation stress by optimizing a three-strain staged compound fermentation process and a specific culture medium.

[0007] On the one hand, the present invention provides an anti-transportation stress additive, which is prepared by staged fermentation of a fermentation medium;

[0008] The fermentation medium contains soybean residue, buckwheat bran powder, rice bran powder, and sugarcane bagasse powder; The staged fermentation process sequentially includes Aspergillus oryzae (… Aspergillus oryzae CICC 40214 fermentation stage, Bacillus licheniformis ( Bacillus licheniformis CICC 10332 fermentation stage and Lactobacillus rhamnosus ( Lactobacillus rhamnosus CICC 6137 fermentation stage.

[0009] Furthermore, in the anti-transportation stress additive, the mass ratio of soybean residue, buckwheat bran powder, rice bran powder, and sugarcane bagasse powder in the fermentation culture medium is 60~70:5~10:3~5:1~5.

[0010] Also provided is a feed to improve the resistance of livestock and poultry to transportation stress, said feed comprising a basal diet and the anti-transportation stress additive as described in claim 1 or 2.

[0011] Furthermore, in the feed, the amount of the anti-transportation stress additive added to the basal diet is 0.02% to 0.03%.

[0012] Furthermore, a method for preparing the anti-transportation stress additive of the present invention is provided, characterized by comprising the following steps: Step 1: Prepare Aspergillus oryzae separately Aspergillus oryzae Seed liquid, Bacillus licheniformis Bacillus licheniformis Seed culture and Lactobacillus rhamnosus Lactobacillus rhamnosus Seed liquid of the Aspergillus oryzae Aspergillus oryzae The accession number is CICC 40214, and the Bacillus licheniformis described therein is... Bacillus licheniformis The accession number is CICC 10332, and the Lactobacillus rhamnosus described therein is Lactobacillus rhamnosus. Lactobacillus rhamnosus Its accession number is CICC 6137; Step 2: Prepare the fermentation medium; Step 3: The Aspergillus oryzae prepared in Step 1... Aspergillus oryzae The seed culture was inoculated into the fermentation medium prepared in step two, and fermented for 1 to 2 days at 28-32℃ and an aeration rate of 1.2-1.5 vvm. Step 4: The Bacillus licheniformis prepared in Step 1 Bacillus licheniformis The seed culture was inoculated into the fermentation medium in step three and fermented for 1 to 2 days at 37°C and an aeration rate of 0.8 to 1.0 vvm. Step 5: The Lactobacillus rhamnosus prepared in Step 1... Lactobacillus rhamnosus The seed culture was inoculated into the fermentation medium in step four and fermented at 37°C for 1-2 days. Step 6: Collect the fermentation product from Step 5, dry and pulverize it to obtain the anti-transportation stress additive. The drying temperature is 44~45℃.

[0013] Furthermore, in the preparation method, the fermentation culture medium in step two comprises soybean residue, buckwheat bran powder, rice bran powder and sugarcane bagasse powder, with a mass ratio of 60~70:5~10:3~5:1~5.

[0014] Furthermore, in the preparation method, the fermentation culture medium in step two is prepared by mixing soybean residue, buckwheat bran powder, rice bran powder and sugarcane bagasse powder in a mass ratio of 60~70:5~10:3~5:1~5, adjusting the moisture content to 25%~30%, and then sterilizing.

[0015] Furthermore, in the preparation method, the Aspergillus oryzae mentioned in step three... Aspergillus oryzae The inoculation amount of the seed culture is 5%~6% of the volume of the fermentation medium; the Bacillus licheniformis mentioned in step four Bacillus licheniformis The inoculation amount of the seed culture is 4%~8% of the volume of the fermentation medium; the *Lactobacillus rhamnosus* mentioned in step five... Lactobacillus rhamnosus The inoculation amount of the seed liquid is 5% to 10% of the volume of the fermentation medium.

[0016] It also provides the application of the anti-transportation stress additives or feeds described in this invention in improving the anti-transportation stress ability of livestock and poultry.

[0017] Furthermore, in the aforementioned application, the livestock and poultry are sheep, pigs, or laying hens.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention utilizes a specific time-sequential, staged fermentation process involving Aspergillus oryzae, Bacillus licheniformis, and Lactobacillus rhamnosus to enable the three strains to exert synergistic effects, resulting in an anti-transport stress additive that synergistically enhances the stress resistance of livestock and poultry. Experiments show that in transport stress models of meat sheep, meat pigs, and laying hens, the additive prepared in this invention can significantly reduce serum cortisol (COR) levels by approximately 27%–39%, increase immunoglobulin G (IgG) levels by approximately 42%–60%, and increase interferon-γ (IFN-γ) levels by approximately 78%–93%, significantly improving the stress resistance of livestock and poultry.

[0019] (2) Through comparative experiments, this invention has demonstrated that the anti-stress effect of the fermentation products obtained by omitting any one of the strains of Aspergillus oryzae, Bacillus licheniformis or Lactobacillus rhamnosus, or by changing the inoculation order of specific strains, is deteriorated to varying degrees.

[0020] (3) In the egg-laying hen transportation stress model, the additive prepared in this invention can increase the egg production rate from 83.17% to 90.87%~91.51% within 100 days after transportation, with a relative increase of 9.3%~10.0%; the feed conversion ratio is reduced from 2.64 to 2.48~2.53, and the feed conversion efficiency is significantly improved. This indicates that this invention can not only alleviate the immediate physiological damage to livestock and poultry caused by transportation stress, but also effectively improve the long-term production performance after stress. Detailed Implementation

[0021] The technical solution of the present invention will be described below with reference to the embodiments. However, the present invention is not limited to the following embodiments.

[0022] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described below with reference to specific embodiments, but the embodiments are not intended to limit the present invention.

[0023] Unless otherwise specified, the experimental and detection methods described in the following embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0024] Lactobacillus rhamnosus Lactobacillus rhamnosus, Preservation number: CICC 6137, hereinafter referred to as Lactobacillus rhamnosus CICC 6137; Aspergillus oryzae Aspergillus oryzae, Preservation number: CICC 40214; Bacillus licheniformis Bacillus licheniformis, Accession number CICC 10332. The above strain was purchased from the China Industrial Microbial Culture Collection Center.

[0025] Example 1 This example describes the preparation of seed culture for Lactobacillus rhamnosus CICC 6137.

[0026] Lactobacillus rhamnosus CICC 6137, stored at -80℃, was inoculated into MRS broth medium and cultured at 37℃ and 60 rpm for 16 h. The culture was then inoculated into fresh MRS broth medium at a volume fraction of 5% and cultured at 37℃ and 100 rpm for 20-24 h to obtain Lactobacillus rhamnosus CICC 6137 seed culture.

[0027] Example 2 This example describes the preparation of Aspergillus oryzae CICC 40214 seed culture.

[0028] The Aspergillus oryzae CICC 40214, stored at -80℃, was streaked onto Czapek's agar slant medium and activated by incubation at 28℃ for 48 h. One loopful of activated Aspergillus oryzae CICC 40214 was then inoculated into a 250 mL shake flask containing 100 mL of Czapek's broth medium and incubated at 28℃ and 230 rpm for 36 h to obtain the Aspergillus oryzae CICC 40214 seed culture.

[0029] Example 3 This example describes the preparation of seed culture for Bacillus licheniformis CICC 10332.

[0030] Bacillus licheniformis CICC 10332, stored at -80℃, was streaked onto nutrient broth agar slant and activated by incubation at 37℃ for 24 hours. One loopful of activated Bacillus licheniformis CICC 10332 was then inoculated into a 250mL shake flask containing 50mL of nutrient broth and incubated at 37℃ on a shaker at 200r / min for 24 hours to obtain the seed culture of Bacillus licheniformis CICC 10332.

[0031] Example 4 This example demonstrates the preparation of a fermentation medium.

[0032] Buckwheat bran, rice bran, and sugarcane bagasse were dried at 70℃ to a moisture content of 18%–20%, then pulverized and passed through a 100-mesh sieve to obtain buckwheat bran powder, rice bran powder, and sugarcane bagasse powder, respectively. Fresh soybean residue was dehydrated to a moisture content of 55% to obtain soybean residue. Soybean residue, buckwheat bran powder, rice bran powder, and sugarcane bagasse powder were mixed evenly in a mass ratio of 70:5:3:5 to prepare a fermentation substrate. Water (30% of the total mass of the fermentation substrate) was added and mixed thoroughly. The mixture was then autoclaved at 121℃ for 30 minutes to obtain the fermentation medium, labeled as Fermentation Medium #1.

[0033] Example 5 This example demonstrates the preparation of a fermentation medium.

[0034] Buckwheat bran, rice bran, and sugarcane bagasse were dried at 65℃ to a moisture content of 18%–20%, then pulverized through a 110-mesh sieve to obtain buckwheat bran powder, rice bran powder, and sugarcane bagasse powder, respectively. Fresh soybean residue was dehydrated to a moisture content of 60% to obtain soybean residue. Soybean residue, buckwheat bran powder, rice bran powder, and sugarcane bagasse powder were mixed evenly in a mass ratio of 65:10:5:2 to prepare a fermentation substrate. Water (28% of the total mass of the fermentation substrate) was added and mixed thoroughly. The mixture was then autoclaved at 121℃ for 30 minutes to obtain the fermentation medium, labeled as Fermentation Medium #2.

[0035] Example 6 This example demonstrates the preparation of a fermentation medium.

[0036] Buckwheat bran, rice bran, and sugarcane bagasse were dried at 60℃ to a moisture content of 18%–20%, then pulverized and passed through a 110-mesh sieve to obtain buckwheat bran powder, rice bran powder, and sugarcane bagasse powder, respectively. Fresh soybean residue was dehydrated to a moisture content of 58% to obtain soybean residue. Soybean residue, buckwheat bran powder, rice bran powder, and sugarcane bagasse powder were mixed evenly in a mass ratio of 60:8:4:1 to prepare a fermentation substrate. Water (25% of the total mass of the fermentation substrate) was added and mixed thoroughly. The mixture was then autoclaved at 121℃ for 30 minutes to obtain the fermentation medium, labeled as Fermentation Medium #3.

[0037] Example 7 This embodiment describes the preparation of an anti-stress additive through compound fermentation.

[0038] The Aspergillus oryzae CICC 40214 seed culture prepared in Example 2 was inoculated into the No. 1 fermentation medium prepared in Example 4 at a volume fraction of 5%. The medium was fermented at 30°C with an aeration rate of 1.5 vvm and stirred once every hour. After fermentation for 1 day, the Bacillus licheniformis CICC 10332 seed culture prepared in Example 3 was added at a volume fraction of 4%. The temperature was raised to 37°C with an aeration rate of 1.0 vvm and stirred once every 12 hours. After fermentation for another 2 days, the Lactobacillus rhamnosus CICC 6137 seed culture prepared in Example 1 was added at a volume fraction of 5%. The medium was then allowed to ferment statically at 37°C for 2 days. After fermentation was completed, the fermentation product was collected and dried at 45°C with a low-temperature forced-air drying process until the moisture content was ≤10%. The product was then pulverized and passed through an 80-mesh sieve to obtain the No. 1 anti-stress additive.

[0039] Example 8 This embodiment describes the preparation of an anti-stress additive through compound fermentation.

[0040] The Aspergillus oryzae CICC 40214 seed culture prepared in Example 2 was inoculated into the No. 2 fermentation medium prepared in Example 5 at a volume fraction of 6%. After fermentation at 30°C with an aeration rate of 1.3 vvm and stirring once every hour for 2 days, the Bacillus licheniformis CICC 10332 seed culture prepared in Example 3 was added at a volume fraction of 8%. The temperature was raised to 37°C with an aeration rate of 0.9 vvm and stirring once every 12 hours. After fermentation continued for 1 day, Lactobacillus rhamnosus CICC 6137 seed culture prepared in Example 1 was added at a volume fraction of 10%. The mixture was allowed to ferment statically at 37°C for 1 day. After fermentation was completed, the fermentation product was collected and dried at 44°C with a low-temperature forced-air drying process until the moisture content was ≤10%. The product was then pulverized and passed through an 80-mesh sieve to obtain the No. 2 anti-stress additive.

[0041] Example 9 This embodiment describes the preparation of an anti-stress additive through compound fermentation.

[0042] The Aspergillus oryzae CICC 40214 seed culture prepared in Example 2 was inoculated into the No. 3 fermentation medium prepared in Example 6 at a volume fraction of 6%. After fermentation at 30°C with an aeration rate of 1.2 vvm and stirring once every hour for 1 day, the Bacillus licheniformis CICC 10332 seed culture prepared in Example 3 was added at a volume fraction of 7%. The temperature was raised to 37°C with an aeration rate of 0.8 vvm and stirring once every 12 hours for 2 days. After fermentation, Lactobacillus rhamnosus CICC 6137 seed culture prepared in Example 1 was added at a volume fraction of 7%. The mixture was allowed to ferment at 37°C for 2 days. After fermentation, the fermentation product was collected and dried at 45°C with a low-temperature forced-air drying process until the moisture content was ≤10%. The product was then pulverized and passed through an 80-mesh sieve to obtain the No. 3 anti-stress additive.

[0043] Comparative Example 1 The difference between this comparative example and Example 9 is that the Aspergillus oryzae CICC 40214 fermentation was omitted.

[0044] The Bacillus licheniformis CICC 10332 seed culture prepared in Example 3 was added to fermentation medium #3 at a volume fraction of 7%. The culture was fermented at 37°C with an aeration rate of 0.8 vvm and stirred once every 12 hours. After 2 days of fermentation, the Lactobacillus rhamnosus CICC 6137 seed culture prepared in Example 1 was added at a volume fraction of 7%. The culture was then allowed to ferment at 37°C for 3 days. After fermentation, the fermentation product was collected and dried at 45°C with a forced air dryer until the moisture content was ≤10%. The product was then pulverized and passed through an 80-mesh sieve to obtain anti-stress additive contrast agent #1.

[0045] Comparative Example 2 The difference between this comparative example and Example 9 is that the fermentation of Bacillus licheniformis CICC 10332 was omitted.

[0046] The Aspergillus oryzae CICC 40214 seed culture prepared in Example 2 was inoculated into the No. 3 fermentation medium prepared in Example 6 at a volume fraction of 6%. The medium was fermented at 30°C with an aeration rate of 1.2 vvm and stirred once per hour. After 2 days of fermentation, the temperature was raised to 37°C, and the Lactobacillus rhamnosus CICC 6137 seed culture prepared in Example 1 was added at a volume fraction of 7%. The medium was then allowed to ferment at 37°C for 3 days. After fermentation, the fermentation product was collected, dried at 45°C with a forced air dryer until the moisture content was ≤10%, and then pulverized through an 80-mesh sieve to obtain the No. 2 anti-stress additive contrast agent.

[0047] Comparative Example 3 The difference between this comparative example and Example 9 is that the fermentation of Lactobacillus rhamnosus CICC 6137 was omitted.

[0048] The Aspergillus oryzae CICC 40214 seed culture prepared in Example 2 was inoculated into the No. 3 fermentation medium prepared in Example 6 at a volume fraction of 6%. The medium was fermented at 30°C with an aeration rate of 1.2 vvm and stirred once every hour for 2 days. Then, the Bacillus licheniformis CICC 10332 seed culture prepared in Example 3 was added at a volume fraction of 7%. The temperature was raised to 37°C with an aeration rate of 0.8 vvm and stirred once every 12 hours. Fermentation was continued for 3 days. The fermentation was then stopped, and the fermentation product was collected. The fermentation product was dried at 45°C with a low-temperature forced air drying until the moisture content was ≤10%. The product was then pulverized and passed through an 80-mesh sieve to obtain the No. 3 anti-stress additive contrast agent.

[0049] Comparative Example 4 The difference between this comparative example and Example 9 is that it does not use staged fermentation, but uses three strains for co-fermentation.

[0050] The Aspergillus oryzae CICC 40214 seed culture prepared in Example 2 was added at a volume fraction of 6%, the Bacillus licheniformis CICC 10332 seed culture prepared in Example 3 was added at a volume fraction of 7%, and the Lactobacillus rhamnosus CICC6137 seed culture prepared in Example 1 was added at a volume fraction of 7% to the No. 3 fermentation medium prepared in Example 6. The medium was fermented at 32°C with an aeration rate of 0.2 vvm and stirred once per hour for 5 days. After fermentation, the fermentation products were collected, dried at 45°C with a forced air dryer until the moisture content was ≤10%, and then pulverized through an 80-mesh sieve to obtain the No. 4 anti-stress additive contrast agent.

[0051] Comparative Example 5 The difference between this comparative example and Example 9 lies in the order of inoculation of the bacterial strains.

[0052] The Bacillus licheniformis CICC 10332 seed culture prepared in Example 3 was added to the No. 3 fermentation medium prepared in Example 6 at a volume fraction of 7%. The medium was fermented at 37°C with an aeration rate of 1.2 vvm and stirred once every 12 hours. After fermentation for 2 days, the Aspergillus oryzae CICC 40214 seed culture prepared in Example 2 was added at a volume fraction of 6%. The medium was fermented at 30°C with an aeration rate of 0.8 vvm and stirred once every hour. After fermentation for another day, the Lactobacillus rhamnosus CICC 6137 seed culture prepared in Example 1 was added at a volume fraction of 7%. The medium was then allowed to ferment statically at 37°C for 2 days. After fermentation, the fermentation product was collected, dried at 45°C with a low-temperature forced-air drying process until the moisture content was ≤10%, and then pulverized through an 80-mesh sieve to obtain the No. 5 anti-stress additive contrast agent.

[0053] Comparative Example 6 The difference between this comparative example and Example 9 lies in the order of inoculation of the bacterial strains.

[0054] The Lactobacillus rhamnosus CICC 6137 seed culture prepared in Example 1 was added to the No. 3 fermentation medium prepared in Example 6 at a volume fraction of 7%. After static fermentation at 37°C for 2 days, Bacillus licheniformis CICC 10332 seed culture prepared in Example 3 was added at a volume fraction of 7%. Fermentation was continued at 37°C with an aeration rate of 0.8 vvm and stirring once every 12 hours. After fermentation continued for 2 days, Aspergillus oryzae CICC 40214 seed culture prepared in Example 2 was added at a volume fraction of 6%. Fermentation was continued at 30°C with an aeration rate of 1.2 vvm and stirring once every hour. After fermentation continued for 1 day, fermentation was stopped, and the fermentation product was collected. The fermentation product was dried at 45°C with a low-temperature forced air drying until the moisture content was ≤10%. The product was then pulverized and passed through an 80-mesh sieve to obtain the No. 6 anti-stress additive contrast agent.

[0055] Application Example 1 Test location: Yuncheng City, Shanxi Province.

[0056] One hundred young mutton sheep of similar age were transported together from Ulanqab City, Inner Mongolia Autonomous Region to Yuncheng City, Shanxi Province, over a 12-hour journey. Two weeks prior to transport, the sheep were randomly divided into 10 groups: a blank control (KC); experimental groups treated with anti-stress additive #1 (Group 1), #2 (Group 2), and #3 (Group 3); and control groups treated with anti-stress additives #1 through #6. Different anti-stress additives were initially added to the basal diet at 0.03% of the basal diet for two consecutive weeks. After transport, blood was collected from the jugular vein, and serum levels of cortisol (COR), immunoglobulin G (IgG), and interferon-γ (IFN-γ) were measured using an ELISA kit from Kelu (Wuhan) Biotechnology Co., Ltd., to assess the anti-stress performance of different additives.

[0057] COR was used to assess the intensity of stress activation, IgG was used to evaluate humoral immune damage, and IFN-γ was used to reflect the level of cellular immunosuppression. The results are shown in Tables 1-4.

[0058] Table 1. Detection results of serum indicators in mutton sheep after treatment with different anti-stress additives.

[0059] Note: The values ​​in the table are averages, and the CV within a group is less than 5%.

[0060] As shown in Table 1, control group #1 lacked the Aspergillus oryzae fermentation step, and its COR was 105.35, significantly higher than that of experimental groups #1-#3; its IgG was 13.55 and IFN-γ was 28.17, both significantly lower than those of experimental groups #1-#3. Control group #2 lacked the Bacillus licheniformis fermentation step, and its COR was 95.32, IgG was 15.98, and IFN-γ was 30.05. Although this was an improvement over control group #1, it still showed a significant difference compared to the experimental groups. Control group #3 lacked the Lactobacillus rhamnosus fermentation step, and its COR was 90.19, the lowest among the six control groups; its IgG was 16.04 and IFN-γ was 27.33. Although the first three indicators were relatively good in the control group, the IFN-γ level was only 27.33. Control group #4 used a three-strain simultaneous fermentation method, with a COR of 98.31, IgG of 15.17, and IFN-γ of 26.11. This group had the lowest IFN-γ level among all control groups. This indicates that when three strains are inoculated and fermented simultaneously, the stress resistance of the final product decreases significantly. Control groups #5 and #6 changed the inoculation order of the strains. The former had a COR of 97.02, IgG of 16.11, and IFN-γ of 31.25; the latter had a COR of 98.13, IgG of 15.97, and IFN-γ of 30.98. The indicators of the two control groups were significantly worse than those of the three experimental groups, indicating that the specific order determined in this invention, namely, "first inoculating with the aerobic fungus Aspergillus oryzae, then with the facultative aerobic Bacillus licheniformis, and finally with the anaerobic Lactobacillus rhamnosus", conforms to the physiological characteristics and metabolic patterns of the three strains. Changing the order disrupts the synergistic relationship between the strains, resulting in a significant decrease in the stress activation of the fermentation products.

[0061] The anti-stress additives #1 to #3 prepared in this invention can effectively alleviate transportation stress in meat sheep. Compared with the blank control group, the COR of the three experimental groups decreased by approximately 32% to 35%, IgG increased by approximately 52% to 59%, and IFN-γ increased by approximately 87% to 93%, with all indicators showing highly significant improvements. The absence of any one of Aspergillus oryzae, Bacillus licheniformis, or Lactobacillus rhamnosus, or alteration of the specific inoculation sequence, leads to varying degrees of deterioration in the anti-stress effect.

[0062] Application Example 2 Test location: Datong City, Shanxi Province.

[0063] One hundred adult fattening pigs of similar age were randomly divided into 10 groups: a blank control (KC); experimental groups treated with anti-stress additive #1 (Group 1), anti-stress additive #2 (Group 2), and anti-stress additive #3 (Group 3); and control groups treated with anti-stress additives #1 through #6 (Groups 1 through #6). Different anti-stress additives were initially added to the basal diet at 0.025% of the basal diet for two consecutive weeks. The pigs were then transported, and after 12 hours of transport, blood was collected from the jugular vein. Serum from each group was separated, and the levels of cortisol (COR), immunoglobulin G (IgG), and interferon-γ (IFN-γ) in the serum were measured using an ELISA kit from Kelu (Wuhan) Biotechnology Co., Ltd., to evaluate the anti-stress performance of different anti-stress additives. The test results are shown in Table 2.

[0064] Table 2. Results of serum markers in pigs after treatment with different anti-stress additives.

[0065] Note: The values ​​in the table are averages, and the CV within a group is less than 5%.

[0066] As shown in Table 2, after feeding transported pigs with the anti-stress additives No. 1 to No. 3 prepared in this invention for 2 weeks, the serum COR decreased by approximately 27% to 30% compared with the blank control group, IgG increased by 49% to 61%, and IFN-γ, which enhances cellular immune function, increased by 78% to 93%. This indicates that the anti-stress additives No. 1 to No. 3 have a significant alleviating effect on transport stress in pigs.

[0067] Application Example 3 Test location: Datong City, Shanxi Province.

[0068] One hundred healthy 17-week-old Hy-Line Brown laying hens of similar weight were randomly divided into 10 groups: a blank control (KC); experimental groups treated with anti-stress additive #1 (Group 1), anti-stress additive #2 (Group 2), and anti-stress additive #3 (Group 3); and control groups treated with anti-stress additives #1 through #6 (Groups 1 through #6). Different anti-stress additives were initially added to the basal diet at 0.02% of the basal diet for two consecutive weeks. The hens were then transferred during the summer, and after 6 hours of transport, blood was collected from the jugular vein. Serum from each group was separated, and the levels of cortisol (COR), immunoglobulin G (IgG), and interferon-γ (IFN-γ) in the serum were measured using an ELISA kit from Kelu (Wuhan) Biotechnology Co., Ltd., to evaluate the anti-stress performance of different anti-stress additives. The test results are shown in Table 3. The daily egg production, daily egg weight, and daily feed consumption of each group of hens were continuously observed and recorded for 100 days after transportation. The egg production rate and feed conversion ratio of each group of hens after transportation were calculated and statistically analyzed, and the results are shown in Table 4. The calculation formula is as follows:

[0069]

[0070] Table 3. Results of serum markers in laying hens after treatment with different anti-stress additives.

[0071] Note: The values ​​in the table are averages, and the CV within a group is less than 5%.

[0072] As shown in Table 3, the anti-stress additives 1# to 3# prepared in this invention have a significant effect on alleviating transportation stress in laying hens. Compared with the blank control group, the cortisol (COR) level in the three experimental groups decreased by about 31% to 40%, the immunoglobulin G (IgG) level increased by about 42% to 47%, and the interferon-γ (IFN-γ) level increased by about 86% to 92%, indicating that the stress intensity was effectively inhibited.

[0073] Comparison of the control groups (groups 1-3) with the missing strains revealed that the absence of any one of *Aspergillus oryzae*, *Bacillus licheniformis*, or *Lactobacillus rhamnosus*, or a change in the inoculation order, led to varying degrees of deterioration in each indicator. This indicates that the timing of co-fermentation of the strains is crucial for the production of active metabolites. Overall, the anti-stress additive treatment groups prepared in Examples 1-3 of this invention showed significantly better performance in all indicators than the six control groups and the blank control group, confirming that the additive prepared by this invention through a specific staged fermentation process can synergistically improve the stress resistance of laying hens in multiple dimensions.

[0074] Table 4. Results of egg production performance tests in laying hens after treatment with different anti-stress additives.

[0075] Table 4 shows that the egg production rate of experimental groups 1# to 3# was 90.87% to 91.51%, which was 9.3% to 10.0% higher than that of the blank control group (83.17%), indicating that the additive of this invention can effectively alleviate the inhibition of egg production performance of laying hens by transportation stress. The egg production rate of the six control groups was 85.31% to 88.51%, all lower than that of the experimental groups. Among them, the egg production rate of control groups 1# and 2# was about 86%, indicating that each strain in the staged fermentation process plays an irreplaceable role in the final recovery of egg production performance. The feed conversion ratio of experimental groups 1# to 3# was 2.48 to 2.53, all lower than that of the blank control group (2.64), indicating higher feed conversion efficiency. The feed conversion ratio of control groups 1# to 6# was 2.55 to 2.62, generally higher than that of the experimental groups. Among them, the feed conversion ratio of control group 4# was 2.63, further confirming that the sequential inoculation of strains is crucial to the generation of the final active product and the economic benefits of breeding. This demonstrates that the additive prepared by the specific staged fermentation process of this invention has significant advantages in improving the production performance of laying hens after transportation stress.

[0076] As described above, the basic principles, main features, and advantages of the present invention have been well described. The above embodiments and specifications are merely descriptions of preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit and scope of the present invention should fall within the protection scope defined by the present invention.

Claims

1. An anti-transportation stress additive, characterized in that, It is prepared by staged fermentation from a fermentation medium; The fermentation medium contains soybean residue, buckwheat bran powder, rice bran powder, and sugarcane bagasse powder; The staged fermentation process sequentially includes Aspergillus oryzae (… Aspergillus oryzae CICC 40214 fermentation stage, Bacillus licheniformis ( Bacillus licheniformis CICC 10332 fermentation stage and Lactobacillus rhamnosus ( Lactobacillus rhamnosus CICC 6137 fermentation stage.

2. The anti-transportation stress additive according to claim 1, characterized in that, The mass ratio of soybean residue, buckwheat bran powder, rice bran powder, and sugarcane bagasse powder in the fermentation medium is 60~70:5~10:3~5:1~5.

3. A feed for improving the resistance of livestock and poultry to transportation stress, characterized in that, The feed comprises a basal diet and the anti-transportation stress additive as described in claim 1 or 2.

4. The feed according to claim 3, characterized in that, The anti-transportation stress additive is added to the basal diet at a rate of 0.02% to 0.03%.

5. A method for preparing an anti-transportation stress additive as described in claim 1 or 2, characterized in that, Includes the following steps: Step 1: Prepare Aspergillus oryzae separately Aspergillus oryzae Seed liquid, Bacillus licheniformis Bacillus licheniformis Seed culture and Lactobacillus rhamnosus Lactobacillus rhamnosus Seed liquid of the Aspergillus oryzae Aspergillus oryzae The accession number is CICC 40214, and the Bacillus licheniformis described therein is... Bacillus licheniformis The accession number is CICC 10332, and the Lactobacillus rhamnosus described therein is Lactobacillus rhamnosus. Lactobacillus rhamnosus Its accession number is CICC 6137; Step 2: Prepare the fermentation medium; Step 3: The Aspergillus oryzae prepared in Step 1... Aspergillus oryzae The seed culture was inoculated into the fermentation medium prepared in step two, and fermented for 1 to 2 days at 28-32℃ and an aeration rate of 1.2-1.5 vvm. Step 4: The Bacillus licheniformis prepared in Step 1 Bacillus licheniformis The seed culture was inoculated into the fermentation medium in step three and fermented for 1 to 2 days at 37°C and an aeration rate of 0.8 to 1.0 vvm. Step 5: The Lactobacillus rhamnosus prepared in Step 1... Lactobacillus rhamnosus The seed culture was inoculated into the fermentation medium in step four and fermented at 37°C for 1-2 days. Step 6: Collect the fermentation product from Step 5, dry and pulverize it to obtain the anti-transportation stress additive. The drying temperature is 44~45℃.

6. The preparation method according to claim 5, characterized in that, The fermentation medium described in step two contains soybean residue, buckwheat bran powder, rice bran powder and sugarcane bagasse powder, with a mass ratio of 60~70:5~10:3~5:1~5.

7. The preparation method according to claim 5, characterized in that, The fermentation medium described in step two is prepared by mixing soybean residue, buckwheat bran powder, rice bran powder and sugarcane bagasse powder in a mass ratio of 60~70:5~10:3~5:1~5, adjusting the moisture content to 25%~30%, and then sterilizing.

8. The preparation method according to claim 5, characterized in that, Aspergillus oryzae mentioned in step three Aspergillus oryzae The inoculation amount of the seed culture is 5%~6% of the volume of the fermentation medium; the Bacillus licheniformis mentioned in step four Bacillus licheniformis The inoculation amount of the seed culture is 4%~8% of the volume of the fermentation medium; the *Lactobacillus rhamnosus* mentioned in step five... Lactobacillus rhamnosus The inoculation amount of the seed liquid is 5% to 10% of the volume of the fermentation medium.

9. The application of the anti-transportation stress additive as described in claim 1 or 2 or the feed as described in claim 3 or 4 in improving the anti-transportation stress ability of livestock and poultry.

10. The application according to claim 9, characterized in that, The livestock and poultry mentioned are sheep, pigs, or laying hens.

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