Broiler feed additive containing chlorella and application thereof

Abstract

The invention discloses a broiler feed additive containing chlorella and application of the broiler feed additive. The additive contains chlorella; the feed also contains one or more of lactobacillus johnsonii, enterococcus faecalis, saccharomyces cerevisiae and bacillus amyloliquefaciens. By adding the additive disclosed by the invention into daily ration, the intestinal tissue form can be improved by regulating antioxidant and immune functions, so that the growth performance of the broiler chicken can be remarkably improved. In the additive, lactobacillus johnsonii BS15 and enterococcus faecalis YP-2 are combined for use, and saccharomyces cerevisiae WHY-7 and bacillus amyloliquefaciens TL are combined for use, so that the average weight of the broiler chicken is increased, the spleen index after LPS stimulation is reduced, and synergistic interaction is achieved.

Description

Technical Field

[0001] This invention belongs to the field of feed additive technology, specifically relating to a broiler feed additive containing Chlorella and its application. Background Technology

[0002] my country is the world's second-largest broiler producer, with broiler output exceeding 11 billion birds in 2023, maintaining a compound annual growth rate of over 3.5%. However, while the industry continues to expand, it also faces technological bottlenecks in improving feed conversion efficiency and managing gut health. Furthermore, under intensive, high-density broiler farming models, low-level feeding and management practices lead to frequent biosecurity and disease problems. Endotoxins produced by pathogens invade the respiratory and digestive tracts through bedding, drinking water, and air, inducing immune stress and severely damaging the overall function and productivity of broilers. This has spurred market demand for functional additives, particularly focusing on the research and development of natural additives that offer comprehensive benefits in improving nutrient utilization, enhancing antioxidant capacity, and regulating gut health.

[0003] In recent years, the application of microalgae in renewable energy, pharmaceutical raw materials, wastewater treatment, and the food and feed industries has been continuously expanding. Microalgae are rich in nutrients, including carbohydrates, essential fatty acids, amino acids, carotenoids, vitamins, and other bioactive compounds. Studies have shown that microalgae, as a feed additive, can lower cholesterol, regulate immune responses, improve intestinal function, promote the colonization of probiotics, improve animal growth and meat quality, and increase feed conversion ratio. Among the many microalgae species, Chlorella is particularly rich in nutrients, with a protein content of 40-60%. It not only contains various amino acids but also abundant vitamins and minerals, and has extremely high production efficiency. Microalgal polysaccharides are natural polysaccharides found in microalgae, with a wide range of sources. The antioxidant, antitumor, immune-enhancing, anti-inflammatory, and antiviral functions of microalgal polysaccharides have been confirmed and applied. Adding microalgal polysaccharides to livestock, poultry, and aquatic feed can improve animal production performance and enhance immune and antioxidant functions. Summary of the Invention

[0004] The purpose of this invention is to provide a broiler feed additive containing Chlorella and its application.

[0005] A broiler feed additive containing Chlorella, wherein the additive contains Chlorella; and also contains one or more of Lactobacillus johnsonii, Enterococcus faecalis, Saccharomyces cerevisiae, and Bacillus amyloliquefaciens.

[0006] The Chlorella is Chlorella ( Chlorella vulgaris ACSXU-QH01, with accession number CCTCC No: M20251501, is classified as Chlorella ACSXU-QH01. Chlorella vulgaris ACSXU-QH01, deposited at the China Center for Type Culture Collection, dated July 8, 2025, address: Wuhan University, Wuhan, China; the described Lactobacillus johnsonii is Lactobacillus johnsonii (… Lactobacillus johnsonii BS15, with accession number CCTCC No: M2013663, was purchased from the China Center for Type Culture Collection; the Enterococcus faecalis mentioned is Enterococcus faecalis (…). Enterococcus faecalis YP-2, with accession number CGMCC No: 29087, was purchased from the China General Microbiological Culture Collection Center; the described brewing yeast is *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyce scerevisiae WHY-7, with accession number CCTCC NO: M 2016550, was purchased from the China Center for Type Culture Collection; the described Bacillus amyloliquefaciens is Bacillus amyloliquefaciens (…). Bacillus Amyloliquefaciens )TL, accession number: CCTCC NO: M2015359, purchased from China Center for Type Culture Collection.

[0007] The feed additive is a powder; the chlorella is a dried powder; the *Lactobacillus johnsonii*, *Enterococcus faecalis*, *Saccharomyces cerevisiae*, and *Bacillus amyloliquefaciens* are freeze-dried powders; the *Lactobacillus johnsonii* in the powder (… Lactobacillus johnsonii BS15 has a viable bacterial count of 1-8 x 10⁻⁸. 8 cfu / g; Enterococcus faecalis ( Enterococcus faecalis YP-2 has a viable count of 1-8 x 10⁻⁸. 8 cfu / g; viable count of Saccharomycetes scerevisiae WHY-7 is 1-5 x 10⁻⁶. 8 cfu / g; Bacillus amyloliquefaciens ( Bacillus Amyloliquefaciens TL viable count is 1-5 x 10⁻⁵. 8 cfu / g.

[0008] The additive also includes a cell protectant, which is one or more of skim milk, fucoidan, agar oligosaccharide, mannitol, and raffinose.

[0009] The preparation method of the broiler feed additive containing Chlorella is carried out according to the following steps: (1) Lactobacillus johnsonii ( Lactobacillus johnsonii BS15, Enterococcus faecalis ( Enterococcus faecalis YP-2, Saccharomycetes scerevisiae, WHY-7, Bacillus amyloliquefaciens ( Bacillus Amyloliquefaciens )TL are activated separately; (2) Inoculate the activated strains from step (1) into a liquid culture medium, ferment at a constant temperature, add sterile PBS to suspend the bacterial cells, centrifuge and wash, and collect the bacterial sludge; (3) Add a cell protectant to the bacterial mud collected in step (2), mix evenly, and freeze dry. The freeze drying operation steps are as follows: lower the temperature of the mixed material to -20℃ to -40℃ within 2-4 hours, adjust the temperature in the freeze dryer chamber to ≤-70℃, the vacuum degree to ≤10Pa, and the freeze drying time to 8-20 hours; mix the freeze-dried material evenly with Chlorella powder to make a feed additive.

[0010] The feed additive can be stored at 4℃ for 2 months and at -20℃ for 1-2 years.

[0011] The Lactobacillus johnsonii ( Lactobacillus johnsonii The specific steps for BS15 activation and fermentation culture are as follows: Select Lactobacillus johnsonii (… Lactobacillus johnsonii A single colony of BS15 was inoculated into MRS liquid medium for activation at 37°C for 12 hours. Then, a 5% inoculum was transferred to MRS liquid medium and incubated statically for 36 hours to obtain the fermentation broth. The MRS liquid medium consisted of: 10.0 g / L peptone, 10.0 g / L beef extract, 4.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 0.2 g / L diammonium citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L Tween 80, and a pH of 6.2.

[0012] The Enterococcus faecalis ( Enterococcus faecalis The specific steps for activating and fermenting YP-2 are as follows: Select Enterococcus faecalis (… Enterococcus faecalis YP-2 single colonies were inoculated into MRS liquid medium for activation at 30℃ for 18 hours. Then, 4% of the colonies were transferred to MRS liquid medium and incubated statically for 48 hours to obtain the fermentation broth. The MRS liquid medium consisted of: 10.0 g / L peptone, 10.0 g / L beef extract, 5.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 0.1 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L Tween 80, and pH 7.0.

[0013] The brewing yeast ( Saccharomyce scerevisiae The specific steps for activating and fermenting WHY-7 are as follows: Select brewing yeast ( Saccharomyce scerevisiae A single colony of WHY-7 was inoculated into YPD liquid medium and activated by shaking at 30°C and 200 rpm for 18 h. Then, 10% of the inoculum was transferred to YPD liquid medium and shaken for 36 h to obtain the fermentation broth. The YPD liquid medium consisted of 10.0 g / L yeast extract, 20.0 g / L peptone, 20.0 g / L glucose, and pH 6.5.

[0014] The Bacillus amyloliquefaciens ( Bacillus Amyloliquefaciens The specific steps for TL and fermentation culture are as follows: Select *Bacillus amyloliquefaciens* (…). Bacillus Amyloliquefaciens TL single colonies were inoculated into MRS liquid medium for activation at 30℃ for 12 hours. Then, 6% of the colonies were transferred to MRS liquid medium and incubated statically for 48 hours to obtain the fermentation broth. The MRS liquid medium consisted of: 10.0 g / L peptone, 10.0 g / L beef extract, 5.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 0.1 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L Tween 80, and pH 6.8.

[0015] The application of the broiler feed additive containing Chlorella in promoting broiler growth.

[0016] The beneficial effects of this invention: Adding the additive of this invention to the diet can significantly improve the growth performance of broilers by regulating antioxidant and immune functions and improving intestinal tissue morphology. The additive contains *Lactobacillus johnsonii* (…). Lactobacillus johnsonii BS15 and Enterococcus faecalis ( Enterococcus faecalis YP-2 is used in combination with Saccharomycetes scerevisiae WHY-7 and Bacillus amyloliquefaciens. Bacillus Amyloliquefaciens The combined use of TL increased the average weight of broilers, reduced the spleen index after LPS stimulation, and had a synergistic effect. Detailed Implementation

[0017] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0018] The following examples describe Lactobacillus johnsonii ( Lactobacillus johnsonii The specific steps for BS15 activation and fermentation culture are as follows: Select Lactobacillus johnsonii (… Lactobacillus johnsonii A single colony of BS15 was inoculated into MRS liquid medium for activation at 37°C for 12 hours. Then, a 5% inoculum was transferred to MRS liquid medium and incubated statically for 36 hours to obtain the fermentation broth. The MRS liquid medium consisted of: 10.0 g / L peptone, 10.0 g / L beef extract, 4.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 0.2 g / L diammonium citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L Tween 80, and a pH of 6.2.

[0019] The Enterococcus faecalis ( Enterococcus faecalis The specific steps for activating and fermenting YP-2 are as follows: Select Enterococcus faecalis (… Enterococcus faecalis YP-2 single colonies were inoculated into MRS liquid medium for activation at 30℃ for 18 hours. Then, 4% of the colonies were transferred to MRS liquid medium and incubated statically for 48 hours to obtain the fermentation broth. The MRS liquid medium consisted of: 10.0 g / L peptone, 10.0 g / L beef extract, 5.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 0.1 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L Tween 80, and pH 7.0.

[0020] The brewing yeast ( Saccharomyce scerevisiae The specific steps for activating and fermenting WHY-7 are as follows: Select brewing yeast ( Saccharomyce scerevisiae A single colony of WHY-7 was inoculated into YPD liquid medium and activated by shaking at 30°C and 200 rpm for 18 h. Then, 10% of the inoculum was transferred to YPD liquid medium and shaken for 36 h to obtain the fermentation broth. The YPD liquid medium consisted of 10.0 g / L yeast extract, 20.0 g / L peptone, 20.0 g / L glucose, and pH 6.5.

[0021] The Bacillus amyloliquefaciens ( Bacillus Amyloliquefaciens The specific steps for TL and fermentation culture are as follows: Select *Bacillus amyloliquefaciens* (…). Bacillus Amyloliquefaciens TL single colonies were inoculated into MRS liquid medium for activation at 30℃ for 12 hours. Then, 6% of the colonies were transferred to MRS liquid medium and incubated statically for 48 hours to obtain the fermentation broth. The MRS liquid medium consisted of: 10.0 g / L peptone, 10.0 g / L beef extract, 5.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 0.1 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L Tween 80, and pH 6.8. Example 1

[0022] A broiler feed additive containing Chlorella, said additive containing Chlorella ( Chlorella vulgaris ACSXU-QH01, Lactobacillus johnsonii ( Lactobacillus johnsonii BS15 and Enterococcus faecalis ( Enterococcus faecalis YP-2.

[0023] The preparation method of the broiler feed additive containing Chlorella is carried out according to the following steps: (1) Lactobacillus johnsonii ( Lactobacillus johnsonii BS15, Enterococcus faecalis ( Enterococcus faecalis YP-2 was activated separately; (2) Inoculate the activated strains from step (1) into a liquid culture medium, ferment at a constant temperature, add sterile PBS to suspend the bacterial cells, centrifuge and wash, and collect the bacterial sludge; (3) Add skim milk to the bacterial sludge collected in step (2), the amount of which accounts for 80% of the mass of the bacterial sludge, mix evenly, and freeze-dry. The freeze-drying operation steps are as follows: lower the temperature of the mixed material to -30℃ within 3 hours, adjust the temperature inside the freeze dryer chamber to -80℃, the vacuum degree to 5Pa, and the freeze-drying time to 14 hours; mix the freeze-dried material evenly with Chlorella powder to make a feed additive; the Lactobacillus johnsonii ( Lactobacillus johnsonii BS15 freeze-dried powder, Enterococcus faecalis ( Enterococcus faecalis The mass ratio of YP-2 freeze-dried powder to Chlorella powder is 1:1:2. Example 2

[0024] A broiler feed additive containing Chlorella, said additive containing Chlorella ( Chlorella vulgaris ACSXU-QH01, brewing yeast ( Saccharomyce scerevisiae WHY-7 and Bacillus amyloliquefaciens ( Bacillus Amyloliquefaciens )TL.

[0025] The preparation method of the broiler feed additive containing Chlorella is carried out according to the following steps: (1) Saccharomycetes scerevisiae WHY-7 and Bacillus amyloliquefaciens ( Bacillus Amyloliquefaciens )TL are activated separately; (2) Inoculate the activated strains from step (1) into a liquid culture medium, ferment at a constant temperature, add sterile PBS to suspend the bacterial cells, centrifuge and wash, and collect the bacterial sludge; (3) Add mannitol and raffinose (mass ratio 1:1) to the bacterial sludge collected in step (2), the amount added is 80% of the bacterial sludge mass, mix evenly, and freeze-dry. The freeze-drying operation steps are as follows: lower the temperature of the mixed material to -20℃ within 2 hours, adjust the temperature inside the freeze dryer chamber to -80℃, the vacuum degree to 8Pa, and the freeze-drying time to 16 hours; mix the freeze-dried material evenly with Chlorella powder to make a feed additive; the brewing yeast ( Saccharomyce scerevisiae WHY-7 lyophilized powder, Bacillus amyloliquefaciens ( Bacillus Amyloliquefaciens The mass ratio of TL freeze-dried powder to Chlorella powder is 1:1:2. Example 3

[0026] A broiler feed additive containing Chlorella, said additive containing Chlorella ( Chlorella vulgaris ACSXU-QH01, Lactobacillus johnsonii ( Lactobacillus johnsonii BS15 and Enterococcus faecalis ( Enterococcus faecalis YP-2.

[0027] The preparation method of the broiler feed additive containing Chlorella is carried out according to the following steps: (1) Lactobacillus johnsonii ( Lactobacillus johnsonii BS15, Enterococcus faecalis ( Enterococcus faecalis YP-2 was activated separately; (2) Inoculate the activated strains from step (1) into a liquid culture medium, ferment at a constant temperature, add sterile PBS to suspend the bacterial cells, centrifuge and wash, and collect the bacterial sludge; (3) Add brown algae oligosaccharide and agar oligosaccharide (mass ratio 1:1) to the fungal sludge collected in step (2), the amount added accounts for 80% of the mass of fungal sludge, mix evenly, and freeze dry. The freeze drying operation steps are as follows: lower the temperature of the mixed material to -40℃ within 3.5h, adjust the temperature inside the freeze dryer chamber to -80℃, the vacuum degree to 6Pa, and the freeze drying time to 16h; mix the freeze-dried material evenly with Chlorella powder to make feed additive.

[0028] The feed additive contains Lactobacillus johnsonii ( Lactobacillus johnsonii The viable count of BS15 was 5.9 x 10⁻⁶. 8 cfu / g; Enterococcus faecalis ( Enterococcus faecalis The viable count of YP-2 was 6.2 x 10⁻⁶. 8 cfu / g.

[0029] Comparative Example 1 A broiler feed additive containing Chlorella, said additive containing Chlorella ( Chlorella vulgaris ACSXU-QH01 and Lactobacillus johnsonii ( Lactobacillus johnsonii BS15.

[0030] The preparation method of the broiler feed additive containing Chlorella is carried out according to the following steps: (1) Lactobacillus johnsonii ( Lactobacillus johnsonii BS15 activation; (2) Inoculate the activated strains from step (1) into a liquid culture medium, ferment at a constant temperature, add sterile PBS to suspend the bacterial cells, centrifuge and wash, and collect the bacterial sludge; (3) Add skim milk to the bacterial sludge collected in step (2), the amount of which accounts for 80% of the mass of the bacterial sludge, mix evenly, and freeze-dry. The freeze-drying operation steps are as follows: lower the temperature of the mixed material to -30℃ within 3 hours, adjust the temperature inside the freeze dryer chamber to -80℃, the vacuum degree to 5Pa, and the freeze-drying time to 14 hours; mix the freeze-dried material evenly with Chlorella powder to make a feed additive; the Lactobacillus johnsonii ( Lactobacillus johnsonii The mass ratio of BS15 freeze-dried powder to Chlorella powder is 2:2.

[0031] Comparative Example 2 A broiler feed additive containing Chlorella, said additive containing Chlorella ( Chlorella vulgaris ACSXU-QH01 and Enterococcus faecalis ( Enterococcus faecalis YP-2.

[0032] The preparation method of the broiler feed additive containing Chlorella is carried out according to the following steps: (1) Enterococcus faecalis ( Enterococcus faecalis YP-2 activation; (2) Inoculate the activated strains from step (1) into a liquid culture medium, ferment at a constant temperature, add sterile PBS to suspend the bacterial cells, centrifuge and wash, and collect the bacterial sludge; (3) Add skim milk to the bacterial sludge collected in step (2), the amount of which accounts for 80% of the mass of the bacterial sludge. Mix evenly and freeze-dry. The freeze-drying operation steps are as follows: lower the temperature of the mixed material to -30℃ within 3 hours, adjust the temperature inside the freeze dryer to -80℃, the vacuum degree to 5Pa, and the freeze-drying time to 14 hours; mix the freeze-dried material evenly with Chlorella powder to make a feed additive; the Enterococcus faecalis ( Enterococcus faecalis The mass ratio of YP-2 freeze-dried powder to Chlorella powder is 2:2.

[0033] Comparative Example 3 A broiler feed additive containing Chlorella, said additive containing Chlorella ( Chlorella vulgaris ACSXU-QH01 and brewer's yeast ( Saccharomyce scerevisiae WHY-7.

[0034] The preparation method of the broiler feed additive containing Chlorella is carried out according to the following steps: (1) Activate Saccharomycetes scerevisiae WHY-7; (2) Inoculate the activated strains from step (1) into a liquid culture medium, ferment at a constant temperature, add sterile PBS to suspend the bacterial cells, centrifuge and wash, and collect the bacterial sludge; (3) Add mannitol and raffinose (mass ratio 1:1) to the bacterial sludge collected in step (2), the amount added is 80% of the bacterial sludge mass, mix evenly, and freeze-dry. The freeze-drying operation steps are as follows: lower the temperature of the mixed material to -20℃ within 2 hours, adjust the temperature inside the freeze dryer chamber to -80℃, the vacuum degree to 8Pa, and the freeze-drying time to 16 hours; mix the freeze-dried material evenly with Chlorella powder to make a feed additive; the brewing yeast ( Saccharomyce scerevisiae The mass ratio of WHY-7 freeze-dried powder to Chlorella powder is 2:2.

[0035] Comparative Example 4 A broiler feed additive containing Chlorella, said additive containing Chlorella ( Chlorella vulgaris ACSXU-QH01 and Bacillus amyloliquefaciens ( Bacillus Amyloliquefaciens )TL.

[0036] The preparation method of the broiler feed additive containing Chlorella is carried out according to the following steps: (1) Bacillus amyloliquefaciens ( Bacillus Amyloliquefaciens TL activation; (2) Inoculate the activated strains from step (1) into a liquid culture medium, ferment at a constant temperature, add sterile PBS to suspend the bacterial cells, centrifuge and wash, and collect the bacterial sludge; (3) Add mannitol and raffinose (mass ratio 1:1) to the bacterial sludge collected in step (2), the amount added is 80% of the bacterial sludge mass, mix evenly, and freeze-dry. The freeze-drying operation steps are as follows: lower the temperature of the mixed material to -20℃ within 2 hours, adjust the temperature inside the freeze dryer chamber to -80℃, the vacuum degree to 8Pa, and the freeze-drying time to 16 hours; mix the freeze-dried material evenly with Chlorella powder to make a feed additive; the Bacillus amyloliquefaciens ( Bacillus Amyloliquefaciens The mass ratio of TL freeze-dried powder to Chlorella powder is 2:2.

[0037] Comparative Example 5 A broiler feed additive containing Chlorella, said additive containing Chlorella ( Chlorella vulgaris ACSXU-QH01, Lactobacillus johnsonii ( Lactobacillus johnsonii BS15 and Enterococcus faecalis ( Enterococcus faecalis YP-2.

[0038] The preparation method of the broiler feed additive containing Chlorella is carried out according to the following steps: (1) Lactobacillus johnsonii ( Lactobacillus johnsonii BS15, Enterococcus faecalis ( Enterococcus faecalis YP-2 was activated separately; (2) Inoculate the activated strains from step (1) into a liquid culture medium, ferment at a constant temperature, add sterile PBS to suspend the bacterial cells, centrifuge and wash, and collect the bacterial sludge; (3) Add brown algae oligosaccharide to the bacterial sludge collected in step (2), the amount of which accounts for 80% of the mass of the bacterial sludge, mix evenly, and freeze dry. The freeze drying operation steps are as follows: lower the temperature of the mixed material to -40℃ within 3.5h, adjust the temperature inside the freeze dryer chamber to -80℃, the vacuum degree to 6Pa, and the freeze drying time to 16h; mix the freeze-dried material evenly with Chlorella powder to make feed additive.

[0039] The feed additive contains Lactobacillus johnsonii ( Lactobacillus johnsonii The viable count of BS15 was 3.8 x 10⁻⁶. 8 cfu / g; Enterococcus faecalis ( Enterococcus faecalis The viable count of YP-2 was 4.6 x 10⁻⁶. 8 cfu / g.

[0040] Comparative Example 6 A broiler feed additive containing Chlorella, said additive containing Chlorella ( Chlorella vulgaris ACSXU-QH01, Lactobacillus johnsonii ( Lactobacillus johnsoniiBS15 and Enterococcus faecalis ( Enterococcus faecalis YP-2.

[0041] The preparation method of the broiler feed additive containing Chlorella is carried out according to the following steps: (1) Lactobacillus johnsonii ( Lactobacillus johnsonii BS15, Enterococcus faecalis ( Enterococcus faecalis YP-2 was activated separately; (2) Inoculate the activated strains from step (1) into a liquid culture medium, ferment at a constant temperature, add sterile PBS to suspend the bacterial cells, centrifuge and wash, and collect the bacterial sludge; (3) Add agar oligosaccharide to the bacterial sludge collected in step (2), the amount of which accounts for 80% of the mass of the bacterial sludge, mix evenly, and freeze dry. The freeze drying operation steps are as follows: lower the temperature of the mixed material to -40℃ within 3.5h, adjust the temperature inside the freeze dryer chamber to -80℃, the vacuum degree to 6Pa, and the freeze drying time to 16h; mix the freeze-dried material evenly with Chlorella powder to make feed additive.

[0042] The feed additive contains Lactobacillus johnsonii ( Lactobacillus johnsonii The viable count of BS15 was 4.2 x 10⁻⁶. 8 cfu / g; Enterococcus faecalis ( Enterococcus faecalis The viable count of YP-2 was 4.2 x 10⁻⁶. 8 cfu / g.

[0043] Experimental example: One-day-old healthy white-feathered broilers were randomly assigned to eight treatment groups, with ten replicates per group. The control and LPS groups were fed a basal diet (referring to the "Broiler Feeding Standard" (NY / T33-2004)). The experimental groups were fed a diet supplemented with 1000 mg / kg of the feed additives prepared in Examples 1-2 and Comparative Examples 1-4. The experiment lasted 21 days, divided into a normal feeding period (days 1-18) and a stress period (days 19-21). The experiment was conducted at the Nankou Pilot Plant of the Chinese Academy of Agricultural Sciences. During the normal feeding period, no treatment was given; during the stress period, the feed additive groups and LPS groups were injected with LPS (0.5 mg / kg) for three consecutive days, while the control group was injected with the same dose of physiological saline for three consecutive days.

[0044] The experiment employed a three-tiered cage rearing method. Prior to the experiment, the poultry house underwent fumigation disinfection for 3 days, followed by ventilation for 7 days. Water lines, feed trays, and feeders in the poultry house were also disinfected. Broilers were fed brown sugar water for days 1-3, room temperature boiled water for days 4-7, and tap water for days 8-21. From day 1-7, chickens were fed six times daily at 07:00, 10:00, 13:00, 16:00, 19:00, and 22:00. From day 8-21, chickens were fed three times daily at 08:00, 14:00, and 20:00. During the trial, the poultry house was kept under normal lighting conditions and was regularly disinfected and cleaned. Broilers were allowed free access to feed and water. For the first 7 days of the trial, the room temperature was kept at around 35°C, and then decreased by 2-3°C each week until the room temperature was kept at around 24°C. The air humidity in the poultry house was kept at 48-60%. On the 7th day, the Newcastle disease attenuated live vaccine was administered via nasal drops or eye drops.

[0045] The broilers were weighed on days 19 and 21 of the experiment, and the average body weight (BW) on days 19 and 21 was calculated.

[0046] After the experiment, three broiler chickens from each group were selected, weighed, and slaughtered. The spleens were then weighed and the spleen index was calculated. The calculation formula is as follows: Spleen index = fresh spleen weight / live body weight.

[0047] After data processing, ANOVA was performed using SPSS software, and Duncan's multiple comparisons were conducted. Statistical results are expressed as mean ± standard error, and P < 0.05 was considered statistically significant. The results are shown in Table 1-2. Table 1 experimental group Average weight in kg at 19 days Average weight in kg at 21 days control group 0.81±0.03 1.08±0.03 LPS Group 0.75±0.01 0.71±0.02* Example 1 0.79±0.04 1.03±0.01 Example 2 0.82±0.01 1.02±0.03 Comparative Example 1 0.76±0.05 0.82±0.01* Comparative Example 2 0.77±0.04 0.85±0.02* Comparative Example 3 0.74±0.02 0.86±0.03* Comparative Example 4 0.76±0.04 0.81±0.01* Note: * indicates that compared with Example 1 group, P<0.05.

[0048] Table 2 experimental group Spleen Index control group 0.075±0.012 LPS Group 0.182±0.013* Example 1 0.086±0.021 Example 2 0.082±0.014 Comparative Example 1 0.157±0.015* Comparative Example 2 0.162±0.014* Comparative Example 3 0.144±0.012* Comparative Example 4 0.166±0.014* Note: * indicates that compared with Example 1 group, P<0.05.

[0049] This study confirms that LPS stress can significantly increase the spleen index of broilers, and that adding the feed additive of this invention to the diet can restore it to normal levels through anti-inflammatory and immunomodulatory pathways.

[0050] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A broiler feed additive containing Chlorella, characterized in that, The additive contains Chlorella; it also contains one or more of Lactobacillus johnsonii, Enterococcus faecalis, Saccharomyces cerevisiae, and Bacillus amyloliquefaciens.

2. The broiler feed additive containing Chlorella according to claim 1, characterized in that, The Chlorella is Chlorella ( Chlorella vulgaris ACSXU-QH01, with accession number CCTCC No: M20251501; the Lactobacillus johnsonii is Lactobacillus johnsonii ( Lactobacillus johnsonii The BS15 accession number is CCTCC No: M2013663; the Enterococcus faecalis is Enterococcus faecalis ( Enterococcus faecalis YP-2, with accession number CGMCC No: 29087; the brewing yeast is brewing yeast ( Saccharomyce scerevisiae WHY-7, with accession number CCTCC NO: M2016550; the Bacillus amyloliquefaciens described is Bacillus amyloliquefaciens ( Bacillus Amyloliquefaciens )TL, accession number: CCTCC NO: M2015359.

3. The broiler feed additive containing Chlorella according to claim 1, characterized in that, The feed additive is a powder; the chlorella is a dried powder; the *Lactobacillus johnsonii*, *Enterococcus faecalis*, *Saccharomyces cerevisiae*, and *Bacillus amyloliquefaciens* are freeze-dried powders; the *Lactobacillus johnsonii* in the powder (… Lactobacillus johnsonii BS15 has a viable bacterial count of 1-8 x 10⁻⁸. 8 cfu / g; Enterococcus faecalis ( Enterococcus faecalis YP-2 has a viable count of 1-8 x 10⁻⁸. 8 cfu / g; viable count of Saccharomyces cerevisiae WHY-7 is 1-5 x 10⁻⁶. 8 cfu / g; Bacillus amyloliquefaciens ( Bacillus Amyloliquefaciens TL viable count is 1-5 x 10⁻⁵. 8 cfu / g.

4. The broiler feed additive containing Chlorella according to claim 1, characterized in that, The additive also includes a cell protectant, which is one or more of skim milk, fucoidan, agar oligosaccharide, mannitol, and raffinose.

5. The method for preparing the broiler feed additive containing Chlorella as described in claim 1, characterized in that, Follow these steps: (1) Lactobacillus johnsonii ( Lactobacillus johnsonii BS15, Enterococcus faecalis ( Enterococcus faecalis YP-2, Saccharomycetes scerevisiae, WHY-7, Bacillus amyloliquefaciens ( Bacillus Amyloliquefaciens )TL are activated separately; (2) Inoculate the activated strains from step (1) into a liquid culture medium, ferment at a constant temperature, add sterile PBS to suspend the bacterial cells, centrifuge and wash, and collect the bacterial sludge; (3) Add a cell protectant to the bacterial mud collected in step (2), mix evenly, and freeze dry. The freeze drying operation steps are as follows: lower the temperature of the mixed material to -20℃ to -40℃ within 2-4 hours, adjust the temperature in the freeze dryer chamber to ≤-70℃, the vacuum degree to ≤10Pa, and the freeze drying time to 8-20 hours; mix the freeze-dried material evenly with Chlorella powder to make a feed additive.

6. The method for preparing the broiler feed additive containing Chlorella according to claim 5, characterized in that, The Lactobacillus johnsonii ( Lactobacillus johnsonii The specific steps for BS15 activation and fermentation culture are as follows: Select Lactobacillus johnsonii (… Lactobacillus johnsonii A single colony of BS15 was inoculated into MRS liquid medium for activation at 37°C for 12 hours. Then, a 5% inoculum was transferred to MRS liquid medium and incubated statically for 36 hours to obtain the fermentation broth. The MRS liquid medium consisted of: 10.0 g / L peptone, 10.0 g / L beef extract, 4.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 0.2 g / L diammonium citrate, 5.0 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L Tween 80, and a pH of 6.

2.

7. The method for preparing the broiler feed additive containing Chlorella according to claim 5, characterized in that, The Enterococcus faecalis ( Enterococcus faecalis The specific steps for activating and fermenting YP-2 are as follows: Select Enterococcus faecalis (… Enterococcus faecalis YP-2 single colonies were inoculated into MRS liquid medium for activation at 30℃ for 18 hours. Then, 4% of the colonies were transferred to MRS liquid medium and incubated statically for 48 hours to obtain the fermentation broth. The MRS liquid medium consisted of: 10.0 g / L peptone, 10.0 g / L beef extract, 5.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 0.1 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L Tween 80, and pH 7.

0.

8. The method for preparing the broiler feed additive containing Chlorella according to claim 5, characterized in that, The specific steps for activating and fermenting the Saccharomycetes scerevisiae WHY-7 are as follows: A single colony of Saccharomycetes scerevisiae WHY-7 is picked and inoculated into YPD liquid medium. The colony is then activated by shaking at 30°C and 200 rpm for 18 hours. Next, a 10% inoculum is transferred to YPD liquid medium and shaken at 30°C and 200 rpm for 36 hours to obtain the fermentation broth. The YPD liquid medium consists of: 10.0 g / L yeast extract, 20.0 g / L peptone, 20.0 g / L glucose, and a pH of 6.

5.

9. The method for preparing the broiler feed additive containing Chlorella according to claim 5, characterized in that, The Bacillus amyloliquefaciens ( Bacillus Amyloliquefaciens The specific steps for TL and fermentation culture are as follows: Select *Bacillus amyloliquefaciens* (…). Bacillus Amyloliquefaciens TL single colonies were inoculated into MRS liquid medium for activation at 30℃ for 12 hours. Then, 6% of the colonies were transferred to MRS liquid medium and incubated statically for 48 hours to obtain the fermentation broth. The MRS liquid medium consisted of: 10.0 g / L peptone, 10.0 g / L beef extract, 5.0 g / L yeast extract, 20.0 g / L glucose, 2.0 g / L dipotassium hydrogen phosphate, 2.0 g / L triammonium citrate, 5.0 g / L sodium acetate, 0.1 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L Tween 80, and pH 6.

8.

10. The application of the broiler feed additive containing Chlorella as described in claim 1 in promoting broiler growth.