A fermentation product of seaweed residue, its preparation process and application

Abstract

This invention discloses a fermented product of seaweed residue, its preparation process, and its application, belonging to the field of seaweed residue fermentation technology. The preparation process of the fermented product of seaweed residue is as follows: (1) Add seaweed residue to a 7% glycine solution and soak it; (2) Take out the seaweed residue, add it to a 0.6% deep eutectic solvent solution, soak it, and dry it; (3) Take the dried seaweed residue, add water, add glucose as a carbon source, add ammonium chloride as a nitrogen source, inoculate with thermophilic mycelium, ferment, and dry to obtain the product. The fermented product of seaweed residue of this invention can significantly improve the growth performance of chicks, significantly improve the immune organ index and serum immune indicators, significantly improve the content of short-chain fatty acids, improve the intestinal microbial environment, and play a positive role in regulating and promoting intestinal health. This invention is of great significance for improving the application value of seaweed residue, enriching feed additives, and improving chicken farming.

Description

Technical Field

[0001] This invention relates to a fermentation product of seaweed residue, its preparation process and application, and belongs to the field of seaweed residue fermentation technology. Background Technology

[0002] Seaweed has a wide range of uses, but its utilization rate is generally low. During industrial production, a large amount of seaweed residue waste is generated. Seaweed residue refers to the seaweed residue or waste left over from aquaculture or seafood processing, which usually includes residual parts of seaweed and impurities such as fragments and fine particles.

[0003] The application of seaweed residue in feed has been well-documented, but its use is limited due to its relatively low protein content compared to traditional feed ingredients. The large amount of indigestible macromolecules also restricts its application in aquaculture. The abundant active substances in seaweed residue suggest its potential for wider application as a feed additive. Currently, research on seaweed residue feed additives is limited due to the difficulty in extracting active substances from seaweed, high industry barriers, and the difficulty or uneconomical removal of harmful byproducts from simple treatment methods such as chemical processing. Fermentation is an effective method for preparing feed additives from seaweed residue. Fermented seaweed residue feed additives offer significant advantages over traditional additives. Fermented seaweed residue contains various nutrients required by animals, such as minerals, vitamins, amino acids, and fatty acids, as well as bioactive substances like polysaccharides and alginate that enhance immunity and provide antioxidant benefits. Fermentation by certain strains of bacteria that produce antimicrobial peptides and other antibacterial substances not only increases the digestibility of seaweed residue components and promotes animal absorption but also improves intestinal health and provides a natural antibiotic alternative.

[0004] Myceliophthora thermophila is a Gram-positive bacterium belonging to the thermophilic microorganisms, which typically live in high-temperature environments. In recent years, research on Myceliophthora thermophila has increased significantly. It possesses advantages such as broad-spectrum biotransformation activity and the ability to promote biodegradation, making it widely applicable in fields such as bioenergy, environmental protection, and industrial production. Summary of the Invention

[0005] In view of the above-mentioned prior art, the present invention provides a seaweed residue fermentation product, its preparation process and application, which belongs to the field of seaweed residue fermentation technology.

[0006] This invention is achieved through the following technical solution: A process for preparing seaweed residue fermentation products includes the following steps: (1) Add the seaweed residue to a 7% glycine solution (by weight) and soak it. (2) Take out the seaweed residue and add it to a 0.6% deep eutectic solvent solution (by weight) for soaking treatment; the deep eutectic solvent is choline chloride and urea, and the molar ratio of the two is 1:1; take out the seaweed residue and dry it; (3) Take 80 g of the dried seaweed residue, add 40 g of pure water and sterilize; add 0.8 g of glucose as carbon source and 0.8 g of ammonium chloride as nitrogen source, inoculate with thermophilic mycelium and ferment; after fermentation, dry the fermentation liquid to obtain fermented seaweed residue, which is the seaweed residue fermentation product.

[0007] Furthermore, the soaking treatment involves soaking at 60°C for 24 hours.

[0008] Furthermore, the concentration of the thermophilic mycelium suspension is 1×10⁻⁶. 6 The concentration of cfu / mL was 0.8 mL.

[0009] Furthermore, the fermentation was carried out at 40°C for 10 days.

[0010] A seaweed residue fermentation product is prepared using the above-mentioned preparation process.

[0011] The application of the seaweed residue fermentation product as an animal feed additive or in the preparation of animal feed additives.

[0012] Furthermore, the animal in question is a chicken, especially a Sanhuang chicken.

[0013] The preparation process of the seaweed residue fermentation product of this invention involves pretreating the seaweed residue with a 7% glycine solution and a 0.6% deep eutectic solvent solution, followed by inoculation with thermophilic mycelium for fermentation. The fermented seaweed residue product has a crude fiber content as low as 9.78%. The seaweed residue fermentation product can be used as an animal feed additive. Feeding experiments on chicks have shown that feeding a basal diet supplemented with 5% seaweed residue fermentation product significantly improves the growth performance of chicks (including the lowest survival rate, average daily weight gain, and feed conversion ratio), significantly improves immune organ indices and serum immune indicators, significantly increases the content of short-chain fatty acids, and improves the intestinal microbial environment, playing a positive role in regulating and promoting intestinal health. This invention provides a fermentation system for seaweed residue fermentation using thermophilic mycelium, and experimentally verifies the efficacy of the fermentation product. This invention is of great significance for improving the application value of seaweed residue, enriching feed additives, and improving chicken farming.

[0014] The various terms and phrases used in this invention have their general meanings known to those skilled in the art. Attached Figure Description

[0015] Figure 1: Schematic diagram of the determination results of crude fiber content in fermented seaweed residue after fermentation by different strains. The letter labeling method is used to indicate significant differences. Different letter labels indicate significant differences at the p≤0.05 level.

[0016] Figure 2 : Schematic diagram of the determination results of crude fiber content in fermented seaweed residue after soaking in glycine solution of different concentrations. The letter labeling method is used to indicate significant differences, and different letter labels indicate significant differences at the p≤0.05 level.

[0017] Figure 3 : Schematic diagram of the determination results of crude fiber content in fermented seaweed residue after soaking in different eutectic solvent solutions. Significant differences are indicated by letter labeling, with different letter labels indicating significant differences at the p≤0.05 level.

[0018] Figure 4 : Schematic diagram of the determination results of crude fiber content in fermented seaweed residue after soaking in eutectic solvent solutions of different concentrations. Significant differences are indicated by letter notation, with different letter notations indicating significant differences at the p≤0.05 level.

[0019] Figure 5 : Schematic diagram of the determination results of crude fiber content in fermented seaweed residue after fermentation with different amounts of bacterial solution. The letter labeling method is used to indicate significant differences, and different letter labels indicate significant differences at the p≤0.05 level.

[0020] Figure 6 : Schematic diagram of the determination results of crude fiber content in fermented seaweed residue after fermentation at different fermentation temperatures. Significant differences are indicated by letter notation, with different letter notations indicating significant differences at the p≤0.05 level.

[0021] Figure 7 : Schematic diagram of the determination results of crude fiber content in fermented seaweed residue after fermentation with different amounts of added carbon source. Significant differences are indicated by letter labeling, and different letter labels indicate significant differences at the p≤0.05 level.

[0022] Figure 8 : Schematic diagram of the determination results of the relative abundance of gut microbiota at the phylum level in chicks, where Firmieutes refers to Firmicutes, Actinobacteriota refers to Actinobacteria, and Proteobacteria refers to Proteobacteria.

[0023] Figure 9: Schematic diagram of the relative abundance of gut microbiota genera in chicks. Among them, unclassified_f_Lachnospiraceae refers to unclassified genera of the Clostridiaceae family, norank_f_norank_o_Clostridia_UCG-0l4 refers to unclassified genera of the Clostridia class, Ruminococcus_torgnes_group refers to the genus Ruminococcus, Norank_f_norank_o_Clostridia_vadinBB60_group refers to another unclassified genus of the Clostridia class, Lachnoclostridium refers to the genus Clostridium woolii, and Others refers to other bacteria. Detailed Implementation

[0024] The present invention will be further described below with reference to embodiments. However, the scope of the present invention is not limited to the following embodiments. Those skilled in the art will understand that various changes and modifications can be made to the present invention without departing from the spirit and scope thereof.

[0025] Unless otherwise specified, the instruments, reagents, and materials used in the following embodiments are all conventional instruments, reagents, and materials already available in the prior art and can be obtained through legitimate commercial channels. Unless otherwise specified, the experimental methods and detection methods used in the following embodiments are all conventional experimental methods and detection methods already available in the prior art.

[0026] The seaweed residue used in this invention was purchased from Qingdao Luoan Biotechnology Co., Ltd.

[0027] The thermophilic filamentous bacteria used in this invention were purchased from the China General Microbiological Culture Collection Center (CICC) with accession number CICC 2441 and accession date January 1, 1983. The biological characteristics of this thermophilic filamentous bacteria are: Gram-positive, filamentous, branched, non-motile; colonies 3–4 mm in size, oval or cylindrical, raised, milky white to light brown, relatively viscous, with neat edges, and the central area is often more raised with wrinkles or radial textures.

[0028] The Rhizopus oryzae, Trichoderma reesei I, Trichoderma reesei II, Bacillus subtilis, and Bacillus velezensis used in this invention are all common strains reported in the prior art for preparing feed from fermented seaweed residue, and were purchased from the China Industrial Microbial Culture Collection Center.

[0029] Example 1: Direct Fermentation Six bacteria, namely Rhizopus oryzae, Trichoderma reesei Ⅰ, Trichoderma reesei Ⅱ, Myceliophthora thermophila, Bacillus subtilis, and Bacillus velezensis, were used to directly ferment seaweed residue. The operation was as follows: Add 80 g of dried seaweed residue to a fermentation box, add 40 g of pure water, and sterilize at 115℃ for 30 min. Cool to room temperature, add 0.5 g of glucose as a carbon source and 0.5 g of ammonium chloride as a nitrogen source, and inoculate with 0.5 mL of each bacterial culture (concentration 1×10⁻⁶). 6 The mixture was prepared by mixing the cfu / mL of the bacteria thoroughly and fermenting it for 10 days at the optimal temperatures for each bacteria (34℃, 30℃, 30℃, 40℃, 37℃, and 37℃, respectively). The fermentation container was shaken regularly every 12 hours to ensure thorough mixing. After fermentation, the fermentation liquid was dried to obtain fermented seaweed residue.

[0030] Example 2: Determination of crude fiber content in seaweed residue Crude fiber content, as a key indicator, plays an irreplaceable role in judging the effectiveness of microbial fermentation in degrading seaweed residue. Changes in crude fiber content directly reflect the ability of microorganisms to decompose the indigestible fibrous components in seaweed residue. Small changes in crude fiber content indicate insufficient degradation by microorganisms, failing to effectively break down the complex fibrous structure into simpler, more usable substances; conversely, large changes in crude fiber content indicate good microbial fermentation, successfully reducing the complexity and recalcitrant nature of the fibers in the seaweed residue.

[0031] Therefore, the crude fiber content in seaweed residue and fermented seaweed residue was determined by the following steps: (1) Accurately weigh 5 g of seaweed residue or fermented seaweed residue, carefully transfer it to a round-bottom flask, add 2-3 glass beads to prevent bumping, pour in 200 mL of boiled 1.25% sulfuric acid solution (mass-volume ratio, unit g / mL), and add a few drops of n-octanol to prevent the formation of bubbles. Cover with a reflux device to ensure that the acid concentration remains constant, heat on a heating mantle, bring to a gentle boil within 5 min, maintain for 30 min, and gently shake the round-bottom flask from time to time to prevent the sample from sticking to the inner wall.

[0032] (2) After heating, remove the round-bottom flask and immediately filter it through a 200-mesh sieve on a vacuum filtration device, and rinse it with boiling water until neutral. Transfer all the filter residue on the sieve to the original round-bottom flask, add 200 mL of freshly prepared 0.23 mol / L potassium hydroxide solution, glass beads and n-octanol, and heat on a heating mantle to boiling for 30 min. Filter through the sieve, wash with boiling water until neutral, and then wash once with ethanol and once with ether. Transfer all insoluble matter to a crucible of known mass.

[0033] (3) Place the crucible in a drying oven at 105℃ and dry for 2 hours. Cool and weigh in a desiccator until constant weight is achieved. Then transfer the crucible and sample to a muffle furnace at 550℃ for ashing, cool and weigh. Substitute the weight into the following formula to obtain the crude fiber content: ; In the formula: G—mass of residue, in grams (g); m—mass of sample, in grams (g).

[0034] The results of the determination of crude fiber content in seaweed residue raw material and fermented seaweed residue after fermentation with different strains are as follows: Figure 1 As shown, "control" refers to seaweed residue as raw material. The crude fiber content in the seaweed residue raw material was determined to be 31.12%. After fermentation with various bacteria, the crude fiber content decreased to varying degrees, but the differences were not significant for four bacteria. However, after fermentation with *Thermophilic pyrophilus*, the crude fiber level decreased the most, to 27.72%, a reduction of 10.9%, which was significantly better than the other five bacteria. This demonstrates that *Thermophilic pyrophilus* has great application potential in the fermentation of seaweed residue.

[0035] Example 3: Glycine Pretreatment and Condition Optimization After fermentation by thermophilic mycelium, the crude fiber content of seaweed residue decreased significantly, but remained relatively high. To further reduce the crude fiber content, glycine was used to treat the seaweed residue, and the pretreatment conditions for glycine were optimized. Glycine pretreatment can act as an ammonia source under alkaline conditions to disrupt the structure of biomass, thereby improving its bioavailability and flavor of fermented seaweed products.

[0036] The steps are as follows: (1) Preparation of glycine solution: Take 10 g, 30 g, 50 g, 70 g and 90 g of glycine respectively, dissolve them in 1000 mL of pure water, and prepare glycine solutions with concentrations of 1%, 3%, 5%, 7% and 9% (weight percentage) respectively.

[0037] (2) Take 6 portions of 100 g each of seaweed residue and add them to glycine solutions of different concentrations and pure water (pure water as control) respectively. Soak them at 60°C for 24 h. After soaking, dry the seaweed residue.

[0038] (3) The dried seaweed residue was fermented using the fermentation method of Example 1. After fermentation, the crude fiber content was determined using the determination method of Example 2.

[0039] The results of the determination of crude fiber content in fermented seaweed residue after soaking in glycine solutions of different concentrations are as follows: Figure 2 As shown, the crude fiber content was significantly reduced after soaking in 1%, 3%, 5%, and 7% glycine solutions. The 7% glycine solution had the most significant effect, reducing the crude fiber content to 20.55%. Soaking in 7% glycine solution was the optimal pretreatment condition for glycine.

[0040] Example 4: Pretreatment and Condition Optimization of Deep Eutectic Solvent After glycine pretreatment, the crude fiber content was significantly reduced, but still remained high. Deep eutectic solvents (DES) were then used for further treatment, and the pretreatment conditions were optimized. Deep eutectic solvents (DES) are low-melting-point mixtures formed by two or more components through hydrogen bonding or other types of interactions. They typically possess good thermal stability, designability, and biocompatibility, making them ideal for biomass processing. DES pretreatment can break down the complex structures in biomass such as plant cell walls.

[0041] The steps are as follows: (1) Referring to Example 3, the seaweed residue was pretreated by soaking in a 7% glycine solution; then, three portions of seaweed residue, each 80 g, were soaked in a 1% (weight percentage) choline chloride-urea solution (the molar ratio of choline chloride to urea is 1:1), a 1% choline chloride-urea solution (the molar ratio of choline chloride to urea is 1:2), and a 1% choline chloride-glycerol solution (the molar ratio of choline chloride to glycerol is 1:1), and soaked at 60°C for 24 h; after soaking, the seaweed residue was dried; the crude fiber content was determined according to step (3) of Example 2.

[0042] The results of the determination of crude fiber content in fermented seaweed residue after soaking in different eutectic solvent solutions are as follows: Figure 3 As shown, after soaking in the eutectic solvents choline chloride-urea (molar ratio of choline chloride to urea is 1:2) and choline chloride-glycerol, the crude fiber content did not change significantly; however, after soaking in the eutectic solvent choline chloride-urea (molar ratio of choline chloride to urea is 1:1), the crude fiber content decreased significantly to 14.83%, indicating that choline chloride-urea (molar ratio of choline chloride to urea is 1:1) was selected as the eutectic solvent.

[0043] (2) Referring to Example 3, the seaweed residue was pretreated by soaking in a 7% glycine solution; then, 6 portions of seaweed residue, each 80 g, were soaked in the glycine solution and pretreated. They were then added to eutectic solvents with mass concentrations of 0%, 0.2%, 0.4%, 0.6%, 0.8%, and 1% respectively in choline chloride-urea (the molar ratio of choline chloride to urea was 1:1), and soaked at 60°C for 24 h. After soaking, the seaweed residue was dried. The crude fiber content was determined according to step (3) of Example 2.

[0044] The results of the determination of crude fiber content in fermented seaweed residue after soaking in eutectic solvent solutions of different concentrations are as follows: Figure 4 As shown, after soaking in 0.2%, 0.4%, 0.6%, 0.8%, and 1% eutectic solvent solutions, the crude fiber content decreased to varying degrees. Among them, the 0.6% eutectic solvent solution had the most significant effect, reducing the crude fiber content to 11.27%. Soaking in 0.6% eutectic solvent solution (with a molar ratio of choline chloride and urea of ​​1:1) was the optimal eutectic solvent pretreatment condition.

[0045] Example 5 Optimization of Fermentation Conditions Based on the optimization results of the above pretreatment conditions, in order to further reduce the crude fiber content, the fermentation conditions were optimized, and the steps are as follows: (a) Single-factor optimization of fermentation conditions (1) Referring to step (2) of Example 4, the seaweed residue was pretreated by soaking in a 7% glycine solution and then pretreated by soaking in a 0.6% eutectic solvent (the molar ratio of choline chloride and urea is 1:1) solution. After pretreatment, the seaweed residue was dried.

[0046] (2) Take 5 portions of 80 g each of the pretreated seaweed residue from (1) above, and ferment them using the fermentation method of Example 1. The difference from Example 1 is that the amount of bacterial solution added is 0.5%, 1.0%, 1.5%, 2.0%, and 2.5% of the seaweed residue mass (i.e., the bacterial solution volumes are 0.4 mL, 0.8 mL, 1.2 mL, 1.6 mL, and 2.0 mL, respectively). After fermentation, the crude fiber content is determined using the determination method of Example 2.

[0047] The results of the determination of crude fiber content in fermented seaweed residue after fermentation with different amounts of bacterial solution are as follows: Figure 5 As shown.

[0048] (3) Take 5 portions of 80 g each of the seaweed residue pretreated in (1) above, and ferment them using the fermentation method of Example 1. The difference from Example 1 is that the fermentation temperatures are 30℃, 35℃, 40℃, 45℃, and 50℃, respectively. After fermentation, the crude fiber content is determined using the determination method of Example 2.

[0049] The results of the determination of crude fiber content in fermented seaweed residue after fermentation at different fermentation temperatures are as follows: Figure 6 As shown.

[0050] (4) Take 5 portions of 80 g each of the pretreated seaweed residue from (1) above, and ferment them using the fermentation method of Example 1. The difference from Example 1 is that the amount of glucose added as the carbon source is 0%, 0.5%, 1.0%, 1.5%, and 2.0% of the seaweed residue mass, respectively (i.e., the amount of glucose added is 0, 0.4 g, 0.8 g, 1.2 g, and 1.6 g, respectively). After fermentation, the crude fiber content is determined using the determination method of Example 2.

[0051] The results of the determination of crude fiber content in fermented seaweed residue after fermentation with different amounts of added carbon source are as follows: Figure 7 As shown.

[0052] (5) Combination Figure 5 , Figure 6 , Figure 7 Based on the measurement results, the optimization range for subsequent response surface methodology experiments was selected as follows: bacterial culture addition amount 0.5%–1.5%, fermentation temperature 35℃–45℃, and carbon source addition amount 0.5%–1.5%.

[0053] (ii) Response surface optimization of fermentation conditions The pretreated seaweed residue described above (I) (1) was used as the optimization range for the addition of 0.5% to 1.5% of bacterial solution, 35℃ to 45℃ of fermentation temperature, and 0.5% to 1.5% of carbon source. Response surface methodology was designed and regression model analysis was performed.

[0054] The experimental results of the response surface central composite design are shown in Table 1.

[0055]

[0056] Based on the analysis results of the regression model, the most suitable fermentation conditions are: bacterial culture addition of 1.053%, fermentation temperature of 40.241℃, carbon source addition of 1.055%, and the predicted crude fiber content under these conditions is 9.748%.

[0057] Considering practical operating conditions, the optimal fermentation process is modified to: 1% bacterial culture addition, 40℃ fermentation temperature, and 1% carbon source addition. In other words, the optimized fermentation method is as follows: The seaweed residue was immersed in a 7% glycine solution at 60℃ for 24 h. The seaweed residue was then removed and added to a 0.6% eutectic solvent solution (the eutectic solvent consisted of choline chloride and urea in a 1:1 molar ratio) at 60℃ for another 24 h. The seaweed residue was then removed and dried. 80 g of the dried seaweed residue was added to a fermentation chamber along with 40 g of pure water and sterilized at 115℃ for 30 min. After cooling to room temperature, 0.8 g of glucose was added as a carbon source, and 0.8 g of ammonium chloride was added as a nitrogen source. 0.8 mL of a thermophilic mycelium culture (1×10⁻⁶) was then inoculated. 6 The mixture was thoroughly mixed (cfu / mL) and fermented at 40℃ for 10 days, with the fermentation container shaken periodically to ensure even mixing. After fermentation, the fermentation liquid was dried to obtain fermented seaweed residue. Three parallel experiments were conducted under these fermentation conditions, and the average crude fiber content in the obtained fermented seaweed residue was 9.78%.

[0058] Example 6: Feeding Experiment of Fermented Seaweed Residue Feed Additive to Chicks Fermented seaweed residue prepared using the optimized fermentation method in Example 5 was used as a feed additive in a feeding experiment on chicks. Eighty one-day-old Sanhuang chicken chicks (from Juxin Poultry Ecological Hatchery) were randomly divided into five groups (control group, positive control group, low-dose group, medium-dose group, and high-dose group), with 16 chicks in each group. The control group was fed a basal diet; the positive control group was fed a basal diet with 3% lincomycin hydrochloride (by weight) added to the drinking water; the fermented feed groups (low, medium, and high-dose groups) had 1%, 5%, and 10% fermented seaweed residue (by weight) added to their basal diet, respectively. The basal diet was corn-wheat type. The chicks were raised in a free-range manner with rice husks laid on the ground, under 24-hour light, and with free access to feed and water. They were vaccinated according to the normal immunization schedule. The experiment lasted for 22 days.

[0059] Example 7: Collection of chick samples After the experiment, the patient fasted for 12 hours, and 10 mL of blood was collected from the carotid artery after ether treatment. The blood was centrifuged at 3500 r / min for 20 min, and the supernatant was transferred to a centrifuge tube using a pipette and stored at -80℃ for later use. After wetting the feathers with water, the abdominal cavity was quickly opened, and the liver, spleen, and bursa of Fabricius were removed and weighed.

[0060] Example 8: Determination of growth indicators in chicks During the experiment, the feed intake of each replicate chick was recorded daily, and the weight was measured on an empty stomach on days 1 and 8. At the same time, the chick survival rate, average daily weight gain and feed conversion ratio were calculated. The results are shown in Tables 2, 3 and 4.

[0061]

[0062]

[0063]

[0064] As shown in Tables 2, 3, and 4, the survival rate of chicks in the positive control group was the lowest, at only 62.5%, while the survival rate in the medium-dose group was the highest, reaching 87.5%. Compared with the control group, the average daily weight gain and feed conversion ratio of the medium-dose group increased by 44.1% and 8.5%, respectively. The results indicate that the addition of an appropriate amount of fermented seaweed residue can significantly improve the growth performance of chicks.

[0065] Example 9: Determination of Immune Indicators in Chicks Assay for interleukins and immunoglobulins: Set up blank wells (blank control wells contain no sample or enzyme-labeled reagent; all other steps are the same), standard wells, and sample wells. Accurately add 50 μL of standard to the enzyme-labeled plate. Add 40 μL of sample diluent to the sample wells, followed by 10 μL of the sample to be tested (final sample dilution is 5-fold). Add the sample to the bottom of the wells, avoiding contact with the well walls, and gently shake to mix. Seal the plate with sealing film and incubate at 37°C for 30 min.

[0066] Dilute the 30-fold concentrated washing buffer 30 times with distilled water and set aside. Carefully remove the sealing film, discard the liquid, and shake off excess water. Fill each well with washing buffer, let stand for 30 seconds, then discard. Repeat this process 5 times, and pat dry. Add 50 μL of enzyme-labeled reagent to each well, except for the blank wells. Seal the plate with the sealing film and incubate at 37°C for 30 min. Carefully remove the sealing film, discard the liquid, and shake off excess water. Fill each well with washing buffer, let stand for 30 seconds, then discard. Repeat this process 5 times, and pat dry. Add 50 μL of chromogenic reagent A to each well, followed by 50 μL of chromogenic reagent B. Gently vortex to mix, and incubate at 37°C in the dark for 10 min. Add 50 μL of stop solution to each well to stop the reaction (the blue color will immediately turn yellow). Zero the plate using a blank control and measure the absorbance (OD value) of each well sequentially at 450 nm. Measurements should be performed within 15 minutes after adding the stop solution.

[0067] Plot a standard curve on graph paper with the concentration of the standard as the x-axis and the OD value as the y-axis. Find the corresponding concentration of the sample from the standard curve based on the OD value of the sample; then multiply by the dilution factor to get the actual concentration of the sample.

[0068] Measurement of immune organ index: Immune organ index (%) = (organ weight / body weight) × 100%.

[0069] The results of the determination of immune organ indices in chicks are shown in Table 5. The results of the determination of serum immune indicators in chicks are shown in Table 6. Among them, the units of IL-1β, IL-4, IL-6, and IL-10 are all ng / L, and the units of IgA and IgG are all μg / mL.

[0070]

[0071]

[0072] Example 10: Determination of short-chain fatty acids in the intestinal tract of chicks (1) Sample preparation: Accurately weigh 50 mg of tissue sample and place it in a 2 mL grinding tube. Add 800 μL of 0.5% phosphoric acid aqueous solution (containing 10 μg / mL internal standard 2-ethylbutyric acid). Grind the sample by cryogenic grinding for 3 min (frequency 50 Hz), followed by sonication for 10 min and centrifugation at 4℃ and 12000 r / min for 15 min. After centrifugation, take 200 μL of supernatant from the sample and transfer it to a 1.5 mL centrifuge tube. Add 200 μL of n-butanol for solvent extraction. Vortex mix for 10 s, sonicate again at low temperature for 10 min, and then centrifuge at 4℃ and 12000 r / min for 5 min. Transfer the supernatant to a sample vial for chromatographic analysis.

[0073] (2) The analysis was performed using an Agilent 8890B-5977 BGC / MSD gas chromatography-mass spectrometry system. Chromatographic conditions: HPFFAP capillary column (30 m × 0.25 mm × 0.25 μm, Agilent J&W Scientific, Folsom, CA, USA), carrier gas was high-purity helium (purity not less than 99.999%), flow rate was 1.0 mL / min, and the injection port temperature was 180℃. The injection volume was 1 μL, split injection was performed, the split ratio was 10:1, and the solvent delay was 2.5 min. The temperature program was as follows: the initial temperature of the column oven was 80℃, the temperature was increased to 120℃ at a rate of 20℃ / min, the temperature was increased to 160℃ at a rate of 5℃ / min, and then the temperature was maintained at 220℃ for 3 min.

[0074] The results of the determination of short-chain fatty acids in the intestines of chicks are shown in Table 7.

[0075]

[0076] As shown in Table 7, the content of short-chain fatty acids such as acetic acid, propionic acid, butyric acid, and hexanoic acid in the medium-dose group was significantly higher than that in other groups, increasing to 1.94 times, 2.10 times, 1.71 times, and 2.50 times that of the control group, respectively.

[0077] Example 11: Determination of the relative abundance of gut microbiota in chicks Cecal contents of 22-day-old chicks from the control group, positive control group, low-dose group, medium-dose group, and high-dose group were collected into sterile cryovials (3 chicks per group). 16S rRNA sequencing was performed by Shanghai Meiji Biomedical Technology Co., Ltd. DNA was extracted using the DNeasy Power Soil kit, and the absorbance of the DNA was measured to determine its concentration. PCR amplification of the V3-V4 region of the bacterial 16S rRNA gene was performed using forward primer 338F (5'-ACTCCTACGGGAGGCAGCA-3') and reverse primer 806R (5'-GGACTACHVGGGTWTCTAAT-3'). The obtained samples were isolated, purified, and quantified. Paired-end sequencing was then performed on the PersonalBio Illumina Miseq platform. Microbiome bioinformatics analysis was performed using QIIME 2. The DADA2 plugin was then used for quality filtering, noise reduction, merging, and chimera removal to obtain non-mononal amplicon sequence variants (ASVs). The abundance of ASVs at the phylum and genus levels of each sample was statistically analyzed, and the ASV abundance data of each sample were compiled into a table and a bar chart was created using Python.

[0078] The results of the determination of the relative abundance of gut microbiota at the phylum level in chicks are as follows: Figure 8 As shown in the figure, the results of the determination of the relative abundance of gut microbiota at the genus level in chicks are as follows: Figure 9 As shown. By Figure 8 , Figure 9 It was observed that in the positive control group, antibiotic treatment resulted in a predominance of Firmicutes bacteria in the gut microbiota, significantly impacting gut microbial diversity and leading to gut microbiota imbalance. Adding fermented seaweed residue as a feed additive resulted in a significant improvement in gut microbiota composition, most notably at moderate dosages. The medium-dose group not only optimized the gut microbiota composition but also showed effects closer to the healthy control group, indicating that medium-dose fermented seaweed residue feed additives can effectively restore a normal gut microbial environment.

[0079] The top six most abundant genera in the gut microbiota of chicks at the genus level were, in descending order: unclassified genera of the family Lachnospiraceae, unclassified genera of the class Clostridia, *Ruminococcus*, another unclassified genus of Clostridia, and *Lachnoclostridium*. In the positive control group, antibiotic treatment resulted in gut microbiota dysbiosis in chicks, with a significant decrease in the relative abundance of *Lachnoclostridium* and *Ruminococcus*. Adding fermented seaweed residue to the feed significantly increased the relative abundance of these two genera, indicating that fermented seaweed residue has a positive effect on regulating and promoting gut health.

[0080] The above embodiments are provided to those skilled in the art to fully disclose and describe how the claimed implementations can be carried out and used, and are not intended to limit the scope of the disclosure herein. Modifications that will be obvious to those skilled in the art will be within the scope of the appended claims.

Claims

1. A process for preparing seaweed residue fermentation products, characterized in that, Includes the following steps: (1) Add the seaweed residue to a 7% glycine solution and soak it for 24 h at 60℃. (2) Take out the seaweed residue and add it to a 0.6% deep eutectic solvent solution for soaking treatment: soak at 60°C for 24 h; the deep eutectic solvent is choline chloride and urea, and the molar ratio of the two is 1:1; take out the seaweed residue and dry it; (3) Take the dried seaweed residue, add water, glucose as a carbon source, and ammonium chloride as a nitrogen source, inoculate with thermophilic mycelium, and ferment; after fermentation, dry the fermentation liquid to obtain fermented seaweed residue, which is the seaweed residue fermentation product.

2. The preparation process of the seaweed residue fermentation product according to claim 1, characterized in that, The specific operation of step (3) is as follows: take 80 g of dried seaweed residue, add 40 g of water, and sterilize; add 0.8 g of glucose as a carbon source and 0.8 g of ammonium chloride as a nitrogen source, inoculate with thermophilic mycelium, and ferment; after fermentation, dry the fermentation liquid to obtain fermented seaweed residue, which is the seaweed residue fermentation product.

3. The preparation process of the seaweed residue fermentation product according to claim 2, characterized in that: The concentration of the thermophilic filamentous fungal culture was 1×10⁻⁶. 6 The concentration of cfu / mL was 0.8 mL.

4. The preparation process of the seaweed residue fermentation product according to claim 2, characterized in that: The fermentation was carried out at 40°C for 10 days.

5. The seaweed residue fermentation product prepared by the preparation process of seaweed residue fermentation product according to any one of claims 1 to 4.

6. The application of the seaweed residue fermentation product according to claim 5 in the preparation of chicken feed additives.

Images