Method for producing water body environment protecting agent by fermenting hermetia illucens larva serous fluid

Abstract

The invention relates to a method for producing a water environment protective agent by fermenting hermetia illucens larva slurry, and belongs to the technical field of microbial fermentation. According to the present invention, the hermetia illucens larvae are adopted as the main raw material, the main raw material is ground into the slurry through the colloid mill, the small amount of the carbon source is supplemented, the Phaffia rhodozyma CGMCC NO.29217, the Bacillus coagulans CGMCC NO.5233 and the Clostridium butyricum CGMCC NO.4729 are subjected to the mixed bacteria synergistic fermentation, and the produced product can be widely used in the aquaculture and other application scenarios. Fermented hermetia illucens larva serous fluid is rich in astaxanthin, a fermented product has obvious acid fragrance and no peculiar smell, the total number of probiotics is larger than or equal to 5.0 * 10 < 8 > cfu / g, the pH value is smaller than 4.50, and the content of small molecule active polypeptide (acid soluble protein) of the fermented product is increased by 2.5-3 times compared with that before fermentation. Bacillus subtilis CGMCC (B) 63501 is used as indicator bacteria to detect that the content of bacteriostatic active substances after fermentation of hermetia illucens larva serous fluid is larger than 1.0 g / kg, pathogenic bacteria such as salmonella, vibrio parahaemolyticus and vibrio cholerae are not detected, and normal-temperature storage can be achieved. The preparation method is low in production cost, easy to operate, safe and free of residues, and the economic benefits of aquaculture can be improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, and relates to an application technology that uses black soldier fly larvae as the main raw material, grinds them into a slurry using a colloid mill, adds a small amount of carbon source, and uses Phaffia rhodozyma CGMCC NO.29217, Bacillus coagulans CGMCC No.5233, and Clostridium butyricum CGMCC No.4729 for fermentation. The resulting product can be widely used in aquaculture and other fields. Background Technology

[0002] Astaxanthin is a red, luteinous carotenoid with strong antioxidant capabilities due to its unique structure, making it widely used in aquaculture and the food industry. Currently, astaxanthin is mainly produced through chemical synthesis, animal and plant extraction, and microbial fermentation. Chemically synthesized astaxanthin involves complex processes, significant pollution, and the simultaneous presence of three stereoisomers, resulting in low animal bioavailability. Compared to chemically synthesized astaxanthin, astaxanthin produced through animal and plant extraction and microbial fermentation has higher bioavailability, stronger coloring ability, and higher biological activity, attracting increasing attention for its development and utilization. Red Pharfoay yeast can synthesize astaxanthin intracellularly, making it one of the most promising and economical natural sources of astaxanthin.

[0003] Black soldier flies (Hemiberlesia spp.) are saprophytic insects belonging to the family Bandidae and the genus Hemiberlesia spp. Their larvae are rich in nutrients such as amino acids, proteins, and minerals, as well as active substances like antimicrobial peptides, chitin, and chitosan. As feed, they can enhance the immunity and disease resistance of farmed animals. In aquaculture, many farmers use black soldier fly larvae as animal protein feed. However, during the process of aquatic animals consuming and dissecting the larvae, a significant amount of nutrients are lost from the larvae, and a large amount of undigested larval skin can be found in their digestive tract. In some ponds, larval skin can even float on the surface. The various digestive enzymes, organic acids, and vitamins produced by microbial fermentation greatly enhance the nutritional value of the insect protein feed. Furthermore, microbial fermentation of compound protein feed can yield physiologically active antimicrobial peptides and improve the bioactivity of health-promoting substances. The fermentation products also contain a certain number of probiotics, which can improve the intestinal microecological environment of animals, playing a significant role in their health and nutrition.

[0004] Patent application CN202410134640.5 discloses a method for processing black soldier fly larvae by removing impurities, dehydrating and sterilizing them, then mixing and pulverizing them with an organic carbon source, fermenting them, and finally drying them to obtain fermented larval powder. This method maximizes the nutritional value of the black soldier fly larvae and increases the content of peptides, functional lipids, and flavor substances in the fermented black soldier fly larvae powder while reducing the content of harmful bacteria. Patent application CN201910436161.8 describes a method for preparing feed protein using microbial fermentation of black soldier fly larvae. This method involves pulping the black soldier fly larvae, mixing them with soybean meal, adding a carbon source, nitrogen source, and microbial inoculum, fermenting them, and then drying and pulverizing them to obtain fermented black soldier fly protein feed. However, the high-temperature heat treatment or drying and pulverizing methods in the above patents lead to the loss of a large amount of bioactive substances.

[0005] Patent application CN202311184508.7 describes a method for processing black soldier fly larvae by scalding and crushing them, adding a concentrated mixed extract of galangal leaves, olive leaves, and areca nut pulp, inoculating with a compound microbial strain, and continuing fermentation. This method is applied to feed to improve shrimp resistance to Aeromonas hydrophila. Patent application CN202110627082.2 discloses a method for processing fermented feed for aquaculture based on black soldier fly larvae. This method involves mincing and homogenizing fresh black soldier fly larvae, mixing them with wheat bran, rice bran, and wheat middlings, adding acidic protease, and mixing to obtain a fermentation medium. This medium is then inoculated with Bacillus fermentation seed liquid, yeast fermentation seed liquid, and lactic acid bacteria fermentation seed liquid, and subjected to anaerobic fermentation under sealed conditions to obtain the finished product. The fermentation process involves synergistic fermentation of bacteria and enzymes, resulting in thorough fermentation. The finished product has a high content of small peptides, is rich in organic acids, and has antibacterial activity. It also has a long storage time, is easy to use, and offers good economic benefits.

[0006] The invention patent with patent number CN201110313933.2 describes the fermentation broth of Bacillus coagulans CGMCC No. 5233, which contains active ingredients such as phenyllactic acid and has a good inhibitory effect on plant pathogens. The invention patent with patent number CN201310546125.X introduces the synergistic fermentation of Bacillus coagulans CGMCC No. 5233 and Clostridium butyricum CGMCC No. 4729, which has mutually beneficial effects. Furthermore, the fermentation process of Clostridium butyricum CGMCC No. 4729 produces a large amount of butyric acid, folic acid, α-ketoisocaproic acid, and other bioactive substances, which have a strong intestinal regulating effect, adjust the intestinal microecological balance, treat related inflammation and diarrhea, and enhance the host's immunity. By using patented strains such as Pharfovia rubescens CGMCC NO.29217, Bacillus coagulans CGMCC No.5233, and Clostridium butyricum CGMCC No.4729 to co-ferment black soldier fly larvae slurry, the accumulation of antimicrobial peptides, astaxanthin, phenyllactic acid, butyric acid, folic acid, and other bioactive substances can be maximized. At the same time, the microorganisms can improve water quality, reduce ammonia nitrogen, decompose residual feed, protect the aquatic environment of aquaculture, and regulate the intestinal microecological balance of farmed animals. Summary of the Invention

[0007] The purpose of this invention is to provide a novel aquatic environmental protection agent by co-fermenting black soldier fly larvae slurry with a mixed culture of *Phaefflera heliotropium* (CGMCC No. 29217), *Bacillus coagulans* (CGMCC No. 5233), and *Clostridium butyricum* (CGMCC No. 4729). The fermented product is rich in various probiotics, astaxanthin, and antimicrobial peptides, exhibiting a predominantly acidic aroma with no off-odors. It has a low pH value, and pathogenic bacteria such as *Salmonella*, *Vibrio parahaemolyticus*, and *Vibrio cholerae* were not detected. Furthermore, it can be stored at room temperature. This preparation method is low-cost, simple to operate, safe, and residue-free, making it widely applicable in aquaculture and other similar applications.

[0008] This invention is achieved through the following technical solution:

[0009] A novel probiotic co-fermentation technology, using black soldier fly larvae as the main raw material, is developed by grinding them into a slurry using a colloid mill, supplementing with a small amount of carbon source, and co-fermenting with mixed bacteria including *Phaeff's erythrosporum* (CGMCC NO. 29217), *Bacillus coagulans* (CGMCC No. 5233), and *Clostridium butyricum* (CGMCC No. 4729). This technology can be widely used in microecological preparations. It includes the cultivation of probiotic strains and the process of co-fermenting the black soldier fly larvae slurry with mixed bacteria. This new water body environmental protection agent can maximize the accumulation of bioactive substances such as antimicrobial peptides, astaxanthin, phenyllactic acid, butyric acid, and folic acid, as well as live probiotics.

[0010] The aforementioned *Phaffia rhodozyma*, named TK1201, with the taxonomic name *Phaffia rhodozyma*, has the accession number CGMCC No. 29217, accession date of December 4, 2023, and is located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The depository is the China General Microbiological Culture Collection Center.

[0011] The Bacillus coagulans described is named TQ33, classified as Bacillus coagulans, with accession number CGMCC No. 5233, accession date: September 9, 2011, accession address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, depositary institution: China General Microbiological Culture Collection Center.

[0012] The *Clostridium butyricum* strain described is named TK2, classified as *Clostridium butyricum*, with accession number CGMCC No. 4729, accession date April 2, 2011, accession address No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and depository institution: China General Microbiological Culture Collection Center.

[0013] The seed culture medium for Pharfia redis CGMCC NO.29217 is as follows: 33-37g glucose, 2.5-3.5g yeast extract, 0.8-1.2g dipotassium hydrogen phosphate, 0.4-0.6g magnesium sulfate, 1L water, pH at natural.

[0014] The seed culture medium for Bacillus coagulans CGMCC No. 5233 is: 20-50 g peptone, 20-50 g glucose, water added to 1 L, pH 7.0-7.2.

[0015] The seed culture medium for Clostridium butyricum CGMCC No. 4729 is as follows: 10-15g beef extract, 5-10g peptone, 3-5g yeast extract, 5-10g glucose, 1-2g soluble starch, 5-10g sodium chloride, 3-5g sodium acetate, 0.5-1g L-cysteine ​​hydrochloride, with water added to 1 L, pH 7.0-7.2.

[0016] Seed culture conditions: Pharfovia rubescens CGMCC NO.29217 was cultured at 23-28℃ on a shaker at 150-210 rpm for 4-8 days; Bacillus coagulans CGMCC No.5233 was cultured at 30-37℃ on a shaker at 150-210 rpm for 20-24 h; Clostridium butyricum CGMCC No.4729 was placed in an anaerobic incubator, with high-purity nitrogen replaced twice, and cultured at 30-37℃ for 20-24 h.

[0017] The black soldier fly larvae used are commercially available frozen larvae or live larvae purchased directly from breeding enterprises. The larvae are pale yellow to yellowish-brown in color. They are washed 3-5 times with clean water to remove blackened larvae and other impurities such as bones and iron products, ensuring proper grinding and minimizing wear on the colloid mill equipment. After screening and washing, the larvae are clean and tidy, then ground into a particle size of ≤40 mesh using a colloid mill. The ground slurry is directly transported through a hose to a sealed fermentation tank, while carbon source and inoculum are continuously added in proportion to prevent oxidation of the black soldier fly larvae slurry.

[0018] The carbon source mentioned includes one or more of glucose, sucrose, brown sugar, molasses or flour, and is not limited to the carbon sources listed herein.

[0019] Furthermore, the composition of the fermented black soldier fly larvae slurry is as follows: black soldier fly larvae slurry accounts for 60%-90%, carbon source (glucose, sucrose, brown sugar, or molasses, etc.) accounts for 5%-15%, and compound microbial strains account for 5%-15%. The material is a light yellow to yellowish-brown, free-flowing, thick slurry with a moisture content of 50-85%.

[0020] Furthermore, the inoculation ratio of the compound strains is 3-6% (v / w) for Pharfovia rubescens CGMCC NO.29217, 1-5% (v / w) for Bacillus coagulans CGMCC No.5233, and 0-5% (v / w) for Clostridium butyricum CGMCC No.4729.

[0021] Furthermore, the fermentation conditions are as follows: fermentation at 25-30℃ without aeration for 2-10 days, liquid volume of 60%-90%, and stirring speed of 120 rpm-300 rpm. The fermentation process utilizes *Phaeophyte Rhodotorula* and *Bacillus coagulans* to consume the residual air in the tank, providing optimal fermentation conditions for the anaerobic fermentation of *Clostridium butyricum*. Simultaneously, *Phaeophyte Rhodotorula* and *Bacillus coagulans* can also undergo anaerobic fermentation.

[0022] The present invention has the following beneficial effects:

[0023] 1) This invention employs *Phaffia rhodozyma* CGMCC No. 29217, *Bacillus coagulans* CGMCC No. 5233, and *Clostridium butyricum* CGMCC No. 4729 for co-fermentation. *Phaffia rhodozyma* and *Bacillus coagulans* can perform both aerobic and anaerobic fermentation, while *Clostridium butyricum* performs anaerobic fermentation. During the fermentation feeding stage, the oxidation reaction of the black soldier fly larvae slurry can be reduced, and during fermentation, the oxidized and darkened material can be reduced to a golden yellow color.

[0024] 2) This invention can retain the antimicrobial peptides and functional oils in the black soldier fly larvae to the greatest extent, and can continuously accumulate bioactive substances such as phenyllactic acid, butyric acid and folic acid during the fermentation process.

[0025] 3) This invention uses multi-strain synergistic fermentation of black soldier fly larvae, resulting in thorough fermentation, high content of small peptides, sour aroma, good palatability, and a 2.5-3 times higher content of small molecule active polypeptides (acid-soluble proteins) compared to before fermentation;

[0026] 4) This invention refers to the national standard for antibiotic testing, using Bacillus subtilis CGMCC(B)63501 as an indicator bacterium to detect the content of antibacterial active substances in fermented black soldier fly larvae slurry >1.0g / kg;

[0027] 5) The fermented black soldier fly larvae slurry of this invention contains astaxanthin, and the astaxanthin content is >10mg / kg as determined by high performance liquid chromatography;

[0028] 6) The fermented black soldier fly larvae slurry of this invention has a low pH and contains abundant antibacterial substances that can effectively reduce the content of pathogenic bacteria. After testing, no pathogenic bacteria such as Salmonella, Vibrio parahaemolyticus, and Vibrio cholerae were detected in the fermented product.

[0029] 7) The product of this invention is rich in probiotics, which can improve water quality, reduce ammonia nitrogen, decompose residual feed, improve the growth environment of intestinal flora, and enhance the body's immunity.

[0030] 8) This invention simplifies the fermentation process while ensuring product quality, has low production costs, is easy to operate, is safe and residue-free, and the fermented product can be stored at room temperature. Attached Figure Description

[0031] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0032] Figure 1 Photos of the fermentation process of black soldier fly larvae slurry.

[0033] Figure 2 Results of fatty acid composition analysis in fermented black soldier fly larvae slurry

[0034] Figure 3 HPLC chromatogram for determination of astaxanthin content in fermented black soldier fly larvae slurry

[0035] Figure 4Graph showing the determination of antibacterial substance content in fermented black soldier fly larvae slurry.

[0036] Figure 5 Results of detection of Vibrio cholerae and Vibrio parahaemolyticus in fermented black soldier fly larvae slurry Detailed Implementation

[0037] The present invention will be further illustrated below with reference to examples. These examples are illustrative and not limiting, and should not be used to limit the scope of protection of the present invention.

[0038] Rhodopseudomonas erythrosporum, Bacillus coagulans, and Clostridium butyricum are all deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession numbers CGMCC No. 29217, CGMCC No. 5233, and CGMCC No. 4729, respectively. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0039] Step 1: Laboratory Seed Preparation

[0040] Rhodopseudomonas erythrosporum CGMCC No. 29217, Bacillus coagulans CGMCC No. 5233, and Clostridium butyricum CGMCC No. 4729 were inoculated from frozen glycerol tubes into culture medium containing liquid seed culture. Rhodopseudomonas erythrosporum CGMCC No. 29217 was cultured at 23-28℃ on a shaker at 150-210 rpm for 4-8 days; Bacillus coagulans CGMCC No. 5233 was cultured at 30-37℃ on a shaker at 150-210 rpm for 20-24 h; and Clostridium butyricum CGMCC No. 4729 was placed in an anaerobic incubator, with high-purity nitrogen replaced twice, and cultured at 30-37℃ for 20-24 h.

[0041] The seed culture medium for Pharfia redis CGMCC NO.29217 is: 33-37g glucose, 2.5-3.5g yeast extract, 0.8-1.2g dipotassium hydrogen phosphate, 0.4-0.6g magnesium sulfate, 1L water, pH natural.

[0042] The seed culture medium for Bacillus coagulans CGMCC No. 5233 is: 20-50 g peptone, 20-50 g glucose, water added to 1 L, pH 7.0-7.2.

[0043] The seed culture medium for *Clostridium butyricum* CGMCC No. 4729 is as follows: 10-15g beef extract, 5-10g peptone, 3-5g yeast extract, 5-10g glucose, 1-2g soluble starch, 5-10g sodium chloride, 3-5g sodium acetate, and 0.5-1g L-cysteine ​​hydrochloride, diluted to 1 L with water, pH 7.0-7.2. After preparing the seed culture media for the three bacteria, pour them into suitable containers and sterilize at 121℃ for 15-25 min.

[0044] Step 2: Preparation of liquid bacterial culture

[0045] Prepare liquid seed culture media for Rhodopseudomonas erythrosporum CGMCC NO.29217, Bacillus coagulans CGMCC No.5233, and Clostridium butyricum CGMCC No.4729 respectively, and put them into 30L, 100L, or 300L liquid fermenters. Incubate at 115-121℃ for 30-20 minutes. Inoculate Rhodopseudomonas erythrosporum CGMCC NO.29217, Bacillus coagulans CGMCC No.5233, or Clostridium butyricum CGMCC No.4729 respectively for single-strain liquid fermentation to prepare inoculum.

[0046] The liquid fermentation conditions for Pharfia redis CGMCC NO.29217 are as follows: automatic temperature control 23-28℃, aeration ratio 1:0.5-1, tank pressure control 5-30kPa, motor speed 100-220r / min, and uncontrolled pH fermentation for 24-48h.

[0047] The liquid fermentation conditions for Bacillus coagulans CGMCC No. 5233 are as follows: automatic temperature control 30-37℃, aeration ratio 1:0.5-1, tank pressure control 5-30kPa, motor speed 100-220r / min, and uncontrolled pH fermentation for 24-48h.

[0048] The liquid fermentation conditions for Clostridium butyricum CGMCC No. 4729 are as follows: automatic temperature control at 30-37℃, no aeration or nitrogen aeration, tank pressure control at 5-30 kPa, motor speed at 100-220 r / min, no pH control or pH 5.5-7.0 fermentation for 24-48 h.

[0049] The seed culture medium for Pharfia redis CGMCC NO.29217 is: 33-37g glucose, 2.5-3.5g yeast extract, 0.8-1.2g dipotassium hydrogen phosphate, 0.4-0.6g magnesium sulfate, 1L water, pH natural.

[0050] The seed culture medium for Bacillus coagulans CGMCC No. 5233 is: 20-50 g peptone, 20-50 g glucose, water added to 1 L, pH 7.0-7.2.

[0051] The seed culture medium for Clostridium butyricum CGMCC No. 4729 is as follows: 10-15g beef extract, 5-10g peptone, 3-5g yeast extract, 5-10g glucose, 1-2g soluble starch, 5-10g sodium chloride, 3-5g sodium acetate, 0.5-1g L-cysteine ​​hydrochloride, with water added to 1 L, pH 7.0-7.2.

[0052] Step 3: Fermentation of black soldier fly larvae and fermentation process

[0053] Black soldier fly larvae are either commercially available frozen larvae or live larvae purchased directly from breeding farms. The larvae are pale yellow to yellowish-brown in color. They are washed 3-5 times with clean water to remove blackened larvae and other impurities such as bones and iron objects, ensuring proper grinding and minimizing wear on the colloid mill. After screening and washing, the larvae are clean and tidy, then ground in a colloid mill to a particle size ≤40 mesh. The ground slurry is directly transported through a hose to a sealed fermentation tank, while carbon source and inoculum are continuously added in proportion to prevent oxidation of the black soldier fly larvae slurry. Carbon sources include one or more of glucose, sucrose, brown sugar, molasses, or flour, and are not limited to those listed herein.

[0054] The composition of fermented black soldier fly larvae slurry is as follows: black soldier fly larvae slurry accounts for 60%-90%, carbon source (glucose, sucrose, brown sugar or molasses, etc.) accounts for 5%-15%, and compound microbial strains account for 5%-15%. The material is a light yellow to yellowish-brown, free-flowing, thick slurry with a moisture content of 50-85%.

[0055] The inoculum ratios for fermentation strains were 3-6% (v / w) for Pharfogel's erythrophage CGMCC NO.29217, 1-5% (v / w) for Bacillus coagulans CGMCC No.5233, and 0-5% (v / w) for Clostridium butyricum CGMCC No.4729.

[0056] Step 4: Sensory evaluation

[0057] Sensory evaluation referenced the enterprise standard "Q / 320411HN043-2024 Microbial Water Quality Improver (Insect Fermented Protein Peptides)".

[0058] Step 5: Determination of viable bacterial count

[0059] The detection of viable bacteria count generally uses the plate count method. For the viable bacteria detection of Bacillus coagulans, refer to the enterprise standard "Q / 320411HN031-2022 Feed Additive Bacillus coagulans".

[0060] For the detection of viable bacteria of Clostridium butyricum, refer to the enterprise standard "Q / 320411HN034-2022 Feed Additive Clostridium butyricum".

[0061] The viable cell count of *Pharrellis rubrum* was tested according to the enterprise standard "Q / 320411HN033-2022 Feed Additive *Pharrellis rubrum*".

[0062] Step 6: pH measurement

[0063] Weigh 10g of the fermented sample and place it in an Erlenmeyer flask. Add 90ml of distilled water. Stir with a magnetic stirrer for 30 minutes, let stand for 10 minutes, and then measure the pH value using a precision pH meter.

[0064] Step 7: Moisture content determination

[0065] Moisture content was determined in accordance with the national standard GB / T 6435-2014, "Determination of Moisture in Feed".

[0066] Step 8: Determination of crude protein and acid-soluble protein

[0067] The determination of crude protein and acid-soluble protein was carried out in accordance with the national standards GB / T 6432-2018 Determination of crude protein in feed by Kjeldahl method and GB / T 22492-2008 Soybean peptide powder, respectively.

[0068] Step 9: Determination of Astaxanthin Content

[0069] Extraction of antioxidant active substances from fermentation broth: The fermented black soldier fly larvae slurry was freeze-dried and pulverized. 1 g of culture was added to 4 mL of DMSO in a 75°C water bath, mixed thoroughly, and then placed in an ultrasonic cleaner for extraction for 20 min. After extraction, 4 mL of anhydrous ethanol was added, shaken to mix, and then placed in an ultrasonic cleaner for extraction for another 20 min. The mixture was then centrifuged at 12000 rpm for 15 min, and the supernatant was collected. The test solution was obtained.

[0070] Astaxanthin determination by liquid chromatography: The astaxanthin content in the above-obtained test solution was determined by high-performance liquid chromatography (HPLC). HPLC conditions: Column: C18 (4.6 × 250 mm, 5 μm), column temperature: 25℃, flow rate: 1 mL / min, injection volume: 20 μL, detection wavelength: 478 nm, mobile phase (volume ratio): V_methanol:V_acetonitrile = 9:1. An HPLC chromatogram of astaxanthin standard was plotted according to this method.

[0071] Step 10: Determination of antibacterial substance content

[0072] The determination of antibacterial substance content was carried out in accordance with the national standard GB / T 21542-2008 Microbiological method for the determination of enramycin in feed, with Bacillus subtilis CGMCC(B)63501 as the indicator bacterium.

[0073] Step 11: Determination of crude fat content

[0074] The determination of crude fat content refers to the national standard GB / T 6433-2025 "Determination of crude fat in feed".

[0075] Step 12: Determination of fatty acid composition

[0076] The determination of fatty acid composition refers to the national standard GB 5009.168-2016 National Food Safety Standard - Determination of Fatty Acids in Food.

[0077] Step 13: Determination of Salmonella content

[0078] The determination of Salmonella content refers to the national standard GB / T 13091-2018 Determination of Salmonella in Feed.

[0079] Step 14: Detection of Vibrio parahaemolyticus content

[0080] The determination of Vibrio parahaemolyticus content refers to the national standard GB 4789.7-2013, "National Food Safety Standard - Microbiological Examination of Food - Examination of Vibrio parahaemolyticus".

[0081] Step 15: Detection of Vibrio cholerae content

[0082] The detection of Vibrio cholerae content is based on the industry standard SN / T 1022-2010 Test Method for Vibrio cholerae in Imported and Exported Food.

[0083] The following describes the determination of some parameters and the optimization of some culture conditions for the method of producing water environmental protection agents using black soldier fly larvae slurry.

[0084] Comparative Case: Natural Fermentation of Black Soldier Fly Larvae Pulp

[0085] Black soldier fly larvae slurry: brown sugar: water = 80:10:10.

[0086] Fermentation of black soldier fly larvae slurry was carried out in a 30L liquid fermentation tank. 21.6kg of fresh black soldier fly larvae were washed five times and placed in a colloid mill hopper. The slurry was then pulped using the finest setting on the colloid mill and fed into the fermentation tank through a hose to reduce oxidation and darkening of the slurry. Simultaneously, the mixed inoculum and brown sugar were slowly added to the fermentation tank. After thorough mixing, fermentation began at a controlled temperature of 28-30℃ for 8 days. The fermentation was carried out in a sealed, aerated environment. The automatic venting tank pressure was set to 25kPa, and the filling coefficient was 90%. Stirring was maintained throughout the fermentation process.

[0087] Naturally fermented black soldier fly larvae slurry is a gray liquid that oxidizes to black on the surface and has a distinct fishy odor. The pH value of the black soldier fly larvae slurry is generally 8.40-8.50, the crude protein content is 10.92g / 100g, the acid-soluble protein content is 2.81g / 100g, and the acid-soluble protein ratio is 25.73%.

[0088] Example 1: Fermentation of black soldier fly larvae slurry by *Phaeophyte rubrum* and *Bacillus coagulans*

[0089] Laboratory strains and liquid seed preparations of *Phaeophyte Rhodotorula* CGMCC No. 29217 and *Bacillus coagulans* CGMCC No. 5233 were obtained through steps 1 and 2.

[0090] Black soldier fly larvae slurry: brown sugar: red Pharf yeast: Bacillus coagulans strain = 90:5:3:2.

[0091] Fermentation of black soldier fly larvae slurry was carried out in a 300L liquid fermentation tank. 243kg of fresh black soldier fly larvae were washed five times and placed in a colloid mill hopper. The slurry was then pulped using the finest setting on the colloid mill and fed into the fermentation tank through a hose to reduce oxidation and darkening of the slurry. Pulping and feeding were carried out simultaneously for approximately 10-15 minutes. At the same time, the valve on the inoculation pipeline was opened, and the mixed inoculum was slowly added to the fermentation tank over a period of 10-12 minutes. After the slurry and inoculum were added, the mixture was stirred and mixed for 20-30 minutes, then brown sugar was added and mixed thoroughly before fermentation began. Fermentation was carried out at a controlled temperature of 28-30℃ for 8 days. The fermentation was conducted in a closed, aerated environment. The automatic venting tank pressure was set to 25kPa, and the filling coefficient was 90%. Stirring was required during the fermentation process to ensure uniform mass and heat transfer and to prevent excessive expansion of the material.

[0092] The fermented black soldier fly larvae slurry is a golden-yellow liquid with a distinct fermented sour aroma and a slight fishy smell. The pH value after fermentation is 5.11, and the total probiotic content is 5.33 × 10⁻⁶. 8 The cfu / g fermentation effect is average.

[0093] Example 2: Co-fermentation of three strains

[0094] Laboratory strains and liquid seed cultures of *Phaeophyte Rhodotorula* CGMCC No. 29217, *Bacillus coagulans* CGMCC No. 5233, and *Clostridium butyricum* CGMCC No. 4729 were obtained through steps 1 and 2.

[0095] Black soldier fly larvae slurry: brown sugar: Pharbitis erythrosporum: Bacillus coagulans: Clostridium butyricum = 90:5:2:2:1.

[0096] Fermentation of black soldier fly larvae slurry was carried out in a 300L liquid fermentation tank. 243kg of fresh black soldier fly larvae were washed five times and placed in a colloid mill hopper. The slurry was then pulped using the finest setting on the colloid mill and fed into the fermentation tank through a hose to reduce oxidation and darkening of the slurry. Pulping and feeding were carried out simultaneously for approximately 10-15 minutes. At the same time, the valve on the inoculation pipeline was opened, and the mixed inoculum was slowly added to the fermentation tank over a period of 10-12 minutes. After the slurry and inoculum were added, the mixture was stirred and mixed for 20-30 minutes, then brown sugar was added and mixed thoroughly before fermentation began. Fermentation was carried out at a controlled temperature of 28-30℃ for 8 days. The fermentation was conducted in a closed, aerated environment. The automatic venting tank pressure was set to 25kPa, and the filling coefficient was 90%. Stirring was required during the fermentation process to ensure uniform mass and heat transfer and to prevent excessive expansion of the material.

[0097] The fermented black soldier fly larvae slurry is a golden-yellow liquid with a distinct fermented sour aroma and a slight fishy smell. The pH value after fermentation is 4.38, and the total probiotic content is 7.58 × 10⁻⁶. 8 The cfu / g concentration indicates good fermentation performance.

[0098] Example 3: Co-fermentation of three strains in a mixed culture, with increased inoculum size and carbon source addition.

[0099] Increasing the inoculum size and carbon source addition can rapidly proliferate the compound microbial strain, allowing it to exert the effect of dominant microbial groups, competitively inhibit pathogens, produce antibacterial substances, and rapidly reduce the pH value of the fermentation slurry. At the same time, excessive carbon source can enrich the flavor of the fermentation slurry and reduce unpleasant odors.

[0100] Laboratory strains and liquid seed cultures of *Phaeophyte Rhodotorula* CGMCC No. 29217, *Bacillus coagulans* CGMCC No. 5233, and *Clostridium butyricum* CGMCC No. 4729 were obtained through steps 1 and 2.

[0101] Black soldier fly larvae slurry: brown sugar: Pharbitis rubescens strain: Bacillus coagulans strain: Clostridium butyricum strain = 80:10:4:4:2.

[0102] Fermentation of black soldier fly larvae slurry was carried out in a 1000L liquid fermentation tank. 720kg of fresh black soldier fly larvae were washed five times and placed in a colloid mill hopper. The slurry was then pulped using the finest setting on the colloid mill and fed into the fermentation tank through a hose to reduce oxidation and darkening of the slurry. Pulping and feeding were carried out simultaneously for approximately 30-40 minutes. At the same time, the valve on the inoculation pipeline was opened, and the mixed inoculum was slowly added into the fermentation tank over a period of 20-30 minutes. After the slurry and inoculum were added, the mixture was stirred for 30-40 minutes, then brown sugar was added and mixed thoroughly before fermentation began. Fermentation was carried out at a controlled temperature of 28-30℃ for 8 days. The fermentation was conducted in a closed, aerated environment. The automatic venting tank pressure was set to 25kPa, and the filling coefficient was 90%. The rotation speed was controlled at 100-150 rpm in the early stages of fermentation, and the speed could be reduced or intermittent stirring (stirring for 10-15 minutes every 2-3 hours) could be used in the later stages.

[0103] The fermented black soldier fly larvae slurry is a golden-yellow liquid (see appendix). Figure 1 It has a distinct fermented sour aroma and no fishy smell. The pH value after fermentation is 3.97, and the total probiotic content is 1.20 × 10⁻⁶. 9 CFU / g; crude protein content was 11.87 g / 100g, acid-soluble protein content was 7.01 g / 100g, and acid-soluble protein accounted for 59.06%; crude fat content was 6.00%, and unsaturated fatty acids accounted for 50.30% of the fat (see appendix). Figure 2 The astaxanthin content was 35 mg / kg. The liquid chromatogram for the astaxanthin content determination is attached. Figure 3 The result of the antibacterial substance content determination was 1.26 g / kg (see attached photo for determination). Figure 4 The coliform bacteria count was <30 MPN / 100g, and Salmonella was not detected (CFU / 25g); the Vibrio cholerae and Vibrio parahaemolyticus counts were both below the detection limit (see appendix). Figure 5 ).

[0104] Furthermore, to meet the needs of different customers, the crude protein content in the product can be reduced, that is, the proportion of black soldier fly larvae slurry can be reduced, and the amount of carbon source or compound strains can be increased. Alternatively, more strains can be added for fermentation to achieve the same fermentation effect as this patent, and all of these should fall within the scope of protection of this patent.

[0105] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the technical solutions of the embodiments of the present invention.

Claims

1. A method for producing a water environmental protection agent by fermenting black soldier fly larvae slurry, comprising using black soldier fly larvae as the main raw material, grinding them into a slurry using a colloid mill, adding a small amount of carbon source, and then co-fermenting with a mixed culture of *Phaeff's rubrum*, *Bacillus coagulans*, and *Clostridium butyricum*. Its characteristics are: The aforementioned Phaffiarhodozyma, named TK1201 and classified as Phaffiarhodozyma, was deposited on December 4, 2023, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO.29217. The Bacillus coagulans, named TQ33, was deposited on September 9, 2011, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 5233. The *Clostridium butyricum* strain, named TK2 and classified as *Clostridium butyricum*, was deposited on April 2, 2011, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC NO. 4729.

2. The composition of the fermented black soldier fly larvae slurry as described in claim 1 is as follows: black soldier fly larvae slurry accounts for 60%-90%, carbon source (glucose, sucrose, brown sugar or molasses, etc.) accounts for 5%-15%, and compound strain accounts for 5%-15%.

3. The method for producing a water environmental protection agent by fermentation of black soldier fly larvae slurry as described in claim 1, characterized in that: The fermentation process involves grinding black soldier fly larvae (fresh or frozen) into a slurry using a colloid mill and directly adding it to the fermentation tank. Simultaneously, a carbon source (glucose, sucrose, brown sugar, or molasses, etc.) and a mixed bacterial culture are slowly added to the fermentation tank in a specific ratio. Fermentation is carried out at 25-30°C without aeration for 2-10 days, with a liquid volume of 60%-90% and a stirring speed of 60-300 rpm. The fermentation process utilizes *Phaefflera rubrum* and *Bacillus coagulans* to consume residual air in the tank, providing optimal fermentation conditions for the anaerobic fermentation of *Clostridium butyricum*. *Phaefflera rubrum* and *Bacillus coagulans* can also perform anaerobic fermentation.

4. The method for producing a water environmental protection agent by fermentation of black soldier fly larvae slurry as described in claim 1 or 3, wherein the inoculation amounts of the strains are as follows: 3-6% (v / w) for Pharfovia rubescens CGMCC NO.29217, 1-5% (v / w) for Bacillus coagulans CGMCC No.5233 and 0-5% (v / w) for Clostridium butyricum CGMCC No.4729.

5. A method for producing a water body environmental protection agent by fermentation of black soldier fly larvae slurry as described in claim 1 or 3, characterized in that: the fermented black soldier fly larvae slurry is rich in astaxanthin, has a sensory color of yellowish-brown to brownish-red, a predominantly sour aroma with no off-odors, a pH < 4.50, and a probiotic content ≥ 5.0 × 10⁻⁶. 8 CFU / g, no pathogenic bacteria such as Salmonella, Vibrio parahaemolyticus, and Vibrio cholerae were detected, and it can be stored at room temperature. The content of small molecule active peptides (acid-soluble proteins) in the fermentation product increased by 2.5-3 times compared with that before fermentation. Using Bacillus subtilis CGMCC(B)63501 as an indicator bacterium, the content of antibacterial active substances in the fermented black soldier fly larvae slurry was >1.0 g / kg.

6. The water environmental protection agent produced by fermenting black soldier fly larvae slurry as described in claim 1 is suitable for application scenarios such as aquaculture.

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