A double bacteria symbiotic culture method and its application in crayfish feed

Abstract

The invention relates to the field of biotechnology, and discloses a double bacteria symbiotic culture method and its application in crayfish feed, using Paenibacillus phyllosphereus Paenibacillus phyllosphaera HYNU-YU148 ferments raw starch to obtain a first fermentation liquid containing glucose, and then Rhodosporidium toroculans is added to the first fermentation liquid to continue fermentation to obtain a second fermentation liquid containing oil, carotenoids and bacterial proteins; then, a sterile sucrose solution is added to the second fermentation liquid to continue fermentation to obtain a double-bacteria fermentation liquid containing oil, carotenoids, bacterial proteins and extracellular polysaccharides. The present invention does not require traditional high-temperature cooking gelatinization and saccharification cooling sugar-making processes, greatly reducing the fermentation production cost. At the same time, after being added to crayfish feed, it can effectively improve the immunity of crayfish, reduce the occurrence of diseases, improve the water resistance of feed, and reduce its pollution to the water environment.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a double-bacteria symbiotic culture method and application thereof in crayfish feed. Background Art

[0002] Crayfish, scientifically known as Procambarus clarkii, are a freshwater crustacean. Their rapid growth, wide diet, and strong adaptability make them widely distributed in my country and give them a distinct competitive advantage. Crayfish, with their delicious meat, are one of the most popular edible shrimp. With rapid economic development, consumer demand has also grown, further stimulating the booming crayfish aquaculture industry. In recent years, crayfish farming has become the fastest-growing shrimp aquaculture industry in my country due to its high economic returns, short production cycles, and low barriers to entry. According to the "China Crayfish Industry Development Report (2023)," the total value of my country's crayfish industry will exceed 450 billion yuan in 2022, with the aquaculture industry's output value approaching 100 billion yuan. The crayfish farming area covers nearly 30 million mu (approximately 1.5 million hectares), with a production of nearly 3 million tons. With the rapid expansion of crayfish farming, problems have become increasingly prominent. For example, submerged feed has poor water resistance, leading to excessive feed entering the aquatic environment and causing water pollution. As the farming process progresses, crayfish become more susceptible to disease and their immune system becomes weaker. Furthermore, crayfish's high demand for fat is not met, resulting in poor fatness in mature shrimp. These factors are hindering the further development of the crayfish industry. Therefore, it is crucial to provide a feed additive that can promote crayfish growth, enhance their immunity, and improve the ability of feed to adhere to water. Summary of the Invention

[0003] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a method for symbiotic cultivation of two bacteria and its application in crayfish feed, through the phyllosphere bacillus Paenibacillus phyllosphaeraeHYNU-YU148 directly ferments raw starch to produce glucose, which is then fermented by Rhodosporidium torulata to accumulate lipids and carotenoids. Sucrose is then added to the fermentation system to further enhance the accumulation of lipids and carotenoids by Rhodosporidium torulata. This also encourages Paenibacillus phyllosphereus HYNU-YU148 to ferment exopolysaccharides, ultimately forming a dual-bacterial fermentation broth. This broth can be directly used in crayfish feed to increase the fat and carotenoid content. The exopolysaccharides in the fermentation broth also serve as a binder. This process directly hydrolyzes raw starch with Paenibacillus phyllosphereus HYNU-YU148 to provide glucose for Rhodosporidium torulata, eliminating the traditional high-temperature cooking, gelatinization, and saccharification / cooling sugar-making processes, significantly reducing fermentation production costs. Furthermore, when added to crayfish feed, it effectively boosts immune and antioxidant capacity, reduces disease risk, and, due to its strong binding properties, improves the water resistance of the feed, reducing aquatic pollution.

[0004] Technical solution: The present invention provides a dual-bacteria symbiotic culture method, using Paenibacillus phyllosphere Paenibacillus phyllosphaerae HYNU-YU148 ferments raw starch to produce a first fermentation broth containing glucose. Rhodosporidium toruloides yeast is then added to the first fermentation broth to continue fermentation to produce a second fermentation broth containing oil, carotenoids, and bacterial proteins. Sterile sucrose solution is then added to the second fermentation broth to continue fermentation, producing a dual-bacteria fermentation broth containing oil, carotenoids, bacterial proteins, and exopolysaccharides.

[0005] Paenibacillus phyllosphere Paenibacillus phyllosphaerae HYNU-YU148 was deposited in Guangdong Provincial Microbiological Culture Collection on October 25, 2021, with the deposit number GDMCC NO.62008.

[0006] Furthermore, the method specifically includes the following steps:

[0007] S1. Paenibacillus phyllosphaerae Inoculate HYNU-YU148 onto a solid plate medium, place the solid plate medium in a constant temperature incubator at 28-32°C for 2-4 days, pick a single colony from the solid plate medium with an inoculating loop and transfer it to a liquid seed medium, incubate the medium in a shaker at 28-32°C, 100-200 rpm for 1-3 days, and use it as a seed solution for later use.

[0008] S2. Inoculate Rhodosporidium toruloides onto a solid plate medium and incubate the plate in a 30°C incubator for 2-3 days. Use an inoculating loop to pick a single colony from the plate and transfer it to a liquid seed medium. Incubate the culture in a shaker at 30°C, 200 rpm, for 24-36 hours to prepare the seed solution.

[0009] S3 S1 cultured Paenibacillus phyllosphere seed solution was accessed into the raw starch fermentation medium to obtain the first fermentation broth;

[0010] S4 S3 fermentation after 1 day, access S2 cultured round red winter spore yeast seed solution, continued fermentation for 4-5 days to obtain a second fermentation broth, the second fermentation broth seed solution inoculation amount by volume ratio of 1-10%;

[0011] S5. Sterilized sucrose solution was added to the second fermentation broth to make the sucrose concentration in the second fermentation broth 2-4 g / 100 mL. Fermentation was continued for 2 days to obtain a dual-bacteria fermentation broth.

[0012] Furthermore, in S2, the components and mass contents of the solid plate culture medium are: peptone 8-10 g / L, yeast extract powder 5-10 g / L, glucose 10-20 g / L, and agar 15-20 g / L.

[0013] Furthermore, in S2, the components and mass contents of the seed liquid culture medium are: peptone 8-10 g / L, yeast extract powder 5-10 g / L, and glucose 10-20 g / L.

[0014] Furthermore, in S1, the components and mass contents of the solid plate culture medium are: peptone 9-11 g / L, yeast extract powder 4.5-5.5 g / L, sodium chloride 4.5-5.5 g / L, soluble starch 9-11 g / L, and agar 15-20 g / L.

[0015] Furthermore, in S1, the components and mass contents of the seed liquid culture medium are: peptone 9-11 g / L, yeast extract powder 4.5-5.5 g / L, sodium chloride 4.5-5.5 g / L, soluble starch 9-11 g / L, and agar 15-20 g / L.

[0016] Furthermore, in S3, the components and mass contents of the raw starch fermentation medium are: raw starch 20-80 g / L, ammonium sulfate 1-3 g / L, yeast extract 2-10 g / L, dipotassium hydrogen phosphate 1-3 g / L, magnesium sulfate heptahydrate 0.5-1.0 g / L, calcium chloride 0.5-1.5 g / L, and ferrous sulfate heptahydrate 0.008-0.012 g / L;

[0017] In S3, the pH of the raw starch fermentation medium was 5.5–6.5;

[0018] In S3, the seed liquid inoculation amount in the first fermentation liquid is 1-10% by volume.

[0019] Furthermore, in S3, the specific conditions of fermentation are: the shaking speed is 100-200 rpm, the fermentation temperature is 28-32°C;

[0020] In S4, the specific conditions of fermentation were as follows: shaking speed of 150-200 r / min and fermentation temperature of 28-30°C;

[0021] In S5, the specific conditions of fermentation are: shaking speed of 150-200 rpm, and fermentation temperature of 28-30°C.

[0022] The present invention also provides a use of the double-bacteria fermentation liquid prepared by any of the above methods in crayfish feed, characterized in that the double-bacteria fermentation liquid is added to the crayfish feed components and mixed to produce pellet feed;

[0023] Alternatively, the double-bacteria fermentation liquid is vacuum-dried at 60-70°C, dissolved in water before use, and added to the crayfish feed components to mix the ingredients for pellet feed production.

[0024] Furthermore, the amount of the double-bacteria fermentation broth added to the crayfish feed is calculated based on the content of extracellular polysaccharides in the double-bacteria fermentation broth in the crayfish feed being 2-8 g / kg.

[0025] Beneficial effects: Compared with the existing technology, the present invention utilizes Paenibacillus phyllosphereus and Rhodosporidium to co-ferment to produce oil, carotenoids, bacterial protein and extracellular polysaccharides, and obtains a dual-bacteria fermentation liquid. Adding it to crayfish feed can effectively increase the oil, protein and carotenoid content in the feed, enhance the crayfish's immunity and reduce the occurrence of diseases. No additional adhesive is required during feed pelleting, and the extracellular polysaccharides in the dual-bacteria fermentation liquid can effectively improve the water resistance of the crayfish feed. At the same time, the present invention directly utilizes raw starch for dual-bacteria synergistic fermentation, reducing the high-temperature steaming and gelatinization and saccharification cooling steps in the glucose production process. It has the advantages of low cost, suitability for industrial large-scale production, and compliance with environmental protection and sustainable development requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the amount of double-bacteria fermentation broth added to crayfish feed in embodiment 6, based on 8g of exopolysaccharide in the double-bacteria fermentation broth / kg of feed, and the result of the increase in oil and carotenoid in the crayfish feed;

[0027] Figure 2 This is a graph showing the dissolution rate of crayfish feed after adding different amounts of double-bacteria fermentation liquid in Implementation Method 6;

[0028] Figure 3 This is a graph showing the softening time of crayfish feed after adding different amounts of double-bacteria fermentation liquid in Implementation Example 6. DETAILED DESCRIPTION

[0029] The present invention is described in detail below with reference to the embodiments.

[0030] The present invention adopts Paenibacillus phyllosphaeraeHYNU-YU148, this strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center (abbreviated as: GDMCC) (Guangdong Provincial Institute of Microbiology, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province) on October 25, 2021, with the deposit number: GDMCC NO.62008.

[0031] Paenibacillus phyllosphere Paenibacillus phyllosphaerae HYNU-YU148 and Rhodosporidium toruloides co-ferment raw starch to produce a dual-bacteria fermentation broth containing oil, carotenoids, bacterial proteins and exopolysaccharides:

[0032] Implementation method 1:

[0033] The co-fermentation of Paenibacillus phyllosphereus and Rhodosporidium toruloides to produce a dual-bacteria fermentation broth containing oil, carotenoids, bacterial proteins and exopolysaccharides comprises the following steps:

[0034] S1. Paenibacillus phyllosphere Paenibacillus phyllosphaerae HYNU-YU148 was inoculated onto a solid plate medium containing 9 g / L peptone, 4.5 g / L yeast extract, 4.5 g / L sodium chloride, 9 g / L soluble starch, and 15 g / L agar. The solid plate medium was incubated in a 28°C incubator for 2 days. A single colony was picked from the solid plate using an inoculating loop and transferred to a liquid seed medium containing 9 g / L peptone, 4.5 g / L yeast extract, 4.5 g / L sodium chloride, and 9 g / L soluble starch. The culture was incubated at 28°C on a shaker at 100 rpm for one day to prepare the seed solution.

[0035] S2. Inoculate Rhodosporidium toruloides onto a solid plate culture medium composed of 8 g / L peptone, 5 g / L yeast extract, 10 g / L glucose, and 15 g / L agar. Incubate the solid plate culture medium in a 30°C incubator for 2 days. Use an inoculating loop to pick a single colony from the solid plate culture medium and transfer it to a liquid seed culture medium composed of 8 g / L peptone, 5 g / L yeast extract, and 10 g / L glucose. Incubate the culture medium at 30°C and a shaker at 150 rpm for 24 hours to prepare the seed solution.

[0036] S3. The Paenibacillus phyllosphere seed solution cultured in S1 was inoculated at a 1% by volume ratio into a raw starch fermentation medium. The composition and weight content of the raw starch fermentation medium were as follows: 20 g / L raw starch, 1 g / L ammonium sulfate, 2 g / L yeast extract powder, 1 g / L dipotassium hydrogen phosphate, 0.5 g / L magnesium sulfate heptahydrate, 0.5 g / L calcium chloride, and 0.008 g / L ferrous sulfate heptahydrate. The pH of the medium was 5.5. The shaker speed was 150 rpm, the fermentation temperature was 28°C, and the fermentation time was 1 day.

[0037] S4. The Rhodosporidium toruloides seed solution cultured in S2 was inoculated into the fermentation solution obtained in S3 at a volume ratio of 1%. After inoculation, the shaker speed was 150 rpm, the fermentation temperature was 28°C, and the fermentation time was 4 days.

[0038] S5. After fermentation in S4 is complete, add sterilized sucrose solution to the fermentation broth to achieve a sucrose concentration of 2 g / 100 mL. Continue fermentation at a shaker speed of 150 rpm and a temperature of 28°C for 2 days to obtain a dual-bacteria fermentation broth.

[0039] After fermentation, the fermentation broth can be mixed directly with the other ingredients in the crayfish feed. Alternatively, the fermentation broth can be vacuum-dried at 60°C, dissolved in water, and then mixed with the other ingredients in the crayfish feed. The mixture should be pelleted. The amount of fermentation broth added to the crayfish feed should be 2-8g of extracellular polysaccharides per kg of feed.

[0040] Implementation 2:

[0041] The co-fermentation of Paenibacillus phyllosphereus and Rhodosporidium toruloides to produce a dual-bacteria fermentation broth containing oil, carotenoids, bacterial proteins and exopolysaccharides comprises the following steps:

[0042] S1. Paenibacillus phyllosphere Paenibacillus phyllosphaerae HYNU-YU148 was inoculated onto a solid plate medium containing 11 g / L peptone, 5.5 g / L yeast extract, 5.5 g / L sodium chloride, 11 g / L soluble starch, and 20 g / L agar. The solid plate medium was incubated in a 32°C incubator for 4 days. A single colony was picked from the solid plate using an inoculating loop and transferred to a liquid seed medium containing 11 g / L peptone, 5.5 g / L yeast extract, 5.5 g / L sodium chloride, and 11 g / L soluble starch. The culture was incubated at 32°C on a shaker at 200 rpm for 3 days to prepare the seed solution.

[0043] S2. Inoculate Rhodosporidium toruloides onto a solid plate culture medium composed of 10 g / L peptone, 10 g / L yeast extract, 20 g / L glucose, and 20 g / L agar. Incubate the solid plate culture medium in a 30°C incubator for 3 days. Use an inoculating loop to pick a single colony from the solid plate culture medium and transfer it to a liquid seed culture medium composed of 10 g / L peptone, 10 g / L yeast extract, and 20 g / L glucose. Incubate the culture medium at 30°C and 200 rpm on a shaker for 36 hours to prepare the seed solution.

[0044] S3. A 10% by volume inoculum of the Paenibacillus phyllosphere seed solution cultured in S1 was inoculated into a raw starch fermentation medium containing the following components by weight: 80 g / L raw starch, 3 g / L ammonium sulfate, 10 g / L yeast extract, 3 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 1.5 g / L calcium chloride, and 0.012 g / L ferrous sulfate heptahydrate. The pH of the medium was 6.5. The shaker speed was 200 rpm, the fermentation temperature was 32°C, and the fermentation time was 1 day.

[0045] S4. The Rhodosporidium toruloides seed solution cultured in S2 was inoculated into the fermentation solution obtained in S3 at a volume ratio of 10%. After inoculation, the shaker speed was 200 rpm, the fermentation temperature was 30°C, and the fermentation time was 5 days.

[0046] S5. After fermentation in S4 is complete, add sterilized sucrose solution to the fermentation broth to achieve a sucrose concentration of 4 g / 100 mL. Continue fermentation at a shaker speed of 200 rpm and a temperature of 30°C for 2 days to obtain a dual-bacteria fermentation broth.

[0047] Implementation 3:

[0048] The co-fermentation of Paenibacillus phyllosphereus and Rhodosporidium toruloides to produce a dual-bacteria fermentation broth containing oil, carotenoids, bacterial proteins and exopolysaccharides comprises the following steps:

[0049] S1. Paenibacillus phyllosphere Paenibacillus phyllosphaeraeHYNU-YU148 was inoculated onto a solid plate culture medium containing 10 g / L peptone, 5 g / L yeast extract, 5 g / L sodium chloride, 10 g / L soluble starch, and 18 g / L agar. The solid plate culture medium was incubated in a 30°C incubator for 3 days. A single colony was picked from the solid plate using an inoculating loop and transferred to a liquid seed culture medium containing 10 g / L peptone, 5 g / L yeast extract, 5 g / L sodium chloride, and 10 g / L soluble starch. The culture medium was incubated at 33°C with a shaker at 150 rpm for 2 days to prepare the seed culture.

[0050] S2. Inoculate Rhodosporidium toruloides onto a solid plate culture medium composed of 9 g / L peptone, 8 g / L yeast extract, 18 g / L glucose, and 18 g / L agar. Incubate the solid plate culture medium in a 30°C incubator for 2 days. Use an inoculating loop to pick a single colony from the solid plate culture medium and transfer it to a liquid seed culture medium composed of 9 g / L peptone, 8 g / L yeast extract, and 18 g / L glucose. Incubate the culture medium at 30°C and 200 rpm on a shaker for 30 hours to prepare the seed solution.

[0051] S3. A 5% by volume inoculum of the Paenibacillus phyllosphere seed solution cultured in S1 was inoculated into a raw starch fermentation medium containing the following components by weight: 50 g / L raw starch, 2 g / L ammonium sulfate, 6 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 0.8 g / L magnesium sulfate heptahydrate, 1 g / L calcium chloride, and 0.01 g / L ferrous sulfate heptahydrate. The pH of the medium was 6. The shaker speed was 180 rpm, the fermentation temperature was 30°C, and the fermentation time was 1 day.

[0052] S4. The Rhodosporidium toruloides seed solution cultured in S2 was inoculated into the fermentation solution obtained in S3 at a volume ratio of 5%. After inoculation, the shaker speed was 180 rpm, the fermentation temperature was 29°C, and the fermentation time was 5 days.

[0053] S5. After fermentation in S4 is complete, add sterilized sucrose solution to the fermentation broth to achieve a sucrose concentration of 3 g / 100 mL. Continue fermenting at a shaker speed of 180 rpm and a temperature of 29°C for 2 days to obtain a dual-bacteria fermentation broth.

[0054] Implementation 4:

[0055] This embodiment is substantially the same as Embodiment 3, except that:

[0056] S3. A 10% by volume inoculum of the Paenibacillus phyllosphere seed solution cultured in S1 was inoculated into a raw starch fermentation medium containing the following components by weight: 20 g / L raw starch, 3 g / L ammonium sulfate, 10 g / L yeast extract, 3 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 1.5 g / L calcium chloride, and 0.012 g / L ferrous sulfate heptahydrate. The pH of the medium was 6.5. The shaker speed was 200 rpm, the fermentation temperature was 30°C, and the fermentation time was 1 day.

[0057] S4. The Rhodosporidium toruloides seed solution cultured in S2 was inoculated into the fermentation solution obtained in S3 at a volume ratio of 10%. After inoculation, the shaker speed was 200 rpm, the fermentation temperature was 30°C, and the fermentation time was 5 days.

[0058] S5. After fermentation in S4 is complete, add sterilized sucrose solution to the fermentation broth to achieve a sucrose concentration of 3 g / 100 mL. Continue fermenting at a shaker speed of 180 rpm and a temperature of 29°C for 2 days to obtain a dual-bacteria fermentation broth.

[0059] Apart from this, this embodiment is exactly the same as embodiment 3 and will not be described in detail here.

[0060] Implementation 5:

[0061] This embodiment is substantially the same as Embodiment 3, except that:

[0062] S3. The Paenibacillus phyllosphere seed solution cultured in S1 was inoculated at a 1% by volume ratio into a raw starch fermentation medium. The composition and weight content of the raw starch fermentation medium were as follows: 80 g / L raw starch, 1 g / L ammonium sulfate, 10 g / L yeast extract powder, 2 g / L dipotassium hydrogen phosphate, 1 g / L magnesium sulfate heptahydrate, 1 g / L calcium chloride, and 0.008 g / L ferrous sulfate heptahydrate. The pH of the medium was 5.5. The shaker speed was 150 rpm, the fermentation temperature was 28°C, and the fermentation time was 1 day.

[0063] S4. The Rhodosporidium toruloides seed solution cultured in S2 was inoculated into the fermentation solution obtained in S3 at a volume ratio of 1%. After inoculation, the shaker speed was 150 rpm, the fermentation temperature was 28°C, and the fermentation time was 4 days.

[0064] S5. After fermentation in S4 is complete, add sterilized sucrose solution to the fermentation broth to achieve a sucrose concentration of 2 g / 100 mL. Continue fermenting at a shaker speed of 200 rpm and a temperature of 30°C for 2 days to obtain a dual-bacteria fermentation broth.

[0065] Apart from this, this embodiment is exactly the same as embodiment 3 and will not be described in detail here.

[0066] Implementation 6:

[0067] Determination of the application performance of double-bacteria fermentation broth in crayfish feed.

[0068] A certain amount of the double-bacteria fermentation liquid prepared in Implementation 5 was taken, and its oil content, carotenoid content, and exopolysaccharide content were measured. The double-bacteria fermentation liquid was then added to the crayfish feed. The amount of double-bacteria fermentation liquid added to the crayfish feed was 2, 5, and 8 g / kg of exopolysaccharide in the double-bacteria fermentation liquid. They were marked as S2, S5, and S8, respectively. The increase in oil and carotenoid content in S2, S5, and S8 was measured. The measurement results are shown in FIG. Figure 1 ;

[0069] The solubility loss rate and softening time of pelletized crayfish feeds S2, S5, and S8 in water were measured. When measuring the solubility loss rate and softening time, the control groups were no binder and 8 g sodium carboxymethyl cellulose binder / kg feed, marked as NC and PC, respectively. The results are shown in the table. Figure 2 and Figure 3 .

[0070] The control crayfish feed formula consisted of: 15% fish meal, 15% soybean meal, 5% rapeseed cake, 5% cotton cake, 18% bran, 12% rice bran, 1% premix, 2% calcium hydrogen phosphate, 4.5% soybean oil, 2% zeolite powder, 0.5% salt, and 20% corn flour. This crayfish feed formula contained 78.8g of fat per kg of feed and 2.45mg of carotenoids per kg of feed.

[0071] Determination of extracellular polysaccharide content: Take 10 mL of the double-bacteria fermentation broth (V1) and add 10 mL of pure water, mix well, centrifuge at 12000 rpm for 20 min, remove the precipitate, then add 80 mL of anhydrous ethanol, mix well, precipitate at 4 ° C for 12 h, centrifuge at 4000 rpm for 10 min, collect the precipitate and vacuum dry to obtain the extracellular polysaccharide, and record its mass as m 1( g), the extracellular polysaccharide content in the double-bacteria fermentation broth is:

[0072] Extracellular polysaccharide content in fermentation broth = ;

[0073] Determination of oil content: 50 mL of the double-bacteria fermentation broth (V2) was freeze-dried, and then 2 mL of 4 mol / L hydrochloric acid solution was added to the freeze-dried product. After thorough mixing, the mixture was placed at room temperature for 30 minutes, and then cooled in a boiling water bath for 3 minutes. After cooling to room temperature, 2 volumes of a chloroform: methanol (2:1, V / V) mixture was added. After thorough mixing, the mixture was centrifuged at 8000 rpm for 5 minutes, and the lower chloroform layer was collected. An equal volume of 0.1% sodium chloride aqueous solution (mass / volume) was added to the collected chloroform layer, and the mixture was shaken and centrifuged at 5000 rpm for 5 minutes. The chloroform layer was collected in a clean and dry glass dish. The weight of the dish was recorded as m2 (g). The dish was placed in a fume hood to evaporate and remove the chloroform. Then, it was dried at 60°C to constant weight and its weight was weighed as m3 (g). The oil content in the double-bacteria fermentation broth is:

[0074] ;

[0075] Determination of carotenoid content: 10 mL of the dual-bacteria fermentation broth (V3) was freeze-dried. 3 mL of hydrochloric acid solution (3 mol / L) was then added to the lyophilized material and the suspended cells were shaken. The mixture was then placed in a boiling water bath for 5 minutes, cooled with ice water, and 2 mL of acetone was added and shaken vigorously. Then, 5 mL of petroleum ether (30-60°C boiling range) and 3 mL of 0.1% sodium chloride aqueous solution (mass / volume) were added. After vigorous shaking, the mixture was centrifuged at 4000 rpm for 10 minutes. The organic phase was collected, fixed to 10 mL, and appropriately diluted (the dilution factor was designated D) to measure its absorbance at 475 nm. The carotenoid concentration in the extract was converted from the absorbance using the β-carotene standard curve, and the concentration was designated C (μg / L). The carotenoid content in the dual-bacteria fermentation broth is:

[0076] ;

[0077] The method for determining the solubility loss rate is as follows: Weigh 10g of sample (mass is recorded as m4) and place it on a cylindrical sieve with a mesh size of 7cm high, 7cm in diameter, and 30 mesh. Place the sieve in a super thermostat at a temperature of 28°C and immerse it to a depth of 5.5cm. After soaking for 5 minutes, slowly lift the sieve out of the water and then slowly sink it back into the water to allow the feed to leave the bottom of the sieve. Repeat this process three times. Collect the feed in the sieve and dry it at 105°C to constant weight (mass is recorded as m5). At the same time, take the unsoaked feed and dry it at 105°C to constant weight to determine its water content (w). Calculate the solubility loss rate (R):

[0078] ;

[0079] The softening time is determined by selecting 30 intact feed pellets of equal length and placing them in a flat-bottomed glass container. Add 100 mL of pure water, immersing all pellets in the water, and starting the timer. After 15 minutes, randomly remove three pellets, quickly remove any surface moisture, and gently press with a dry spoon to check for a hard core. Then, randomly remove three pellets every 2-5 minutes and repeat this gentle pressing with a spoon until no hard core remains. This time is the softening time.

[0080] Experimental results show that the addition of the dual-bacteria fermentation broth produced by the present invention effectively increases the fat content in crayfish feed. When the dual-bacteria fermentation broth is added to the feed at a concentration of 8g of exopolysaccharides per kg of feed, the fat content in the feed increases by 7.6g per kg of feed. Crayfish have a 7% to 10% higher fat requirement in their feed than common freshwater fish. Adding the dual-bacteria fermentation broth can increase the fat content in the feed, satisfying the crayfish's fat requirement and making them plumper. Furthermore, the results show that the addition of the dual-bacteria fermentation broth can increase the carotenoid content in the feed. Carotenoids are known to have strong antioxidant and immune-enhancing properties. Therefore, adding the dual-bacteria fermentation broth can enhance the immune system of crayfish and reduce their disease risk. In addition, it can be seen from the experimental results that after adding the double-bacteria fermentation liquid, under the action of the extracellular polysaccharides in the double-bacteria fermentation liquid, the dissolution rate of the crayfish feed is significantly lower than that of the feed without adding an adhesive, and its softening time is also greatly improved. As the amount of double-bacteria fermentation liquid added increases, the amount of extracellular polysaccharides added in the double-bacteria fermentation liquid increases, the dissolution rate gradually decreases, and the softening time gradually increases. Compared with the feed with sodium carboxymethyl cellulose added as an adhesive, when the double-bacteria fermentation liquid is added to the feed at an amount of 5g / kg of extracellular polysaccharides in the double-bacteria fermentation liquid, its performance is comparable. The experimental results show that the new crayfish feed double-bacteria fermentation liquid provided by the present invention can effectively increase the content of oil and carotenoids in the feed, thereby improving the quality of crayfish and improving its immune ability. At the same time, it can significantly reduce the dissolution rate of crayfish feed in water, prolong the softening time, and improve its stability in water.

[0081] The above embodiments are intended only to illustrate the technical concepts and features of the present invention. Their purpose is to enable those skilled in the art to understand the contents of the present invention and implement them accordingly. They are not intended to limit the scope of protection of the present invention. Any equivalent changes or modifications made in accordance with the spirit of the present invention are intended to be covered by the scope of protection of the present invention.

Claims

1. A dual bacteria symbiotic culture method, characterized in that: Paenibacillus phyllosphere Paenibacillus phyllosphaerae HYNU-YU148 ferments raw starch to obtain a first fermentation liquid containing glucose, and then Rhodosporidium toroculans is added to the first fermentation liquid to continue fermentation to obtain a second fermentation liquid containing oil, carotenoids and bacterial proteins; then sterile sucrose solution is added to the second fermentation liquid to continue fermentation to obtain a double-bacteria fermentation liquid containing oil, carotenoids, bacterial proteins and extracellular polysaccharides; Paenibacillus phyllosphere Paenibacillus phyllosphaerae HYNU-YU148 was deposited in Guangdong Provincial Microbiological Culture Collection Center on October 25, 2021, with the deposit number GDMCC NO.62008.

2. The dual bacteria symbiotic culture method according to claim 1, characterized in that: The following steps are involved: S1. Paenibacillus phyllosphaerae HYNU-YU148 was inoculated onto a solid plate medium, which was placed in a constant temperature incubator at 28-32°C for 2-4 days. A single colony was picked from the solid plate medium with an inoculation loop and placed in a liquid seed medium, which was shaken at 28-32°C and 100-200 rpm for 1-3 days as seed liquid for later use; S2. Inoculate Rhodosporidium toroculans onto a solid plate medium, place the solid plate medium in a 30°C constant temperature incubator and culture for 2-3 days, pick a single colony from the solid plate medium with an inoculation loop and transfer it to a liquid seed medium, and culture it in a shaking incubator at 30°C and 200 rpm for 24-36 hours as a seed solution for later use; S3. The seed solution of Paenibacillus phyllosphere cultured in S1 was inoculated into the raw starch fermentation medium to obtain the first fermentation broth; S4 S3 fermentation 1 day after access S2 cultured round red winter spore yeast seed solution, continued fermentation for 4-5 days to obtain a second fermentation liquid, the second fermentation liquid seed liquid inoculation amount by volume ratio of 1-10%; S5. Add sterilized sucrose solution to the second fermentation broth to make the sucrose mass concentration in the second fermentation broth 2-4 g / 100 mL, and continue fermentation for 2 days to obtain a dual-bacteria fermentation broth.

3. The dual bacteria symbiotic culture method according to claim 2, characterized in that: In S2, the components and mass contents of the solid plate culture medium are: peptone 8-10 g / L, yeast extract powder 5-10 g / L, glucose 10-20 g / L, and agar 15-20 g / L.

4. The dual bacteria symbiotic culture method according to claim 2, characterized in that: In S2, the components and mass contents of the seed liquid culture medium are: peptone 8-10 g / L, yeast extract powder 5-10 g / L, and glucose 10-20 g / L.

5. The dual bacteria symbiotic culture method according to claim 2, characterized in that: In S1, the components and mass contents of the solid plate culture medium are: peptone 9-11 g / L, yeast extract powder 4.5-5.5 g / L, sodium chloride 4.5-5.5 g / L, soluble starch 9-11 g / L, and agar 15-20 g / L.

6. The dual bacteria symbiotic culture method according to claim 2, characterized in that: In S1, the components and mass contents of the seed liquid culture medium are: peptone 9-11 g / L, yeast extract powder 4.5-5.5 g / L, sodium chloride 4.5-5.5 g / L, soluble starch 9-11 g / L, and agar 15-20 g / L.

7. The dual bacteria symbiotic culture method according to claim 2, characterized in that: In S3, the components and mass contents of the raw starch fermentation medium are: raw starch 20-80 g / L, ammonium sulfate 1-3 g / L, yeast extract 2-10 g / L, dipotassium hydrogen phosphate 1-3 g / L, magnesium sulfate heptahydrate 0.5-1.0 g / L, calcium chloride 0.5-1.5 g / L, ferrous sulfate heptahydrate 0.008-0.012 g / L; In S3, the pH of the raw starch fermentation medium was 5.5-6.5; In S3, the seed liquid inoculation amount in the first fermentation liquid is 1-10% by volume.

8. The dual bacteria symbiotic culture method according to claim 2, characterized in that: In S3, the specific conditions of fermentation are: the shaking speed is 100-200 rpm, and the fermentation temperature is 28-32°C; In S4, the specific conditions of fermentation were: shaking speed of 150-200 r / min, fermentation temperature of 28-30°C; In S5, the specific conditions of fermentation are: shaking speed of 150-200 rpm, and fermentation temperature of 28-30°C.

9. Use of the double-bacteria fermentation liquid prepared by the method according to any one of claims 1 to 8 in crayfish feed, characterized in that: The double-bacteria fermentation liquid is added to the crayfish feed components to mix the feed and produce pellet feed; Alternatively, the double-bacteria fermentation liquid is vacuum dried at 60-70°C, dissolved in water when used, and added to the crayfish feed components to mix with the feed to produce pellet feed.

10. The use of the double-bacteria fermentation liquid in crayfish feed according to claim 9, characterized in that: The amount of the double-bacteria fermentation liquid added to the crayfish feed is calculated based on the content of extracellular polysaccharides in the double-bacteria fermentation liquid in the crayfish feed being 2-8 g / kg.

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