Bacillus siamensis with grease degradation function and application of bacillus siamensis

Abstract

The invention discloses bacillus siamensis with a grease degradation function and application of the bacillus siamensis. The bacillus siamensis SM01-01 with a function of efficiently degrading grease is separated from a soil sample of a pine forest in Taishan Mountain and is screened through grease degradation activity verification, and the preservation number of the strain is CGMCC (China General Microbiological Culture Collection Center) NO.25550. The invention further discloses a preparation method of the bacillus siamensis SM01-01. The bacillus siamensis SM01-01 has the function of degrading the grease in the fish protein raw stock, the degradation rate reaches up to 89.76% under the optimal condition, and the problems of secondary fermentation and stinking caused by grease residues in the fish protein raw stock are fully solved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to a siam bacillus with oil degradation function and application thereof. Background Art

[0002] At present, the team of fish protein fertilizer production is becoming stronger and stronger. Fish protein fertilizer, like seaweed fertilizer and chitin fertilizer, has become a major product for the utilization of marine biological resources, and has gradually occupied the traditional fertilizer market. At present, the raw material for the production of fish protein fertilizer is fish protein pulp, which is obtained by bacterial hydrolysis or enzymatic hydrolysis technology; during the production process, the protein is gradually decomposed into small molecules to play a role; and the oil in the fish meat cannot be fully decomposed, which leads to the secondary fermentation of miscellaneous bacteria to produce hydrogen sulfide, volatile fatty acids and other substances with strong irritating odors, which causes the fish protein pulp to stink. How to effectively degrade the oil in fish protein pulp has become an urgent problem that every fish protein fertilizer production source company needs to solve. At the same time, after the secondary fermentation of fish protein pulp, it is difficult to become a high-quality raw material and becomes an oily waste liquid.

[0003] Traditional physical and chemical methods cannot effectively solve the problem of high oil content in fish protein puree, and are also likely to affect the efficacy of fish protein puree. Microbial technology has become a new way to solve the above problems. Bacillus siamensis, also translated as Sim Bacillus, is a new species published in the genus Bacillus in the past decade. At present, the research on Bacillus siamensis is mainly concentrated in the field of biocontrol and plant growth promotion, and no research has been seen in the field of oil degradation. Summary of the invention

[0004] In view of the above problems, the present invention separated and screened a strain of Bacillus siamensis SM01-01 with the function of highly efficient oil degradation, optimized its degradation conditions, and successfully applied it to the degradation of oil in raw protein puree, fully solving the secondary fermentation and odor problems caused by oil residue in fish protein puree, and showing broad application prospects in the green degradation and transformation of high-oil wastewater.

[0005] The above-mentioned object of the present invention is achieved by the following technical scheme: The present invention isolates and screens Bacillus siamensis SM01-01 with high-efficiency oil degradation function from Taishan pine forest soil samples, and obtains the strain through oil degradation activity verification and screening. The strain has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee on August 19, 2022 (deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing), and the deposit number is CGMCC NO.25550. After the strain grows on beef extract peptone agar medium for 2 to 3 days, the colony is irregularly round, opaque milky white, with wrinkles on the edges, dry and uneven surface, and the central wrinkles are wavy. The strain is rod-shaped under microscopic examination, which will be slightly shortened in the later stage; spores are produced, and they are terminal spores; Gram staining is positive.

[0006] The invention also provides a fermentation bacterial liquid of Bacillus siamensis SM01-01.

[0007] The method for preparing the above-mentioned Siamese Bacillus SM01-01 fermentation liquid comprises the following steps:

[0008] (1) Seed solution acquisition: First, activate Bacillus siamensis SM01-01, then inoculate it into a seed solution culture medium, and culture it at a constant temperature of 25 to 35° C. with shaking for 10 to 15 hours to prepare a seed solution;

[0009] (2) Large-scale fermentation culture: The seed liquid is inoculated into a large-scale fermentation medium at a rate of 1-5%, and fermented at 25-35° C. for 16-20 h to obtain a fermentation culture liquid of Bacillus siamensis SM01-01.

[0010] The formula of the seed liquid culture medium (mass ratio) is: 0.5% glucose, 0.5% peptone, 0.3% beef powder, 0.5% sodium chloride, and the rest is water, and the pH is adjusted to 7.3±0.1.

[0011] The formula of the above-mentioned large-scale fermentation medium (mass ratio) is: 2% tributyrin, 0.9% glucose, 0.8% urea, 0.2% peptone, 0.1% dipotassium hydrogen phosphate trihydrate, 0.1% ammonium sulfate, 0.05% magnesium sulfate heptahydrate, and the rest is water, with a pH of 7.2-7.5.

[0012] The present invention also provides the use of the above-mentioned Bacillus siamensis SM01-01 or its fermented bacterial liquid in the degradation of oil. The optimal conditions for degradation of oil (fish oil) are: at 30°C, the fermented bacterial liquid is inoculated with a 2% inoculum into a liquid culture medium containing fish oil, the pH value is 6.5, and the degradation rate of oil is the highest after 48 hours of reaction.

[0013] The present invention also provides the use of Bacillus siamensis SM01-01 or its fermented bacterial liquid in degrading the oil in fish protein slurry. The degradation rate is 89.76% in 48 hours, which is 32 times that of the blank control group.

[0014] Compared with the prior art, the advantages of the present invention are:

[0015] 1. The present invention isolates Bacillus siamensis SM01-01 from a soil sample of a pine forest in Mount Tai and obtains the bacillus siamensis SM01-01 having a high-efficiency oil degradation function through oil degradation activity verification and screening. It is also the first time to discover that Bacillus siamensis SM01-01 has the function of degrading oil in fish protein puree. Under optimal conditions, the degradation rate is as high as 89.76%, which is 32 times that of the blank control group.

[0016] 2. The fermentation liquid of Bacillus siamensis SM01-01 is easy to obtain and simple to use. It fully solves the problems of secondary fermentation and odor caused by oil residue in fish protein pulp; and shows broad application prospects in the green degradation and transformation of high-oil wastewater. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is the fish oil standard curve;

[0018] Figure 2 The colony morphology is that of Bacillus siamensis SM01-01;

[0019] Figure 3 The bacterial morphology of Bacillus siamensis SM01-01;

[0020] Figure 4 It is the spore morphology of Bacillus siamensis SM01-01;

[0021] Figure 5 The effect of temperature on the oil degradation rate of Bacillus siamensis SM01-01;

[0022] Figure 6 The effect of pH on the oil degradation rate of Bacillus siamensis SM01-01;

[0023] Figure 7 The effect of inoculum size on the oil degradation rate of Bacillus siamensis SM01-01;

[0024] Figure 8 This is the effect of fermentation time on the oil degradation rate of Bacillus siamensis SM01-01. DETAILED DESCRIPTION

[0025] The following will combine the contents in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Example 1: Isolation and screening of strain SM01-01

[0027] The strain SM01-01 involved in the present invention is separated from soil samples of a pine forest in Mount Tai, Tai'an City. The collected samples are respectively subjected to enrichment culture, primary screening and secondary screening in sequence to obtain the desired strain.

[0028] 1. Isolation and screening of oil-degrading bacteria

[0029] (1) Enrichment culture of oil-degrading bacteria

[0030] Take 10g of the collected sample and place it in a conical flask containing 90mL of sterile water, homogenize at 180rpm for 30min; take 1mL of the homogenized liquid and add it to the enrichment medium, and culture it at 30℃ and 180rpm / min for 24h. When the fish oil degrades, take 10mL of the enrichment liquid and add it back to the new enrichment medium. Repeat this three times to obtain a stable enrichment liquid of oil-degrading bacteria, which is stored for later use.

[0031] Enrichment medium: 0.2 g yeast extract, 0.5 g sodium chloride, 3.5 g disodium hydrogen phosphate, 1.5 g potassium dihydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 10 mL fish oil, dilute to 1000 mL with distilled water, adjust pH to 7.0. Sterilize at 121°C for 20 min and store for later use.

[0032] (2) Preliminary screening of oil-degrading bacteria

[0033] Take 10 mL of the oil-degrading bacterial enrichment solution obtained in (1) above and dilute it to 10 with sterile water by density gradient dilution method. -5 , 10 -6 , 10 -7 For the three concentration gradients, 100 μL of the dilution was respectively taken and evenly spread on the neutral red medium with fish oil as the only carbon source, and the medium was cultured in a constant temperature incubator at 30°C. The strains were selected according to the colony morphology and the color development of the neutral red medium, and further separated and purified to obtain single colonies, which were stored for later use.

[0034] Neutral red medium: 5g beef extract, 5g sodium chloride, 10g peptone, 1mL 1.6% neutral red aqueous solution, 10g fish oil, 20g agar powder, distilled water to 1000mL, adjust pH to 7.0. Sterilize at 121℃ for 20min and store for later use.

[0035] (3) Rescreening of oil-degrading bacteria

[0036] The oil-degrading bacteria purified in step (2) were spotted in the center of the tributyrin plate using the spot grafting method and cultured in an incubator at 30°C for 48 h. The size of the transparent circle and the colony diameter were observed, and the strain with a larger ratio of transparent circle to colony diameter was selected for oil degradation rate determination.

[0037] Tributyrin solid medium: 5 g beef extract, 5 g sodium chloride, 10 g peptone, 20 mL tributyrin emulsion (V 三丁酸甘油酯 :V 2％聚乙烯醇 =1:3), agar powder 20g, distilled water to 1000mL, adjust pH to 7.0. Sterilize at 121℃ for 20min and store for later use.

[0038] (4) Determination of oil degradation rate

[0039] Drawing of standard curve: Dissolve fish oil in petroleum ether to prepare fish oil-petroleum ether standard solutions with different concentrations of 0, 5, 10, 15, 20, 25, 30, 35, and 40 mg / mL, measure the absorbance of each solution at a wavelength of 210 nm, and draw a standard curve for fish oil.

[0040] The strains screened out in the rescreening were inoculated into beef extract peptone liquid medium and cultured at 30°C for 48 hours to obtain the seed solution of the corresponding strain. Then, 1% inoculation was inoculated into fish oil liquid medium. After 48 hours of culture, 10 mL of petroleum ether was added to the conical flask for preliminary dilution and extraction. The extract was slightly shaken and then pipetted with a pipette after standing. D was measured by ultraviolet spectrophotometer. 210nm value, determine the oil content, and calculate the oil degradation rate.

[0041] The calculation formula is:

[0042] Grease degradation rate P = (1-W2 / W1) × 100%

[0043] W2: fish oil content after degradation; W1: initial fish oil content; all experiments were set up in triplicate and the data are expressed as mean ± SD.

[0044] Fish oil liquid culture medium: 2g ammonium sulfate, 2g dipotassium hydrogen phosphate, 2g sodium dihydrogen phosphate, 5g sodium chloride, 20g fish oil, distilled water to 1L, pH 7.0. Sterilize at 121℃ for 20min and store for later use.

[0045] 2. Results Analysis

[0046] (1) The initial screening results showed that after enrichment culture, 45 strains showed red color in the neutral red medium. After excluding strains with consistent colony morphology through colony morphology observation, a total of 8 strains were screened and numbered SM01-01 to SM01-08.

[0047] (2) The results of the rescreening showed that after the strains obtained from the initial screening were inoculated on tributyrin plates for culture, transparent circles of different sizes appeared. Among them, SM01-01, SM01-02, and SM01-08 had the best oil degradation effects, with the ratios of transparent circle to colony diameter being 3.23±0.02, 2.72±0.04, and 2.64±0.07, respectively (see Table 1).

[0048] Table 1 Results of the ratio of transparent zone to colony diameter of different strains

[0049]

[0050] Note: Titer assessment: ratio <1.0: -; 1.0≤ratio<2.0: +; 2.0≤ratio<3.0: ++; ratio ≥3.0: +++.

[0051] (3) Draw a standard curve for fish oil (refer to Figure 1 ), and obtain the oil degradation rate.

[0052] The results of the oil degradation rate test showed that after 48 hours of culture in the fish oil liquid medium, the oil degradation rates of SM01-01, SM01-02 and SM01-08 were 68.8%, 54.8% and 52.7% respectively (see Table 2). This result shows that strain SM01-01 can effectively degrade fish oil.

[0053] Table 2 Statistics of oil degradation rate of three strains

[0054]

[0055] Example 2: Identification of strain SM01-01

[0056] 1. Morphological identification

[0057] (1) Colony culture characteristics: After the strain grows on beef extract peptone agar medium for 2 to 3 days, the colonies are irregularly round, opaque milky white, with wrinkles on the edges, a dry and uneven surface, and wavy convex wrinkles in the center (refer to Figure 2 ).

[0058] (2) Microscopic observation: The bacteria are rod-shaped (refer to Figure 3 ), which will become slightly shorter in the later stage; spores are produced, and they are terminal spores (ref. Figure 4 ); Gram stain positive.

[0059] From the above results, it can be preliminarily determined that strain SM01-01 is a Bacillus bacterium.

[0060] 2. Physiological and biochemical identification

[0061] According to the Manual of Identification of Common Bacteria Systems, the strain SM01-01 was subjected to physiological and biochemical identification. The results are shown in Table 3:

[0062] Table 3 Physiological and biochemical characteristics of strain SM01-01

[0063]

[0064] Note: Positive: +; Negative: -

[0065] The strain SM01-01 is positive for oxidase and catalase; it can hydrolyze gelatin, starch and cellulose; it is positive for nitrate reduction and VP test; it is negative for hydrogen sulfide test and methyl red test. Based on the physiological and biochemical characteristics of the strain SM01-01, it can be preliminarily identified as a bacterium of the genus Bacillus.

[0066] 3. Molecular Identification

[0067] The 16S rDNA gene sequence of the strain is as follows (SEQ-1):

[0068]

[0069] Through morphological, physiological and biochemical and molecular identification, strain SM01-01 was identified as Bacillus siamensis. The strain was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on August 19, 2022, with the deposit number CGMCC NO.25550.

[0070] Example 3: Preparation of Siamese Bacillus SM01-01 fermentation broth

[0071] The preparation process of Siamese Bacillus SM01-01 fermentation liquid is as follows:

[0072] (1) Seed liquid acquisition: Under sterile conditions, SM01-01 was streaked on NA medium and activated at 30°C, while observing whether there was any bacterial contamination. After activation, a loop of bacterial moss was taken with an inoculation loop and inoculated into the seed liquid medium, and cultured at 30°C and 180 rpm for 12 h to prepare the SM01-01 seed liquid.

[0073] (2) Large-scale fermentation culture: Infuse water to 60-70% of the tank volume, add materials according to the large-scale fermentation medium formula, wait until the raw materials are completely dissolved, adjust the pH to 7.2-7.3, add 0.1% soybean oil defoamer, and sterilize at 121°C, 0.1-0.12MPa for 30 min; after sterilization, add the seed liquid to the fermentation tank (500L) at a 5% inoculation rate, and ferment and culture at 30°C for 16-20h. The bacterial count can reach about 10 billion / ml, which is the SM01-01 fermentation liquid.

[0074] The formula of seed liquid culture medium (mass ratio) is as follows: 0.5% glucose, 0.5% peptone, 0.3% beef powder, 0.5% sodium chloride, and the rest is water, and the pH is adjusted to 7.3±0.1.

[0075] Formula of large-scale fermentation medium (mass ratio): 2% tributyrin, 0.9% glucose, 0.8% urea, 0.2% peptone, 0.1% dipotassium hydrogen phosphate trihydrate, 0.1% ammonium sulfate, 0.05% magnesium sulfate heptahydrate, and the rest is water, with a pH of 7.2-7.5.

[0076] Example 4: Optimization of oil degradation conditions by Siamese Bacillus SM01-01

[0077] 1. Experimental Methods

[0078] In order to improve the efficiency of SM01-01 bacterial solution in degrading oil, we optimized the main factors affecting the degradation efficiency of the strain. The main test methods are as follows:

[0079] (1) Temperature optimization

[0080] The fermentation temperature was set at 20°C, 25°C, 30°C, 35°C, and 40°C, and 2 mL of bacterial solution was inoculated into a fish oil medium (pH 7.0) containing 20 g / L fish oil at an inoculum size of 2%, and the culture was performed at 180 rpm for 48 hours. The fermentation liquid was extracted with 50 mL of petroleum ether, and its oil degradation rate was detected.

[0081] Fish oil liquid culture medium: 2g ammonium sulfate, 2g dipotassium hydrogen phosphate, 2g sodium dihydrogen phosphate, 5g sodium chloride, 20g fish oil, distilled water to 1L, pH 7.0. Sterilize at 121℃ for 20min and store for later use.

[0082] (2) pH optimization

[0083] Based on the optimum temperature, 2 mL of bacterial solution was inoculated into the fish oil medium containing 20 g / L fish oil according to the inoculation amount of 2%, the pH value was adjusted to 5.5, 6, 6.5, 7, 7.5, and the fermentation was carried out at 180 rpm for 48 hours. The fermentation liquid was extracted with 50 mL of petroleum ether to detect its oil degradation rate.

[0084] (3) Optimization of inoculum size

[0085] Based on the optimum temperature and pH, the inoculation amount was 1%, 2%, 3%, 4%, and 5% respectively inoculated into the fish oil medium containing 20g / L fish oil, and the fermentation culture was carried out at 180rpm for 48h. The fermentation broth was extracted with 50mL petroleum ether to detect its oil degradation rate.

[0086] (4) Fermentation time optimization

[0087] Based on the above optimal conditions, the fermentation culture was carried out at 180 rpm for 24 h, 36 h, 48 h, 60 h and 72 h respectively. The fermentation broth was extracted with 50 mL of petroleum ether to detect its oil degradation rate.

[0088] 2. Results Analysis

[0089] (1) The results of the temperature optimization test show (such as Figure 5 As shown in Figure 2, when the temperature is 30°C, the strain SM01-01 has the best oil degradation effect, with a degradation rate of 68.5%; as the temperature further increases, the degradation rate gradually decreases. This result shows that the optimal temperature for the strain SM01-01 to exert its degradation effect is 30°C.

[0090] (2) The results of pH optimization test showed that ( Figure 6As shown in Figure 2, under the optimal temperature conditions, when the initial pH was 6.5, strain SM01-01 had the best oil degradation effect, with a degradation rate of 71.2%. This result shows that the optimal pH for strain SM01-01 to exert its degradation effect is 6.5, and it can work better in a slightly acidic environment.

[0091] (3) The results of the inoculum optimization test showed that ( Figure 7 As shown in the figure, under the optimal temperature and pH conditions, with the increase of inoculation amount, the degradation rate of oil by strain SM01-01 first increased and then decreased; when the inoculation amount was 2%, the degradation rate of oil reached 72.6%. Both high and low inoculation amounts would affect the degradation efficiency of SM01-01.

[0092] (4) The fermentation time optimization results prove (such as Figure 8 As shown): Under the optimal temperature, pH and inoculum size conditions, when the fermentation time was 48h, the strain SM01-01 had the highest oil degradation efficiency of 74.5%.

[0093] The above results fully demonstrate that the optimal conditions for strain SM01-01 to exert its oil degradation function are: temperature 30°C, pH 6.5, inoculation amount 2%, and fermentation time 48h.

[0094] Example 5: Application of Siamese Bacillus SM01-01 in the process of fish protein paste production to oil degradation

[0095] After the fish meat is crushed, it can be enzymatically hydrolyzed to produce fish protein paste, but the presence of a large amount of oil in the production process leads to a strong fishy and foul smell. In order to test whether Siamese Bacillus SM01-01 bacterial liquid can effectively degrade the oil in fish protein paste, the following experiment was conducted.

[0096] 1. Test methods

[0097] Preparation method of enzymatic fish protein slurry: (1) Raw material selection: fresh whole fish is preferred, as it has a stable protein content, can ensure the stability of the indicators of different batches of finished products, and the fresh fish protein is not corrupt. (2) Raw material cleaning: the frozen fish raw materials transported from the cold storage are cleaned 1 to 2 times to remove surface impurities and salt. (3) Raw material crushing: the cleaned fish raw materials are fully crushed in the crushing workshop to make fish slurry. (4) Feeding: the crushed fish slurry is pumped into the fermentation tank, and a certain amount of water is added. The feed-liquid ratio is about 7:3, and the feed amount is 60 to 70% of the tank capacity. (5) Enzymatic hydrolysis: after the feeding process is completed, the temperature is raised to 90°C for 0.5 hours, and then it is cooled to 50°C to 55°C. Adjust the pH value between 6 and 8, add enzymatic solution (animal-derived protein hydrolase, including protease ≥ 20000U / g, cellulase> 50U / g, pre-dissolve the hydrolase), add 2-5kg / t, keep at 50℃~55℃ for 2-4 hours, and keep the speed at 50-60r / min during the enzymatic hydrolysis. (6) Inactivation of enzymes: When inactivating enzymes, it is necessary to take samples to detect the decomposition of fish meat. No fish meat is visible, and a small amount of fish bones are left. Smell the odor. After meeting the requirements, heat to above 90℃ and inactivate the enzyme for 10 minutes. (7) Filtration after the can is out of the tank: After the enzyme inactivation is completed, filter out the fish bones and some other undecomposed substances, which is the fish protein slurry.

[0098] 100 mL of enzymatically hydrolyzed fish protein pulp was evenly sampled and placed in a 250 mL conical flask. A blank control group CK and a treatment group T1 were set up. Each treatment group was repeated three times. The initial oil concentrations were detected to be 30.2 g / L and 32.4 g / L, respectively. The pH was adjusted to 6.5. 2 mL of Siamese Bacillus SM01-01 fermentation liquid was inoculated into the T1 group. The degradation rate was determined after 48 hours of reaction at 30°C. The degradation rate was determined by the same method as above. The control group was not inoculated with bacterial liquid. Each group was repeated three times.

[0099] 2. Results Analysis

[0100] The results showed that the degradation rate of the blank control group was only 2.8% at 48 hours, while after fermentation with Siamese Bacillus SM01-01, the oil degradation rate was 89.76% at 48 hours, and the degradation efficiency was 32 times that of the control group. This result fully demonstrates that in the production process of fish protein puree, adding Siamese Bacillus SM01-01 for oil degradation can effectively remove residual oil and improve the quality of fish protein puree.

[0101] Table 4 Statistics of oil degradation rate of two groups

[0102]

Claims

1. A Bacillus siamensis SM01-01, the deposit number of which is CGMCCNO.25550.

2. The fermentation liquid of Bacillus siamese SM01-01 according to claim 1.

3. The method for preparing the siam Bacillus SM01-01 fermentation liquid according to claim 2, characterized in that: The following steps are involved: (1) Seed solution acquisition: first, activate Siamese Bacillus SM01-01, then inoculate it into a seed solution culture medium, and culture it at a constant temperature of 25 to 35° C. with shaking for 10 to 15 hours to prepare a seed solution; (2) Large-scale fermentation culture: The seed liquid is inoculated into a large-scale fermentation medium at a rate of 1-5%, and fermented at 25-35° C. for 16-20 h to obtain a fermentation culture liquid of Bacillus siame SM01-01.

4. The method for preparing the fermentation liquid of Bacillus siamese SM01-01 as claimed in claim 3, characterized in that: The seed liquid culture medium comprises: 0.5% glucose, 0.5% peptone, 0.3% beef powder, 0.5% sodium chloride, and the rest is water, and the pH is adjusted to 7.3±0.

1.

5. The method for preparing the fermentation liquid of Bacillus siamese SM01-01 as claimed in claim 3, characterized in that: The large-scale fermentation medium comprises: 2% tributyrin, 0.9% glucose, 0.8% urea, 0.2% peptone, 0.1% dipotassium hydrogen phosphate trihydrate, 0.1% ammonium sulfate, 0.05% magnesium sulfate heptahydrate, and the rest is water, with a pH of 7.2-7.

5.

6. Use of the siam bacillus SM01-01 according to claim 1 or the fermentation liquid according to claim 2 in degrading oils and fats.

7. The use according to claim 6, characterized in that: The degraded oil is fish oil, and the degradation conditions are: at 30°C, the fermentation bacteria liquid is inoculated into a liquid culture medium containing fish oil at a 2% inoculation rate, the pH value is 6.5, and the degradation time is 48 hours.

8. Use of the Siamese Bacillus SM01-01 according to claim 1 or the fermented bacterial liquid according to claim 2 in degrading oil in fish protein puree.

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