A strain of Siamese Bacillus with oil degradation function and its application

Abstract

The present invention discloses a Siamese Bacillus strain with oil-degrading capabilities and its applications. The strain, isolated from soil samples from a pine forest in Mount Tai and screened for oil-degrading activity, yielded Siamese Bacillus siamensis SM01-01, which is highly efficient in oil degradation. The strain is deposited with CGMCC No. 25550. Siamese Bacillus siamensis SM01-01 is capable of degrading oil in fish protein slurry. Under optimal conditions, the degradation rate reaches 89.76%, effectively resolving the issues of secondary fermentation and odor generation caused by residual oil in the fish protein slurry.

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] Currently, the fish protein fertilizer production team is growing stronger and stronger. Like seaweed fertilizer and chitin fertilizer, fish protein fertilizer has become a major product in the utilization of marine biological resources and is gradually taking over the traditional fertilizer market. Currently, the raw material for fish protein fertilizer production is fish protein pulp, which is obtained through bacterial or enzymatic hydrolysis. During the production process, the protein is gradually broken down into small molecules to exert its effects. However, the oil in the fish flesh cannot be fully decomposed, which leads to secondary fermentation by miscellaneous bacteria, producing substances with strong pungent odors such as hydrogen sulfide and volatile fatty acids, causing the fish protein pulp to stink. How to effectively degrade the oil in fish protein pulp has become an urgent problem for every fish protein fertilizer production source company. Furthermore, after secondary fermentation, the fish protein pulp becomes an oily waste liquid, making it difficult to use as a high-quality raw material.

[0003] Traditional physical and chemical methods are ineffective in addressing the high oil content in fish protein concentrate and can also affect its efficacy. Microbial technology has emerged as a novel approach to addressing this issue. Bacillus siamensis, also known as Sim's Bacillus, is a new species of the genus Bacillus described in the past decade. Currently, research on Bacillus siamensis primarily focuses on biocontrol and plant growth promotion, with little research on its application in oil degradation. Summary of the Invention

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

[0005] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The present invention isolates Bacillus siamensis SM01-01 with high-efficiency oil degradation function from Taishan pine forest soil samples and screens through oil degradation activity verification. The strain has been deposited in the General Microbiology Center of China Culture Collection of Microorganisms (deposit address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on August 19, 2022, with a deposit number of 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, a dry and uneven surface, and the central wrinkles are wavy and protruding. The bacteria of the strain are rod-shaped under microscopic examination, which will become slightly shorter in the later stage; spores are produced, and they are terminal spores; Gram staining is positive.

[0006] The present 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 preparation: First, activate Bacillus siamensis SM01-01, then inoculate it into a seed solution culture medium, and culture it at a constant temperature of 25-35°C with shaking for 10-15 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 hours to obtain a fermentation liquid of Bacillus siamensis SM01-01.

[0010] The above seed liquid culture medium formula (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 above-mentioned large-scale fermentation medium formula (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 aforementioned Bacillus siamensis SM01-01 or its fermented liquid for oil degradation. The optimal conditions for oil (fish oil) degradation are: at 30°C, a 2% inoculum of the fermented liquid is inoculated into a liquid culture medium containing fish oil, and the pH is 6.5. The highest oil degradation rate is achieved 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 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 isolated Bacillus siamensis SM01-01 from a soil sample from a pine forest in Mount Tai and obtained it through screening and oil degradation activity verification, and found for the first time 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 is shown;

[0020] Figure 4 This is the spore-forming form 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 be combined with the contents of the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described 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 making creative efforts 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 was isolated from a soil sample from a pine forest in Mount Tai, Tai'an City. The collected samples were enriched, cultured, pre-screened and re-screened 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] Place 10g of the collected sample in a conical flask containing 90mL of sterile water and homogenize at 180rpm for 30 minutes. Add 1mL of the homogenized liquid to the enrichment medium and incubate at 30°C and 180rpm for 24 hours. If the fish oil shows signs of degradation, add 10mL of the enrichment liquid to fresh enrichment medium. Repeat this process three times to obtain a stable enrichment of oil-degrading bacteria. Store this for future 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 until ready for use.

[0032] (2) Initial 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% by density gradient dilution with sterile water. -5 , 10 -6 , 10 -7 For the three concentration gradients, 100 μL of the dilution solution 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: 5 g beef extract, 5 g sodium chloride, 10 g peptone, 1 mL 1.6% neutral red aqueous solution, 10 g fish oil, 20 g agar powder, distilled water to 1000 mL, adjust pH to 7.0. Sterilize at 121°C for 20 min 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 used to determine the oil degradation rate.

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

[0038] (4) Determination of oil degradation rate

[0039] Preparation 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 selected from the rescreening were inoculated into beef extract peptone liquid medium and cultured at 30°C with shaking for 48 hours to obtain the seed solution of the corresponding strain. Then, a 1% inoculum 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 mixture was gently shaken and allowed to stand. The petroleum ether extract was then pipetted and the D was measured using a UV 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: 2 g ammonium sulfate, 2 g dipotassium hydrogen phosphate, 2 g sodium dihydrogen phosphate, 5 g sodium chloride, 20 g fish oil, dilute to 1 L with distilled water, pH 7.0. Sterilize at 121°C for 20 min and store until ready for use.

[0045] 2. Results Analysis

[0046] (1) The results of the initial screening showed that after enrichment culture, 45 strains showed red color in the neutral red medium. After excluding strains with the same colony morphology through observation of colony morphology, 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: Potency 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 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, dry and uneven surface, and wavy folds 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 (refer to Figure 4 ); Gram staining was 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, 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] Strain SM01-01 was positive for oxidase and catalase; it could hydrolyze gelatin, starch, and cellulose; it was positive for nitrate reduction and VP tests; and it was negative for hydrogen sulfide and methyl red tests. Based on its physiological and biochemical characteristics, strain SM01-01 was preliminarily identified as a Bacillus bacterium.

[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 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 onto NA medium and activated at 30°C while observing for bacterial contamination. After activation, a loop of bacterial moss was taken with an inoculating loop and inoculated into the seed liquid medium. The culture was shaken at 30°C and 180 rpm for 12 h to prepare the SM01-01 seed liquid.

[0073] (2) Large-scale fermentation culture: Add water to 60-70% of the tank volume, add materials according to the large-scale fermentation culture 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 minutes; after sterilization, add the seed liquid at a 5% inoculum amount to the fermentation tank (500L), and ferment and culture at 30°C for 16-20 hours. The bacterial count can reach about 10 billion / ml, which is the SM01-01 fermentation culture liquid.

[0074] The seed liquid culture medium formula (mass ratio) is as follows: glucose 0.5%, peptone 0.5%, beef powder 0.3%, sodium chloride 0.5%, 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] Fermentation temperatures were set at 20°C, 25°C, 30°C, 35°C, and 40°C. 2 mL of bacterial culture was inoculated at a 2% inoculum into fish oil medium (pH 7.0) containing 20 g / L of fish oil. Fermentation was performed at 180 rpm for 48 hours. The fermentation broth was extracted with 50 mL of petroleum ether, and the oil degradation rate was measured.

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

[0082] (2) pH optimization

[0083] At the optimal temperature, 2 mL of bacterial culture was inoculated into fish oil medium containing 20 g / L fish oil at a 2% inoculum size. The pH was adjusted to 5.5, 6, 6.5, 7, and 7.5, and the culture was incubated at 180 rpm for 48 hours. The fermentation broth was extracted with 50 mL of petroleum ether, and the oil degradation rate was measured.

[0084] (3) Optimization of inoculum size

[0085] Based on the optimal temperature and pH, the culture medium was inoculated with 1%, 2%, 3%, 4%, and 5% of the inoculum, respectively, into a fish oil culture medium containing 20g / L of fish oil. The culture was cultured at 180 rpm for 48 hours. The fermentation broth was extracted with 50mL of petroleum ether, and the oil degradation rate was measured.

[0086] (4) Fermentation time optimization

[0087] Based on the above optimal conditions, the fermentation culture was cultured 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 the oil degradation rate.

[0088] 2. Results Analysis

[0089] (1) The results of the temperature optimization test show that (such as Figure 5 As shown in Figure 2, strain SM01-01 showed the best oil degradation at 30°C, with a degradation rate of 68.5%. This degradation rate gradually decreased as the temperature increased. This result indicates that the optimal temperature for strain SM01-01 to achieve its degradation effect is 30°C.

[0090] (2) The results of pH optimization test showed that (e.g. Figure 6As shown in Figure 2, under optimal temperature conditions, when the initial pH was 6.5, strain SM01-01 was most effective at degrading oil, achieving a degradation rate of 71.2%. This result indicates that strain SM01-01's optimal degradation effect is at a pH of 6.5, and that it is more effective in slightly acidic environments.

[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, the oil degradation rate of strain SM01-01 first increased and then decreased with the increase of inoculum size; when the inoculum size was 2%, the oil degradation rate reached 72.6%. Both high and low inoculum sizes would affect the degradation efficiency of SM01-01.

[0092] (4) The fermentation time optimization results proved (such as Figure 8 As shown): Under the optimal temperature, pH and inoculum size conditions, when the fermentation time was 48 h, 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 size 2%, and fermentation time 48h.

[0094] Example 5: Application of Siamese Bacillus SM01-01 in the Degradation of Oil in the Production of Fish Protein Puree

[0095] Fish protein concentrate can be produced by enzymatic hydrolysis after pulverization. However, the presence of large amounts of fat during the production process results in a strong fishy and foul odor. To test whether the Siamese Bacillus SM01-01 bacterial solution can effectively degrade the fat in fish protein concentrate, the following experiment was conducted.

[0096] 1. Test methods

[0097] Preparation method of enzymatically hydrolyzed fish protein slurry: (1) Raw material selection: Fresh whole fish is preferred, as it has a stable protein content and can ensure the stability of the finished product indicators of different batches. In addition, the fresh fish protein is free of corruption. (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 material-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 the temperature is lowered to 50°C to 55°C. Adjust the pH value between 6 and 8, add enzymatic solution (animal-derived protein hydrolyzing enzyme, including protease ≥ 20,000 U / g, cellulase > 50 U / g, pre-dissolve the hydrolyzing enzyme), add 2 to 5 kg / t, keep at 50℃ to 55℃ for 2 to 4 hours, and keep the speed at 50 to 60 r / min during the enzymatic hydrolysis process. (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 remain. Smell the odor. After meeting the requirements, heat to above 90℃ and inactivate the enzyme for 10 minutes. (7) Filtration of the tank: After the inactivation of enzymes 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 added to 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 added with bacterial liquid. Each group was repeated three times.

[0099] 2. Results Analysis

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

[0101] Table 4 Statistics of oil degradation rates of the two groups

[0102]

Claims

1. A Bacillus siamensis SM01-01 strain, the deposit number of which is CGMCC NO.25550.

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

3. The method for preparing the fermentation solution of Bacillus siamensis SM01-01 according to claim 2, characterized in that: The following steps are involved: (1) Seed solution preparation: First, activate Bacillus siamensis SM01-01, then inoculate it into a seed solution culture medium, and culture it at a constant temperature of 25-35°C with shaking for 10-15 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 liquid of Bacillus siamensis SM01-01.

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

1.

5. The method for preparing the fermentation liquid of Bacillus siamensis SM01-01 according to claim 3, wherein: 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 remainder 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 at 2% into the liquid culture medium containing fish oil, the pH value is 6.5, and the degradation time is 48 hours.

8. Use of the siam bacillus SM01-01 according to claim 1 or the fermentation liquid according to claim 2 in degrading oil in fish protein puree.

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