Preparation process and application of enterococcus hirsutei IDO5 freeze-dried powder

Abstract

The invention discloses a preparation process and application of enterococcus hirsutei I DO5 freeze-dried powder. The preparation process has the following beneficial effects that (1) compared with the prior art, the preparation process of the enterococcus hirsutei I DO5 freeze-dried powder disclosed by the invention is simpler and easier to operate when being used for preparing enterococcus hirsutei I DO5 into immobilized pellets; (2) the enterococcus hirsutei I DO5 is prepared into freeze-dried powder, and the physiological activity of the enterococcus hirsutei I DO5 strain can be better reserved at low temperature and low pressure in the freeze-drying process; the enterococcus hirsuti I DO5 freeze-dried powder can be stably stored for a long time, so that the requirement of frequent supplement is reduced, and the enterococcus hirsuti I DO5 freeze-dried powder is more suitable for a long-term use breeding scene (3) the enterococcus hirsutei I DO5 freeze-dried powder is good in deodorization effect, and the degradation rates of indole, skatole and ammonia gas in the pig manure wastewater can reach 60% or above; and (4) the surface morphology of the enterococcus hisei I DO5 freeze-dried powder can be effectively improved through the protective agent solution, the freezing resistance of cells is enhanced, the integrity of a cell membrane structure is maintained, and the freeze-drying survival rate of the enterococcus hisei I DO5 is increased.

Description

Technical Field

[0001] The invention relates to the technical field of environmental protection, and in particular to a preparation process and application of Enterococcus hirae IDO5 freeze-dried powder. Background Art

[0002] If livestock manure is discharged on site without treatment, it will cause serious non-point source pollution around the city. In addition, the odor pollution caused by livestock manure seriously threatens the health of farmed animals and surrounding residents. Therefore, how to minimize the pollution of livestock and poultry farming odor to the environment has become a major issue that must be faced and solved in the sustainable development of the farming industry.

[0003] Odor emissions from livestock and poultry farms mainly come from livestock and poultry houses, manure storage sites, composting rooms and sewage pools. The main odor components of odor substances in livestock and poultry manure are ammonia, sulfide, volatile fatty acids, indole and skatole (scientific name 3-methylindole). Among them, indole and skatole are the main malodorous pollutants in livestock and poultry manure. They are metabolites of nitrogen-containing compounds such as protein and amino acids fermented by hindgut microorganisms. They are a typical nitrogen pollutant with a very strong fecal odor. The environmental pollution and public health problems caused by them have always been the focus of attention in the breeding industry. Therefore, how to reduce the odor pollution such as ammonia, indole and skatole emitted by the breeding industry is a technical problem that needs to be solved urgently in the process of livestock and poultry breeding.

[0004] The applicant previously screened out a deodorizing strain capable of degrading odor substances such as indole and skatole from pig manure samples degraded by black soldier fly larvae. The strain was identified as Enterococcus hirae and named Enterococcus hirae IDO5. However, in practical applications, the composition of aquaculture manure and aquaculture wastewater is complex. In order to improve the activity of Enterococcus hirae IDO5 in aquaculture manure and wastewater, enhance the resistance of microorganisms to adverse environmental influences, and improve the degradation efficiency of odor substances, it is necessary to use a simple preparation process to prepare Enterococcus hirae IDO5 into an economical and efficient freeze-dried powder of Enterococcus hirae IDO5, so as to meet the targeted needs of the treatment of odor pollution caused by aquaculture manure, aquaculture wastewater, etc., and realize the effective treatment of the odorous gas generated by aquaculture waste. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the shortcomings of the prior art, the present invention provides a preparation process and application of Enterococcus hirae IDO5 freeze-dried powder, which can effectively degrade and remove ammonia, indole and skatole in odor to reduce odor pollution caused by livestock and poultry breeding. In addition, the preparation process of Enterococcus hirae IDO5 freeze-dried powder is simple, and the prepared Enterococcus hirae IDO5 freeze-dried powder is easy to transport and store for a long time.

[0007] (II) Technical solution

[0008] To solve the above technical problems, the present invention provides the following technical solution: A preparation process of Enterococcus hirae IDO5 freeze-dried powder, comprising the following steps:

[0009] S1. Prepare a culture medium for strain culture;

[0010] S2. Inoculate Enterococcus hirae IDO5 into the culture medium to prepare a bacterial liquid;

[0011] S3. Centrifuge the bacterial liquid prepared in step S2 to obtain bacterial sludge;

[0012] S4. Resuspend the bacterial sludge obtained in step S3 with a protective agent solution and transfer it to a freeze-drying bottle, and further perform freeze-drying on the freeze-drying bottle to obtain Enterococcus hirae IDO5 freeze-dried powder.

[0013] Preferably, the culture medium is LB medium, and the LB medium includes liquid LB medium and solid LB medium.

[0014] Preferably, the liquid LB medium comprises the following raw materials: peptone 10.0 g / L, yeast powder 5.0 g / L, NaCl 10.0 g / L; the solid LB medium comprises the following raw materials: peptone 10.0 g / L, yeast powder 5.0 g / L, NaCl 10.0 g / L, agar powder 10.0 g / L.

[0015] Preferably, step S2 specifically comprises: Take 1 cryopreservation tube of Enterococcus hirae IDO5, streak inoculate it on the sterilized solid LB medium, and culture it at 37 °C for 24-48 h; after observing no contaminants, pick a single colony on the solid LB medium and inoculate it into 100 mL of liquid LB medium, culture it at 37 °C and 200 r / min for 24 h, and then transfer it to 100 mL of liquid LB medium for subculture at an inoculation amount of 5% for 24 h to prepare the bacterial liquid.

[0016] Preferably, step S3 specifically comprises: Take 2 mL of the bacterial liquid for centrifugation, discard the supernatant and collect the bacterial cells, resuspend and wash the bacterial cells with sterile physiological saline, and then perform centrifugation again to obtain the bacterial sludge.

[0017] Preferably, step S4 specifically comprises: Resuspend the bacterial sludge with 2 mL of sterilized protective agent solution and transfer it to a freeze-drying bottle, first place the freeze-drying bottle in a 4 °C refrigerator for precooling for 2 h, then place it in a -80 °C freezer for pre-freezing for 12 h, and finally place the pre-frozen freeze-drying bottle in a pre-cooled vacuum freeze-dryer for freeze-drying at -54 °C and below 25.9 Pa for 72 h to obtain Enterococcus hirae IDO5 freeze-dried powder.

[0018] Preferably, in step S3, the centrifugation speed conditions are: centrifuging at 8000 r / min for 5 min, or centrifuging at 4000 r / min for 10 min.

[0019] Preferably, the cryoprotectant solution comprises the following raw materials: lactose, inulin, sorbitol, maltose.

[0020] Preferably, the cryoprotectant solution comprises the following raw materials at the following concentrations: lactose 7.5 g / 100 mL, inulin 15 g / 100 mL, sorbitol 5 g / 100 mL, maltose 4 g / 100 mL.

[0021] To solve the above technical problems, the present invention provides another technical solution as follows: applying the freeze-dried powder of Enterococcus hirae IDO5 prepared by the above preparation process to the treatment of breeding manure or breeding wastewater.

[0022] (III) Beneficial effects

[0023] Compared with the prior art, the present invention provides a preparation process and application of a freeze-dried powder of Enterococcus hirae IDO5, having the following beneficial effects: (1) In the present invention, Enterococcus hirae IDO5 is inoculated into a culture medium to prepare a bacterial solution, the bacterial solution is further centrifuged to obtain a bacterial sludge, and finally the bacterial sludge is freeze-dried to obtain the freeze-dried powder of Enterococcus hirae IDO5. The preparation process of the freeze-dried powder of Enterococcus hirae IDO5 in the present invention is simpler and easier to operate than the existing preparation processes such as preparing Enterococcus hirae IDO5 into immobilized microspheres; (2) In the present invention, by preparing Enterococcus hirae IDO5 in the form of a freeze-dried powder, the low temperature and low pressure during the freeze-drying process can better retain the physiological activity of the Enterococcus hirae IDO5 strain, and the freeze-dried powder can also increase the contact area between the deodorizing microorganism Enterococcus hirae IDO5 and the odor substances, enhancing the resistance of the microorganism to the influence of adverse environments, thereby better ensuring the use effect of Enterococcus hirae IDO5; in addition, the freeze-dried powder of Enterococcus hirae IDO5 can be stably stored for a long time, reducing the need for frequent replenishment, and is more suitable for long-term use in breeding scenarios; (3) The deodorizing effect of the freeze-dried powder of Enterococcus hirae IDO5 is good, and the degradation rates of indole, skatole, and ammonia in pig manure wastewater can all reach more than 60%. When applied to livestock and poultry breeding manure and breeding wastewater, it can effectively reduce the concentrations of odor substances such as ammonia, indole, and skatole, thereby effectively reducing the odor pollution caused by livestock and poultry breeding; (4) In the present invention, the cryoprotectant solution can effectively improve the surface morphology of the freeze-dried powder of Enterococcus hirae IDO5, enhance the cryoresistance of cells, maintain the integrity of the cell membrane structure, and improve the freeze-drying survival rate of Enterococcus hirae IDO5. Description of the drawings

[0024] Figure 1 It is a process flow diagram of a preparation process of a freeze-dried powder of Enterococcus hirae IDO5 according to the present invention;

[0025] Figure 2 This is the trend chart of the optimal combination of the IDO5 protectant for Enterococcus hirae in the present invention;

[0026] Figure 3 These are the scanning electron microscope images of the freeze-dried powder of Enterococcus hirae IDO5 without and with the addition of the composite protectant;

[0027] Figure 4 This is the graph showing the effect of different addition amounts of the freeze-dried powder of Enterococcus hirae IDO5 on the degradation rate of skatole;

[0028] Figure 5 This is the graph showing the effect of different addition amounts of the freeze-dried powder of Enterococcus hirae IDO5 on the degradation rate of indole;

[0029] Figure 6 This is the graph showing the effect of different addition amounts of the freeze-dried powder of Enterococcus hirae IDO5 on the degradation rate of ammonia. Detailed implementation manners

[0030] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0031] The present invention provides a preparation process for the freeze-dried powder of Enterococcus hirae IDO5, including the following steps:

[0032] S1. Prepare a culture medium for strain cultivation.

[0033] Preferably, the culture medium is LB medium, and the LB medium includes liquid LB medium and solid LB medium.

[0034] The liquid LB medium includes the following raw materials: peptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L; the solid LB medium includes the following raw materials: peptone 10.0 g / L, yeast extract 5.0 g / L, NaCl 10.0 g / L, agar powder 10.0 g / L.

[0035] In step S1, dissolve according to the ratio of peptone 10.0 g / L, yeast extract 5.0 g / L, and NaCl 10.0 g / L in distilled water, adjust the pH of the culture medium to 7.3, and sterilize at 121 °C for 15 min to obtain the liquid LB medium. The solid LB medium is the LB agar plate, and the solid LB medium is based on the raw material formula of the liquid LB medium plus agar powder. The LB medium and its preparation process of the present invention are all prior arts and will not be elaborated here too much.

[0036] S2. Inoculate Enterococcus hirae IDO5 into a culture medium to prepare a bacterial solution.

[0037] It should be noted that Enterococcus hirae IDO5 of the present invention is the Enterococcus hirae IDO5 recorded in the patent document with the authorization announcement number CN118516342B and the invention title "Preparation method and application of immobilized pellets of a deodorizing bacterium". This Enterococcus hirae IDO5 is a deodorizing strain screened from a pig manure sample degraded by black soldier fly larvae, which can degrade odor substances such as indole and skatole. Enterococcus hirae IDO5 was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on March 28, 2024. The deposit address is on the 5th floor of the Experimental Building, No. 100 compound, Xianlie Middle Road, Yuexiu District, Guangzhou. The deposit number is GDMCC NO.64457, and the taxonomic name is: Enterococcus hirae IDO5.

[0038] Preferably, step S2 specifically includes: Take 1 cryotube of Enterococcus hirae IDO5 (for example, a 2 ml cryotube), streak inoculate it on a sterilized solid LB medium (i.e., LB agar plate), and culture it at 37 °C for 24 - 48 h; After observing no contaminants, pick a single colony on the solid LB medium and inoculate it into 100 mL of liquid LB medium. After culturing at 37 °C and 200 r / min for 24 h, further transfer it to 100 mL of liquid LB medium for subculture at an inoculation amount of 5% for 24 h, thus preparing the bacterial solution. In addition, preferably, a microplate reader can be used to measure the viable cell count, and after determining that the concentration of the bacterial solution is approximately 10 9 cfu / mL, carry out subsequent experiments.

[0039] S3. Centrifuge the bacterial solution prepared in step S2 to obtain bacterial sludge.

[0040] Preferably, step S3 specifically includes: Take 2 mL of the bacterial solution prepared in the above step S2 and centrifuge it at a rotation speed of 8000 r / min for 5 min (or 4000 r / min for 10 min). Discard the supernatant and collect the bacterial cells. After resuspending and washing the bacterial cells with sterile physiological saline, centrifuge them again under the same rotation speed conditions to obtain the bacterial sludge required for freeze-drying experiments.

[0041] S4. Resuspend the bacterial sludge obtained in step S3 with a protective agent solution and transfer it to a freeze-drying bottle. Further, freeze-dry the freeze-drying bottle to obtain the freeze-dried powder of Enterococcus hirae IDO5.

[0042] Preferably, step S4 specifically includes: resuspending the bacterial sludge obtained by centrifugation in step S3 with 2 mL of sterilized protective agent solution, transferring it to a freeze-drying bottle, first placing the freeze-drying bottle in a 4°C refrigerator for precooling for 2 h, further placing it in a -80°C freezer for pre-freezing for 12 h, and finally placing the pre-frozen freeze-drying bottle in a pre-cooled vacuum freeze-dryer for freeze-drying at -54°C and below 25.9 Pa for 72 h to obtain the freeze-dried powder of Enterococcus hirae IDO5.

[0043] Preferably, the protective agent solution includes raw materials with the following concentrations: lactose 7.5 g / 100 mL, inulin 15 g / 100 mL, sorbitol 5 g / 100 mL, maltose 4 g / 100 mL; the protective agent solution is prepared by dissolving the above raw materials in distilled water according to the ratio.

[0044] Next, single-factor test analysis, orthogonal test analysis, verification of the survival rate effect of the freeze-dried powder of Enterococcus hirae IDO5, and microscopic morphology analysis of the freeze-dried powder of Enterococcus hirae IDO5 are carried out on the protective agent solution of the present invention.

[0045] (1) Single-factor test analysis: The single-factor test is used to investigate the influence of different types and concentrations of single protective agents on the survival rate of Enterococcus hirae IDO5 strains after freeze-drying. Keeping the test conditions such as pre-freezing, vacuum freeze-drying, rehydration, and viable cell counting unchanged, taking the survival rate of the strains after freezing as the index, the conditions with the highest survival rate of the strains after freeze-drying in various protective agents are screened out. The freeze-drying protective agents include sodium glutamate, skim milk powder, trehalose, sorbitol, lactose, mannitol, maltose, vitamin C, and inulin. Five concentration levels are set for each factor as shown in Table 1 below, and the survival rate of Enterococcus hirae IDO5 is measured after freeze-drying.

[0046] Table 1 Single factor level table Table1 Single factors and levels

[0047]

[0048] The results of the freeze-drying survival rate of Enterococcus hirae IDO5 are shown in Table 2 below. There are differences in the protective effects of various protective agents at different concentrations. Among the 9 protective agents, when the lactose content is 7.5 g / 100 mL, the survival rate of Enterococcus hirae IDO5 in the freeze-dried powder is the highest, which is 67.12%.

[0049] Table 2 Influence of single cryoprotectant on the freeze-drying survival rate of Enterococcus hirae IDO5 Table2 Survival rate of Enterococcus hirae GDIAS-5 in presence of single cryoprotectant

[0050]

[0051] The test results showed that lactose exhibited a high protection rate among all factors and levels. Especially when the concentration was 10 g / 100 mL and 15 g / 100 mL, the survival rates of IDO5 were 67.12% and 65.75% respectively, with the best protection effect, which was significantly higher than that of sorbitol, inulin, and maltose (P<0.05). The protection effect of sorbitol was proportional to its concentration. When the concentration was 5 g / 100 mL, the survival rate of IDO5 reached 56.52%. Maltose had a good protection effect at 5 g / 100 mL, and the highest survival rate of IDO5 was 51.30%. The protection effect of inulin at different concentration levels was relatively stable compared with other factors, and the survival rate of IDO5 was between 41.74% and 58.04%.

[0052] In summary, inulin, sorbitol, lactose, and maltose had particularly significant protection effects on the freeze-dried powder of Enterococcus hirae IDO5 at different concentrations, especially with a high protection rate at high concentrations, indicating their good application potential as protectants for the freeze-dried powder of Enterococcus hirae IDO5.

[0053] (2) Orthogonal test analysis: As shown in Table 1 above, based on the results of the single-factor test, the present invention selected inulin (A), sorbitol (B), lactose (C), and maltose (D) as the four factors for the orthogonal test for subsequent experimental studies. Using the protection rate of Enterococcus hirae IDO5 as the evaluation index, an L9(34) orthogonal test was carried out. As shown in Table 3 below, -1, 0, and 1 were used to represent the low, medium, and high levels of each variable. The experiment was designed using Design-Expert, and the experimental data was orthogonally analyzed. By calculating the regression equation, analyzing the contour plot and three-dimensional surface plot, the optimal raw material ratio of the composite protectant was determined.

[0054] Table 3 Factors and levels of the orthogonal testTable3 Response surface experimentfactorsandlevels

[0055]

[0056] In the range analysis method, the range R value was used to judge the influence degree of the above four factors on the protection rate. The larger the R value, the more significant the influence of the factor on the protection rate of Enterococcus hirae IDO5, and thus the higher the importance of the factor to the experimental evaluation index. At the same time, the parameter k represented the influence of different levels of each factor on the evaluation index, helping to identify the optimal combination scheme. In this experiment, with the goal of a higher protection rate, k was taken as the maximum value of each column.

[0057] The results of the range analysis are shown in Table 4 below. The primary and secondary relationships of the influence of each factor on the protection rate are: C lactose > A inulin > B sorbitol > D maltose. This indicates that C lactose is the key factor affecting the protection rate of Enterococcus hirae IDO5, followed by A inulin and B sorbitol, and the influence of D maltose is relatively small. The optimal level combination of each factor is A2B3C1D2, that is, the optimal process parameters for the protective agent solution of the Enterococcus hirae IDO5 freeze-dried powder are: A inulin concentration 15 g / 100 mL, B sorbitol concentration 5 g / 100 mL, C lactose concentration 7.5 g / 100 mL, and D maltose concentration 4 g / 100 mL. The specific range analysis data and results are shown in Table 4 below, and the trend diagram of the optimal combination is as Figure 2 shown.

[0058] Table 4 Intuitive analysis table of protection rate

[0059]

[0060] It can be seen from Table 4 that when the other three factors remain unchanged, the protection rate of Enterococcus hirae IDO5 shows different trends with the changes of the four factors. Specifically, as the inulin concentration increases, the protection rate first rises and then falls, and the rising and falling rates are basically the same; as the sorbitol concentration increases, the protection rate first falls and then rises; the increase in lactose concentration leads to a rapid decline in the protection rate; while the increase in maltose concentration shows a trend that the protection rate starts to decline after remaining stable for a period of time.

[0061] The results of the variance analysis are shown in Table 5 below. A inulin, B sorbitol, C lactose, and D maltose have a significant impact on the protection rate of the test results (P < 0.05). Among them, the significance order of the four factors for the protection rate is C lactose > A inulin > B sorbitol > D maltose, and this result is consistent with the ranking in the range analysis method. This result further verifies the importance of lactose in affecting the protection rate of Enterococcus hirae IDO5. The specific analysis results and data are shown in Table 5 below.

[0062] Table 5 Variance analysis table

[0063]

[0064] Note: F 0.1 (2,2) = 9.00, F 0.05 (2,2) = 19.00, F 0.01 (2,2) = 99.00; * indicates significant difference (P < 0.05); ** indicates significant difference (P < 0.01)

[0065] (3)Effect verification of the survival rate of Enterococcus hirae IDO5 freeze-dried powder: It can be seen from the range (Table 4) and variance analysis (Table 5) of the test results that the order of the effects of A inulin, B sorbitol, C lactose, and D maltose on the protection rate of Enterococcus hirae IDO5 is the same, which is C lactose > A inulin > B sorbitol > D maltose. In addition, the optimal combination A2B3C1D2 predicted by the orthogonal test has appeared in the orthogonal test design table, and the corresponding protection rate is the highest value. Therefore, in order to verify the stability and reliability of this predicted combination, three repeated tests were carried out. The results show that the average protection rate reaches 74.31%, further confirming the optimal parameters of the Enterococcus hirae IDO5 protectant in the process of the present invention: A inulin concentration 15g / 100ml, B sorbitol concentration 5g / 100ml, C lactose concentration 7.5g / 100ml, and D maltose concentration 4g / 100ml. The specific verification results are shown in Table 6 below.

[0066] Table 6 Analysis table of results of different test combinations

[0067]

[0068] (4)Microscopic morphology analysis of Enterococcus hirae IDO5 freeze-dried powder: Based on scanning electron microscopy, the microscopic structures of Enterococcus hirae IDO5 bacterial powder (i.e., Enterococcus hirae IDO5 freeze-dried powder) without and with the addition of the composite protectant of IDO5 strain (i.e., the raw material formula corresponding to the optimal concentration of the protectant solution used in the above step S4: lactose 7.5g / 100mL, inulin 15g / 100mL, sorbitol 5g / 100mL, maltose 4g / 100mL) were analyzed. The relevant comparison diagrams are shown in Figure 3 ( Figure 3 In it, note: (A) Morphology of Enterococcus hirae IDO5 freeze-dried powder without the addition of the above composite protectant magnified 300 times; (B) Morphology of the freeze-dried powder without the addition of the composite protectant magnified 1000 times; (C) Morphology of the freeze-dried powder without the addition of the composite protectant magnified 5000 times; (D) Morphology of the freeze-dried powder with the addition of the composite protectant magnified 300 times; (E) Morphology of the freeze-dried powder with the addition of the composite protectant magnified 1000 times; (F) Morphology of the freeze-dried powder with the addition of the composite protectant magnified 5000 times). From Figure 3 A and Figure 3 D comparison, it can be seen that Enterococcus hirae IDO5 is surrounded by inulin, sorbitol, lactose, and maltose and is completely covered under the coating. From Figure 3 B and Figure 3 E comparison, it can be seen that the surface of Enterococcus hirae IDO5 bacterial powder is relatively flat, smooth, the structure is compact, the overall contour is clear, and the bacteria body is tightly combined with the composite protectant. From Figure 3 C and Figure 3Comparing with Group F, the cell morphology of Enterococcus hirae IDO5 is plump and intact, without any damage or shriveling. Only a very small number of cells show shrinkage, which proves that the optimized composite protectant can effectively improve the surface morphology of the bacterial powder, enhance the cryoresistance of cells, maintain the integrity of the cell membrane structure, and improve the freeze-drying survival rate of Enterococcus hirae IDO5.

[0069] In addition, preferably, before step S4 and after the above step S3, the viable count of the bacterial sludge before drying can be measured: the bacterial sludge sample obtained by centrifugation is resuspended with 2 mL of sterile normal saline and transferred to a sterile test tube, shaken well, 100 μL of the bacterial suspension is pipetted into 900 μL of sterile normal saline, and the operation is repeated after sufficient shaking; diluted to 106-1010 times, 100 μL is taken and evenly spread on an LB agar plate, and the LB agar plate is inverted and cultured in a constant temperature incubator at 37 °C for 24-48 h. After single colonies grow on the LB agar plate, the plate with the number of colonies between 30 and 300 is selected for colony counting calculation.

[0070] The freeze-dried powder of Enterococcus hirae IDO5 prepared by the present invention can be applied to the treatment of breeding manure or breeding wastewater. The freeze-dried powder of Enterococcus hirae IDO5 can effectively reduce the concentrations of odor substances such as ammonia, indole, and skatole in livestock and poultry breeding manure and breeding wastewater, thereby effectively reducing the odor pollution caused by livestock and poultry breeding.

[0071] Next, the effects of different addition amounts of the freeze-dried powder of Enterococcus hirae IDO5 on the degradation of skatole, indole, and ammonia in pig manure wastewater are analyzed: (1) Effect on the degradation rate of skatole: As Figure 4 shown, with the increase in the addition amount of the freeze-dried powder of Enterococcus hirae IDO5, the degradation rate of skatole gradually increases. When the addition concentration of the freeze-dried powder of Enterococcus hirae IDO5 reaches 20 g / L, the degradation rate approaches the maximum value. (2) Effect on the degradation rate of indole: As Figure 5 shown, the degradation effect of the freeze-dried powder of Enterococcus hirae IDO5 on indole shows a significant upward trend with the increase in the addition amount of the bacterial powder (P<0.05). The degradation rate of indole is relatively low when the addition amount of the bacterial powder is from 1 g / L to 10 g / L, but with the increase in the addition amount to 15 g / L and above, the removal effect increases significantly (P<0.05), indicating that the degradation of indole is proportional to the addition amount of the freeze-dried powder of Enterococcus hirae IDO5, and the addition amount above 15 g / L has a better effect. (3) Effect on the degradation rate of ammonia: As Figure 6As shown in the figure, the degradation rate of ammonia also increases with the increase in the addition amount of Enterococcus hirae IDO5 freeze-dried powder, and the addition amount of 15 g / L and above has a better effect. To sum up, the addition amount of Enterococcus hirae IDO5 freeze-dried powder in the present invention has a significant impact on the removal effect of odor substances in pig manure wastewater. Appropriately increasing the addition amount of the bacterial powder can improve the degradation effect of several main odor substances, but the effect tends to be stable after reaching a certain addition amount. The degradation rates of indole, skatole, and ammonia in pig manure wastewater by Enterococcus hirae IDO5 freeze-dried powder can all reach more than 60%.

[0072] Compared with the prior art, the present invention provides a preparation process and application of Enterococcus hirae IDO5 freeze-dried powder, which has the following beneficial effects: (1) In the present invention, Enterococcus hirae IDO5 is inoculated into a culture medium to prepare a bacterial liquid, the bacterial liquid is further centrifuged to obtain bacterial sludge, and finally the bacterial sludge is freeze-dried to obtain Enterococcus hirae IDO5 freeze-dried powder. The preparation process of Enterococcus hirae IDO5 freeze-dried powder in the present invention is simpler and easier to operate than the existing preparation processes such as preparing Enterococcus hirae IDO5 into immobilized microspheres; (2) In the present invention, by preparing Enterococcus hirae IDO5 into the form of freeze-dried powder, the low temperature and low pressure during the freeze-drying process can better retain the physiological activity of Enterococcus hirae IDO5 strain, and the freeze-dried powder can also increase the contact area between the deodorizing microorganism Enterococcus hirae IDO5 and odor substances, enhancing the influence of microorganisms to resist adverse environments, so as to better ensure the use effect of Enterococcus hirae IDO5; in addition, Enterococcus hirae IDO5 freeze-dried powder can be stored stably for a long time, reducing the need for frequent replenishment, and is more suitable for long-term use in breeding scenarios; (3) The deodorizing effect of Enterococcus hirae IDO5 freeze-dried powder is good, and the degradation rates of indole, skatole, and ammonia in pig manure wastewater can all reach more than 60%. When applied to livestock and poultry manure and breeding wastewater, it can effectively reduce the concentrations of odor substances such as ammonia, indole, and skatole, thereby effectively reducing the odor pollution caused by livestock and poultry breeding; (4) In the present invention, the protective agent solution can effectively improve the surface morphology of Enterococcus hirae IDO5 freeze-dried powder, enhance the cryoresistance of cells, maintain the integrity of the cell membrane structure, and improve the freeze-drying survival rate of Enterococcus hirae IDO5.

[0073] It should be noted that the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or elements inherent to such process, method, article or device. Without further limitation, an element defined by the phrase "comprising a..." does not exclude the presence of additional identical elements in the process, method, article or device including the element.

[0074] Although embodiments of the present invention have been shown, those of ordinary skill in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A preparation process of Enterococcus hirae IDO5 freeze-dried powder, characterized in that: The following steps are involved: S1. preparing a culture medium for strain culture; S2, inoculating Enterococcus hirae IDO5 into the culture medium to prepare a bacterial solution; S3, centrifuging the bacterial solution prepared in step S2 to obtain bacterial sludge; S4. Resuspend the bacterial sludge obtained in step S3 using a protective agent solution and transfer the suspension to a freeze-drying bottle, and further freeze-dry the freeze-drying bottle to obtain freeze-dried powder of Enterococcus hirae IDO5.

2. The preparation process of Enterococcus hirae IDO5 freeze-dried powder according to claim 1, characterized in that: The culture medium is LB culture medium, and the LB culture medium includes liquid LB culture medium and solid LB culture medium.

3. The preparation process of Enterococcus hirae IDO5 freeze-dried powder according to claim 2, characterized in that: The liquid LB culture medium includes the following raw materials: 10.0 g / L peptone, 5.0 g / L yeast powder, and 10.0 g / L NaCl; the solid LB culture medium includes the following raw materials: 10.0 g / L peptone, 5.0 g / L yeast powder, 10.0 g / L NaCl, and 10.0 g / L agar powder.

4. The preparation process of Enterococcus hirae IDO5 freeze-dried powder according to claim 2, characterized in that: The step S2 specifically includes: taking one cryopreservation tube of the Enterococcus hirsutus IDO5, streaking and inoculating it on the sterilized solid LB culture medium, and culturing it at 37° C. for 24-48 hours; after observing that there are no foreign bacteria, picking a single colony on the solid LB culture medium and inoculating it into 100 mL of the liquid LB culture medium, culturing it at 37° C. and 200 r / min for 24 hours, and then further transferring it to 100 mL of the liquid LB culture medium with an inoculum size of 5% for expansion and culturing for 24 hours, thereby preparing the bacterial liquid.

5. The preparation process of Enterococcus hirae IDO5 freeze-dried powder according to claim 4, characterized in that: The step S3 specifically includes: taking 2 mL of the bacterial solution and centrifuging it, discarding the supernatant to collect the bacterial cells, resuspending and washing the bacterial cells with sterile saline, and centrifuging again to obtain the bacterial sludge.

6. The preparation process of Enterococcus hirae IDO5 freeze-dried powder according to claim 5, characterized in that: The step S4 specifically includes: using 2 mL of the sterilized protective agent solution to resuspend the bacterial sludge and then transfer it to the freeze-dried bottle, first placing the freeze-dried bottle in a 4°C refrigerator for pre-cooling for 2 hours, then placing it in a -80°C freezer for pre-freezing for 12 hours, and finally placing the pre-frozen freeze-dried bottle in a pre-cooled vacuum freeze dryer for freeze-drying at -54°C and below 25.9 Pa for 72 hours to obtain the Enterococcus hirae IDO5 freeze-dried powder.

7. The preparation process of Enterococcus hirae IDO5 freeze-dried powder according to claim 5, characterized in that: In step S3, the centrifugal speed condition is: 8000 r / min for 5 min, or 4000 r / min for 10 min.

8. The preparation process of Enterococcus hirae IDO5 freeze-dried powder according to claim 1, characterized in that: The protective agent solution comprises the following raw materials: lactose, inulin, sorbitol and maltose.

9. The preparation process of Enterococcus hirae IDO5 freeze-dried powder according to claim 8, characterized in that: The protective agent solution includes raw materials with the following concentrations: lactose 7.5 g / 100 mL, inulin 15 g / 100 mL, sorbitol 5 g / 100 mL, and maltose 4 g / 100 mL.

10. An application of Enterococcus hirae IDO5 freeze-dried powder, characterized in that: The Enterococcus hirae IDO5 freeze-dried powder prepared according to any one of claims 1 to 9 is applied to the treatment of aquaculture manure or aquaculture wastewater.

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