Preparation process and application of Enterococcus hirae IDO5 freeze-dried powder

Abstract

The present invention discloses a preparation process and application of Enterococcus hirae I DO5 freeze-dried powder, which has the following beneficial effects: (1) the preparation process of Enterococcus hirae I DO5 freeze-dried powder of the present invention is simpler and easier to operate than the existing preparation process of preparing Enterococcus hirae I DO5 into immobilized pellets in the prior art; (2) by preparing Enterococcus hirae I DO5 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 the Enterococcus hirae I DO5 strain; the Enterococcus hirae I DO5 freeze-dried powder 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 Enterococcus hirae I DO5 freeze-dried powder has a good deodorizing effect, and the degradation rate of indole, skatole and ammonia in pig manure wastewater can reach more than 60%; (4) the protective agent solution can effectively improve the surface morphology of the Enterococcus hirae I DO5 freeze-dried powder, enhance the cell's anti-freezing ability, maintain the integrity of the cell membrane structure, and improve the freeze-dried survival rate of Enterococcus hirae I DO5.

Description

Technical Field

[0001] The present 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 untreated, it can cause serious non-point source pollution around cities. Furthermore, the odor pollution caused by livestock manure poses a serious threat to the health of farmed animals and surrounding residents. Therefore, minimizing the environmental impact of livestock and poultry odor pollution has become a major issue that must be addressed and resolved for the sustainable development of the livestock 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 the odorous 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. Both are metabolites of nitrogen-containing compounds such as protein and amino acids fermented by hindgut microorganisms. They are typical nitrogen pollutants 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 a deodorizing strain capable of degrading odorous 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 actual applications, the composition of livestock manure and aquaculture wastewater is complex. In order to improve the activity of Enterococcus hirae IDO5 in livestock manure and wastewater, enhance the microbial resistance to adverse environmental influences, and improve the degradation efficiency of odorous substances, it is necessary to use a simple preparation process to prepare Enterococcus hirae IDO5 into an economical and efficient Enterococcus hirae IDO5 freeze-dried powder. This can meet the targeted needs of treating odor pollution caused by livestock manure, aquaculture wastewater, etc., and effectively treat the odorous gases generated by livestock waste. Summary of the Invention

[0005] (1) Technical problems solved

[0006] In response to the shortcomings of the existing technology, the present invention provides a preparation process and application of Enterococcus hirae IDO5 freeze-dried powder, which can effectively degrade and remove ammonia, indole, skatole, etc. in odor, thereby reducing odor pollution caused by livestock and poultry farming. 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] (2) 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. preparing a culture medium for strain culture;

[0010] S2, inoculating Enterococcus hirae IDO5 into a culture medium to prepare a bacterial solution;

[0011] S3, centrifuging the bacterial solution 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 freeze-dry the freeze-drying bottle to obtain freeze-dried powder of Enterococcus hirae IDO5.

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

[0014] Preferably, the liquid LB medium includes the following raw materials: peptone 10.0 g / L, yeast powder 5.0 g / L, NaCl 10.0 g / L; the solid LB medium includes 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 includes: taking one frozen tube of Enterococcus hirae IDO5, streaking it on a sterilized solid LB 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 medium and inoculating it into 100 mL of liquid LB medium, culturing it at 37°C and 200 r / min for 24 hours, and then further transferring it to 100 mL of liquid LB medium with an inoculum size of 5% and expanding the culture for 24 hours to prepare a bacterial liquid.

[0016] Preferably, step S3 specifically includes: taking 2 mL of bacterial liquid and centrifuging it, discarding the supernatant to collect the bacterial cells, resuspending and washing the bacterial cells with sterile saline, and centrifuging it again to obtain bacterial sludge.

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

[0018] Preferably, in step S3, the centrifugal speed condition is: 8000 r / min for 5 min, or 4000 r / min for 10 min.

[0019] Preferably, the protective agent solution includes the following raw materials: lactose, inulin, sorbitol, and maltose.

[0020] Preferably, the protective agent solution comprises 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.

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

[0022] (3) Beneficial effects

[0023] 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) The present invention prepares a bacterial solution by inoculating Enterococcus hirae IDO5 into a culture medium, further centrifuging the bacterial solution to obtain bacterial mud, and finally freeze-drying the bacterial mud to obtain Enterococcus hirae IDO5 freeze-dried powder. The preparation process of Enterococcus hirae IDO5 freeze-dried powder of the present invention is simpler and easier to operate than the existing preparation process of preparing Enterococcus hirae IDO5 into immobilized pellets in the prior art; (2) The present invention prepares Enterococcus hirae IDO5 into the form of freeze-dried powder, and the low temperature and low pressure of the freeze-drying process can better retain the physiological activity of the Enterococcus hirae IDO5 strain, and the freeze-dried powder can also expand the contact between the deodorizing microorganism Enterococcus hirae IDO5 and odorous substances. contact area, enhancing the resistance of microorganisms to adverse environmental influences, thereby better ensuring the use effect of Enterococcus hirae IDO5; in addition, Enterococcus hirae IDO5 freeze-dried powder 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) Enterococcus hirae IDO5 freeze-dried powder has a good deodorizing effect, and the degradation rate of indole, skatole, and ammonia in pig manure wastewater can reach more than 60%. It can be applied to livestock and poultry breeding manure and breeding wastewater to effectively reduce the concentration of odor substances such as ammonia, indole, and skatole, thereby effectively reducing the odor pollution caused by livestock and poultry breeding; (4) The present invention can effectively improve the surface morphology of Enterococcus hirae IDO5 freeze-dried powder through the protective agent solution, enhance the cell's anti-freezing ability, maintain the integrity of the cell membrane structure, and improve the freeze-dried survival rate of Enterococcus hirae IDO5. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a process flow chart for preparing a freeze-dried powder of Enterococcus hirae IDO5 of the present invention;

[0025] Figure 2 This is a trend diagram of the optimal combination of the protective agent for Enterococcus hirae IDO5 of the present invention;

[0026] Figure 3 The scanning electron micrographs of freeze-dried powder of Enterococcus hirae IDO5 without and with compound protective agents added are shown;

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

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

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

[0030] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. 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 making creative efforts are within the scope of protection of the present invention.

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

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

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

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

[0035] In step S1, 10.0 g / L of peptone, 5.0 g / L of yeast extract, and 10.0 g / L of NaCl are dissolved in distilled water, the pH of the culture medium is adjusted to 7.3, and sterilized at 121°C for 15 minutes to obtain liquid LB culture medium. Solid LB culture medium is an LB agar plate, which is a liquid LB culture medium with agar powder added to the raw material formula. The LB culture medium of the present invention and its preparation process are both prior art and will not be described in detail here.

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

[0037] It should be noted that the Enterococcus hirae IDO5 of the present invention is the Enterococcus hirae IDO5 recorded in the patent document with authorization announcement number CN118516342B and invention name "A method for preparing immobilized pellets of deodorizing bacteria and its application". The Enterococcus hirae IDO5 is a deodorizing strain screened out from pig manure samples degraded by black soldier fly larvae. It can degrade odorous substances such as indole and skatole. Enterococcus hirae IDO5 was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on March 28, 2024. The deposit address is 5th Floor, Dayuan Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City. The deposit number is GDMCCNO.64457, and the classification name is: Enterococcus hirae (Enterococcus hirae) IDO5.

[0038] Preferably, step S2 specifically includes: taking a cryopreservation tube (for example, a 2ml cryopreservation tube) of Enterococcus hirae IDO5, streaking and inoculating it on a sterilized solid LB medium (i.e., LB agar plate), and culturing it at 37°C for 24-48h; after observing that there are no foreign bacteria, picking a single colony on the solid LB medium and inoculating it into 100mL of liquid LB medium, culturing it at 37°C and 200r / min for 24h, and then further transferring it to 100mL of liquid LB medium with an inoculum size of 5% and expanding the culture for 24h to prepare a bacterial solution. In addition, preferably, the number of viable bacteria can be determined by an enzyme-labeled instrument to determine that the bacterial solution concentration is approximately 10 9 cfu / mL and then proceed to the subsequent experiments.

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

[0040] Preferably, step S3 specifically includes: taking 2 mL of the bacterial solution prepared in the above step S2 and centrifuging it at a speed of 8000 r / min for 5 min (or 4000 r / min for 10 min), discarding the supernatant to collect the bacteria, resuspending and washing the bacteria with sterile saline, and centrifuging again at the same speed to obtain the bacterial slurry required for the freeze-drying experiment.

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

[0042] Preferably, step S4 specifically includes: using 2 mL of sterilized protective agent solution to resuspend the bacterial sludge obtained by centrifugation in the above step S3 and then transfer it to a freeze-dried bottle, first placing the freeze-dried bottle in a 4°C refrigerator for pre-cooling for 2 hours, and further 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 at -54°C and below 25.9 Pa for freeze-drying for 72 hours to obtain the freeze-dried powder of Enterococcus hirae IDO5.

[0043] Preferably, the protective agent solution comprises 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; the protective agent solution is prepared by dissolving the above raw materials in distilled water according to proportions.

[0044] The protective agent solution of the present invention was subjected to single factor test analysis, orthogonal test analysis, effect verification of the survival rate of Enterococcus hirae IDO5 freeze-dried powder, and microscopic morphology analysis of Enterococcus hirae IDO5 freeze-dried powder.

[0045] (1) Single factor test analysis: A single factor test was used to investigate the effects of different types and concentrations of single protective agents on the survival rate of Enterococcus hirae IDO5 strains after freeze-drying. The experimental conditions such as pre-freezing, vacuum freeze-drying, rehydration, and viable bacteria count were kept unchanged. The survival rate of the strains after freezing was used as an indicator to screen out the conditions with the highest survival rate of the strains after freeze-drying among various protective agents. Freeze-drying protective agents include sodium glutamate, skim milk powder, trehalose, sorbitol, lactose, mannitol, maltose, vitamin C, and inulin. Five concentration levels were set for each factor, as shown in Table 1 below. The survival rate of Enterococcus hirae IDO5 was measured after freeze-drying.

[0046] Table 1 Single factors and levels

[0047]

[0048] The results of the freeze-dried survival rate of Enterococcus hirae IDO5 are shown in Table 2 below. The protective effects of various protectants at different concentrations were different. Among the nine protectants, the survival rate of Enterococcus hirae IDO5 in the freeze-dried powder was the highest, at 67.12%, when the lactose content was 7.5 g / 100 mL.

[0049] Table 2 Survival rate of Enterococcus hirae GDIAS-5 in presence of single cryoprotectant

[0050]

[0051] The results showed that lactose exhibited a high protective effect across all factors and levels, with the highest IDO5 survival rates reaching 67.12% and 65.75% at concentrations of 10 g / 100 mL and 15 g / 100 mL, respectively, representing the most effective protection. This was significantly higher than sorbitol, inulin, and maltose (P < 0.05). The protective effect of sorbitol was proportional to its concentration, reaching 56.52% at a concentration of 5 g / 100 mL. Maltose exhibited a greater protective effect at 5 g / 100 mL, with the highest IDO5 survival rate reaching 51.30%. Compared to the other factors, the protective effect of inulin was relatively stable across different concentration levels, with IDO5 survival rates ranging from 41.74% to 58.04%.

[0052] In summary, inulin, sorbitol, lactose and maltose had significant protective effects on Enterococcus hirae IDO5 lyophilized powder at different concentrations, especially at high concentrations, which showed that they had good application potential as protective agents for Enterococcus hirae IDO5 lyophilized powder.

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

[0054] Table 3 Response surface experiment factors and levels

[0055]

[0056] In the range analysis method, the range R value is used to determine the impact of the four factors mentioned above on the protection rate. A larger R value indicates a more significant impact of the factor on the protection rate against E. hirae IDO5, and therefore, a higher importance of the factor to the test evaluation metric. Furthermore, the parameter k represents the impact of different levels of each factor on the evaluation metric, helping to identify the optimal combination. Since this experiment aims for a high protection rate, kk takes the maximum value in 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, which shows that C lactose is the key factor affecting the protection rate of Enterococcus hirae IDO5, followed by A inulin and B sorbitol, and D maltose has a smaller effect. The optimal level combination of each factor is A2B3C1D2, that is, the optimal process parameters of the protective agent solution of Enterococcus hirae IDO5 freeze-dried powder are: A inulin concentration 15g / 100mL, B sorbitol concentration 5g / 100mL, C lactose concentration 7.5g / 100mL and D maltose concentration 4g / 100mL. The specific range analysis data and results are shown in Table 4 below, and the trend chart of the optimal combination is shown below. Figure 2 shown.

[0058] Table 4 Protection rate intuitive analysis table

[0059]

[0060] As can be seen in Table 4, when the other three factors remain unchanged, the protection rate against E. hirae IDO5 exhibits different trends as the four factors change. Specifically, with increasing inulin concentration, the protection rate first increases and then decreases, with the rates of increase and decrease being roughly the same. With increasing sorbitol concentration, the protection rate first decreases and then increases. Increasing lactose concentration leads to a rapid decrease in the protection rate. In contrast, increasing maltose concentration shows a trend of the protection rate stabilizing for a period of time before beginning to decline.

[0061] The results of the variance analysis are shown in Table 5 below. Inulin (A), sorbitol (B), lactose (C), and maltose (D) significantly impacted the protection rate (P < 0.05). The order of significance for the four factors on the protection rate was lactose (C) > inulin (A) > sorbitol (B) > maltose (D), consistent with the ranking results from the range analysis method. This result further confirms the importance of lactose in influencing the protection rate against E. hirae IDO5. Detailed 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) Verification of the effect of Enterococcus hirae IDO5 freeze-dried powder survival rate: It can be seen from the range of the test results (Table 4) and the variance analysis (Table 5) that the order of influence of A inulin, B sorbitol, C lactose and D maltose on the protection rate of Enterococcus hirae IDO5 is consistent, which is C lactose>A inulin>B sorbitol>D maltose. In addition, the optimal combination A2B3C1D2 predicted by the orthogonal experiment has appeared in the orthogonal experiment design table, and the corresponding protection rate is the highest value. Therefore, in order to verify the stability and reliability of the predicted combination, three repeated experiments were carried out. The results showed that the average protection rate reached 74.31%, further confirming the optimal parameters of the Enterococcus hirae IDO5 protective agent in the process of the present invention: A inulin concentration of 15g / 100ml, B sorbitol concentration of 5g / 100ml, C lactose concentration of 7.5g / 100ml and D maltose concentration of 4g / 100ml. The specific verification results are detailed in Table 6 below.

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

[0067]

[0068] (4) Micromorphological analysis of Enterococcus hirae IDO5 freeze-dried powder: The microstructure of Enterococcus hirae IDO5 powder (i.e., Enterococcus hirae IDO5 freeze-dried powder) without and with the addition of the composite protective agent of the IDO5 strain (i.e., the raw material formula corresponding to the optimal concentration of the protective agent solution used in step S4 above: lactose 7.5 g / 100 mL, inulin 15 g / 100 mL, sorbitol 5 g / 100 mL, maltose 4 g / 100 mL) was analyzed using scanning electron microscopy. The relevant comparison figure is shown in Figure 3 ( Figure 3 Note: (A) The morphology of the freeze-dried powder of Enterococcus hirae IDO5 without the addition of the above-mentioned composite protective agent after 300-fold magnification; (B) The morphology of the freeze-dried powder without the addition of the composite protective agent after 1000-fold magnification; (C) The morphology of the freeze-dried powder without the addition of the composite protective agent after 5000-fold magnification; (D) The morphology of the freeze-dried powder with the addition of the composite protective agent after 300-fold magnification; (E) The morphology of the freeze-dried powder with the addition of the composite protective agent after 1000-fold magnification; (F) The morphology of the freeze-dried powder with the addition of the composite protective agent after 5000-fold magnification). Figure 3 A and Figure 3 D shows that E. hirae IDO5 is surrounded by inulin, sorbitol, lactose and maltose, all covered by the coating. Figure 3 B and Figure 3 E comparison shows that the surface of Enterococcus hirae IDO5 powder is relatively flat and smooth, with a compact structure and clear overall outline, and the bacteria are tightly combined with the composite protective agent. Figure 3 C and Figure 3From the comparison of F, it can be seen that the bacterial morphology of Enterococcus hirae IDO5 is full and complete, without any damage or shriveling, and only a very small number of bacteria are wrinkled. This proves that the optimized composite protective agent can effectively improve the surface morphology of bacterial powder, enhance the cell's resistance to freezing, maintain the integrity of the cell membrane structure, and improve the freeze-drying survival rate of Enterococcus hirae IDO5.

[0069] In addition, preferably, after the above-mentioned step S3 and before step S4, the number of live bacteria in the bacterial sludge before drying can also be determined: the bacterial sludge sample obtained by centrifugation is resuspended with 2 mL of sterile physiological saline and transferred to a sterile test tube, fully shaken, 100 μL of bacterial suspension is drawn and dropped into 900 μL of sterile physiological saline, and the operation is repeated after sufficient shaking; diluted to 106-1010 times, 100 μL is taken on the LB agar plate, spread evenly, and the LB agar plate is inverted and incubated in a constant temperature incubator at 37°C for 24-48 hours. When a single colony grows on the LB agar plate, the plate with a colony count of 30-300 is selected for colony counting.

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

[0071] The following is an analysis of the effects of different amounts of Enterococcus hirae IDO5 freeze-dried powder added on the degradation of skatole, indole and ammonia in pig manure wastewater: (1) Effect on skatole degradation rate: Figure 4 As shown in the figure, with the increase of the amount of Enterococcus hirae IDO5 freeze-dried powder added, the degradation rate of skatole gradually increased. When the concentration of Enterococcus hirae IDO5 freeze-dried powder added reached 20g / L, the degradation rate was close to the maximum value. (2) Effect on indole degradation rate: Figure 5 As shown in the results, the degradation effect of Enterococcus hirae IDO5 freeze-dried powder on indole showed a significant upward trend with the increase of the amount of bacterial powder added (P<0.05). The degradation rate of indole was relatively low when the amount of bacterial powder added was 1g / L to 10g / L, but as the amount added increased to 15g / L and above, the removal effect increased significantly (P<0.05), indicating that the degradation of indole was proportional to the amount of Enterococcus hirae IDO5 freeze-dried powder added, and the effect was better when the amount added was above 15g / L. (3) Effect on ammonia degradation rate: Figure 6As shown, the degradation rate of ammonia also increases with the increase in the addition amount of Enterococcus hirae IDO5 lyophilized powder, and the effect is better when the addition amount is 15g / L or above. In summary, the addition amount of Enterococcus hirae IDO5 lyophilized powder of the present invention has a significant impact on the removal effect of odorous substances in pig manure wastewater. Appropriately increasing the addition amount of bacterial powder can improve the degradation effect of several major odorous substances, but the effect tends to be stable after reaching a certain addition amount. The degradation rate of Enterococcus hirae IDO5 lyophilized powder for indole, skatole, and ammonia in pig manure wastewater can 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) The present invention prepares a bacterial solution by inoculating Enterococcus hirae IDO5 into a culture medium, further centrifuging the bacterial solution to obtain bacterial mud, and finally freeze-drying the bacterial mud to obtain Enterococcus hirae IDO5 freeze-dried powder. The preparation process of Enterococcus hirae IDO5 freeze-dried powder of the present invention is simpler and easier to operate than the existing preparation process of preparing Enterococcus hirae IDO5 into immobilized pellets in the prior art; (2) The present invention prepares Enterococcus hirae IDO5 into the form of freeze-dried powder, and the low temperature and low pressure of the freeze-drying process can better retain the physiological activity of the Enterococcus hirae IDO5 strain, and the freeze-dried powder can also expand the contact between the deodorizing microorganism Enterococcus hirae IDO5 and odorous substances. contact area, enhancing the resistance of microorganisms to adverse environmental influences, thereby better ensuring the use effect of Enterococcus hirae IDO5; in addition, Enterococcus hirae IDO5 freeze-dried powder 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) Enterococcus hirae IDO5 freeze-dried powder has a good deodorizing effect, and the degradation rate of indole, skatole, and ammonia in pig manure wastewater can reach more than 60%. It can be applied to livestock and poultry breeding manure and breeding wastewater to effectively reduce the concentration of odor substances such as ammonia, indole, and skatole, thereby effectively reducing the odor pollution caused by livestock and poultry breeding; (4) The present invention can effectively improve the surface morphology of Enterococcus hirae IDO5 freeze-dried powder through the protective agent solution, enhance the cell's anti-freezing ability, maintain the integrity of the cell membrane structure, and improve the freeze-dried survival rate of Enterococcus hirae IDO5.

[0073] It should be noted that the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprises a ..." does not preclude the presence of other identical elements in the process, method, article, or apparatus that includes the element.

[0074] Although embodiments of the present invention have been shown, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the invention, and that the scope of the 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, resuspending the bacterial sludge obtained in step S3 with a protective agent solution and transferring the suspension to a freeze-drying bottle, further freeze-drying the freeze-drying bottle to obtain freeze-dried powder of Enterococcus hirae IDO5; The culture medium is LB culture medium, the Enterococcus hirae IDO5 deposit number is GDMCC NO.64457, and the protective agent solution is prepared by dissolving 7.5 g / 100 mL of lactose, 15 g / 100 mL of inulin, 5 g / 100 mL of sorbitol, and 4 g / 100 mL of maltose in distilled water in proportion.

2. The preparation process of Enterococcus hirae IDO5 freeze-dried powder according to claim 1, characterized in that: 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 frozen tube of the Enterococcus hirae IDO5, streaking the tube on the sterilized solid LB medium, and culturing at 37°C for 24-48 hours; after observing that there are no foreign bacteria, picking a single colony on the solid LB medium and inoculating it into 100 mL of the liquid LB medium, culturing at 37°C and 200 r / min for 24 hours, and then further transferring the colony to 100 mL of the liquid LB medium at a 5% inoculum size and expanding the culture for 24 hours to prepare 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 transferring 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 at -54°C and 25.9 Pa for freeze-drying for 72 hours to obtain the Enterococcus hirae IDO5 freeze-dried powder.

7. The process for preparing the freeze-dried powder of Enterococcus hirae IDO5 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. 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 7 is used to reduce the concentrations of ammonia, indole and skatole in livestock and poultry manure and aquaculture wastewater.

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