Application of Pseudomonas aeruginosa GXun-2 in the deproteinization of crayfish shell

Abstract

The application provides application of pseudomonas aeruginosa GXun-2 in deproteinization of small crayfish shell and belongs to the technical field of microorganisms.The application provides application of pseudomonas aeruginosa GXun-2 in deproteinization of small crayfish shell, wherein the small crayfish shell comprises by-products generated in the processing of small crayfish; the by-products comprise shrimp shells, shrimp heads and shrimp tails.The application overcomes the defects of poor deproteinization effect and long cycle in the deproteinization process of small crayfish shell by existing microorganisms, and has the advantages of fast deproteinization speed, good deproteinization effect, rich amino acid content in deproteinization liquid and the like when pseudomonas aeruginosa GXun-2 is used for deproteinization of small crayfish shell, so that the deproteinization cycle can be significantly shortened, the deproteinization effect is improved, the environmental pollution caused by small crayfish shell is alleviated, and the reutilization of waste protein resources is realized.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to the application of Pseudomonas aeruginosa GXun-2 in the deproteinization of crayfish shells. Background Technology

[0002] Besides live and frozen whole crayfish, crayfish can also be processed into products such as crayfish tails, crayfish meat, and crayfish paste. The main byproducts generated during processing are the crayfish heads and shells. Since crayfish meat accounts for a small proportion of the whole crayfish weight, byproducts generated after processing account for approximately 70-85% of the whole crayfish's weight. Most of these byproducts are discarded, causing environmental pollution and resource waste, and therefore require further processing and utilization. In the dry weight of crayfish byproducts, chitin content is 20-30%, protein is 25-30%, ash is 25-35%, and other components account for about 5%.

[0003] Currently, shrimp shell treatment mainly involves landfill, incineration, and chemical treatment. Landfill and incineration not only pollute the environment but also waste resources. Chemical methods, primarily used for chitin extraction, not only generate large amounts of difficult-to-treat acidic and alkaline wastewater but also damage the product structure, and the abundant protein resources in shrimp shells cannot be utilized, resulting in resource waste. However, using microorganisms to deproteinize shrimp shells is a gentle and pollution-free process that allows for the utilization of protein resources, and it has gradually replaced chemical methods, becoming a current research hotspot.

[0004] Currently reported microorganisms that can be used for deproteinization of shrimp shells include Bacillus, Pseudomonas, and Vibrio. Belem et al. utilized... Lactobacillus sp. B2 fermentation of shrimp and crab shells to obtain chitin, with a deproteinization rate of 56% (AlbinoBF, Arias L, Gomez J, et al. Chitin and L(+)-lactic acid production from crab). Callinectes bellicosus wastes by fermentation of Lactobacillus sp. B2 using sugar cane molasses as carbon source. Bioprocess Biosyst Eng, 2012, 35: 1193–1200.). Jung et al. used Lactobacillus paracasei KCTC-3074 and Serratia marcescensFS-3 was used in a study on deproteinization of shrimp shells using a combination of two bacteria, achieving a deproteinization rate of 68.9% (Jung WJ, Jo GH, Kuk JH, et al. Production of chitin from red crab shell waste by successivefermentation with Lactobacillus paracasei KCTC-3074 and Serratia marcescens FS-3. Carbohydrate Polymers, 2007, 68 (4): 746-750.). Zhang et al. utilized highly compatible... Bacillus subtilis and Acetobacter pasteurianus The fermentation process involves two steps, first using... B. subtilis Add after 3 days of fermentation. A. pasteurianus Fermentation continued for 2 days, and the protease activity in the fermentation broth was 96.9 U / mL (Zhang Q, Duan LR, Li YC, et al. Positive effects and mechanism of ultrasound onchitin preparation from shrimp shells by co-fermentation. Ssrn ElectronicJournal, 2022: 106066.). Kan Jia used Bacillus thuringiensis LX-W4 and Enterococcus faecalis LX18-9 for co-fermentation to study the deproteinization of crayfish shells. The fermentation time was 4 days, and the deproteinization rate reached 95.21% (Kan Jia, Jiang Ning, Wang Xin, et al. Screening of protease-producing Bacillus thuringiensis and its application in chitin preparation [J]. Food and Fermentation Industries, 2025, 51(22): 247-253.). The above studies show that although significant progress has been made in the research on deproteinizing shrimp shells using microorganisms, there are still a series of problems, such as low enzyme activity of strains, low deproteinization rate, long processing cycle, and unclear composition of degradation products. It is urgent to select superior strains, optimize related processes, and study the composition and application of degradation products. Summary of the Invention

[0005] The purpose of this invention is to provide the application of *Pseudomonas aeruginosa* GXun-2 in the deproteinization of crayfish shells, overcoming the shortcomings of existing microbial methods in the deproteinization process of crayfish shells, such as poor deproteinization effect and long cycle. This invention utilizes *Pseudomonas aeruginosa* GXun-2 for crayfish shell deproteinization, which has advantages such as rapid deproteinization speed, good effect, and rich amino acid content in the deproteinization solution, significantly shortening the deproteinization cycle and improving the deproteinization effect.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of Pseudomonas aeruginosa GXun-2 in the deproteinization of crayfish shells.

[0007] Preferably, the crayfish shell includes by-products generated during crayfish processing; the by-products include crayfish shells, crayfish heads, and crayfish tails.

[0008] This invention provides a method for preparing deproteinized crayfish shells using Pseudomonas aeruginosa GXun-2, comprising the following steps: (1) Preparation of seed culture containing Pseudomonas aeruginosa GXun-2; (2) Prepare a fermentation medium containing crayfish shells; (3) The seed liquid obtained in step (1) is inoculated into the fermentation medium obtained in step (2) for culture. After centrifugation, the liquid part is taken to obtain the crayfish shell deproteinization liquid.

[0009] Preferably, the concentration of Pseudomonas aeruginosa GXun-2 in the seed solution in step (1) is 0.5 × 10⁻⁶. 9 cfu / mL ~1.5×10 9 cfu / mL.

[0010] Preferably, the fermentation medium in step (2) uses water as a solvent and contains wet crayfish shells or dried crayfish shells; the pH of the fermentation medium is 6.5~8.5; When the fermentation medium contains wet crayfish shells, the mass-to-volume ratio of the wet crayfish shells to water is 1-3 g: 0.8-1.2 mL, and the water content of the wet crayfish shells is 70-85%. When the fermentation medium contains dried crayfish shells, the mass-to-volume ratio of the dried crayfish shells to water is 0.2~0.6g:0.8~1.2mL.

[0011] Preferably, in step (3), the seed liquid is inoculated into the fermentation medium at a volume ratio of 0.5 to 5.0%; the culture temperature is 25 to 42°C, the rate is 100 to 300 rpm, and the time is 1 to 5 days.

[0012] This invention provides a deproteinized crayfish shell product prepared by the method described above.

[0013] This invention provides an application of the deproteinized crayfish shell product in promoting plant growth.

[0014] Preferably, the plants include wheat and corn.

[0015] This invention provides an amino acid fertilizer, comprising the deproteinized crayfish shell product.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention selects *Pseudomonas aeruginosa*, which produces a high amount of protease. Pseudomonas aeruginosa GXun-2, as a crayfish shell deproteinization strain, has had its fermentation medium composition and ratio optimized, and its culture conditions regulated to ensure the nutritional conditions and external environment required for the rapid growth and metabolism of Pseudomonas aeruginosa GXun-2. It has a large cell yield and protease expression level, the process is simple, improves production efficiency and reduces costs, and has great application potential.

[0017] This invention uses Pseudomonas aeruginosa GXun-2 to deproteinize crayfish shells, which can degrade the proteins in the shells into active ingredients such as amino acids and small peptides. The resulting deproteinized crayfish shell products can not only be used to prepare amino acid water-soluble fertilizers, but also to extract bioactive components to make plant immune agents, which has broad application prospects in the agricultural field.

[0018] This invention overcomes the shortcomings of existing microbial deproteinization processes for crayfish shells, such as poor deproteinization effect and long cycle. Using Pseudomonas aeruginosa GXun-2 for crayfish shell deproteinization has the advantages of fast deproteinization speed, good effect, and rich amino acid content in the deproteinization solution. It can significantly shorten the deproteinization cycle, improve the deproteinization effect, help alleviate the environmental pollution caused by crayfish shells, and realize the reuse of waste protein resources. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0020] Figure 1 The growth of different strains on casein plates in Example 1; Figure 2 This demonstrates the effect of different strains on the degradation of crayfish shells in Example 1; Figure 3 The results of Pseudomonas aeruginosa GXun-2 desquamating crayfish shell proteins under different conditions in Example 2; Figure 4 This demonstrates the effect of Pseudomonas aeruginosa GXun-2 on the degradation of crayfish shells at different concentrations in Example 3. Figure 5 The degradation effect of Pseudomonas aeruginosa GXun-2 on crayfish shells at different fermentation times in Example 4; Figure 6The effect of crayfish shell deproteinization solution at different dilution ratios on wheat seedling growth in Example 6; Figure 7 The effect of different dilution ratios of crayfish shell deproteinization solution on the growth of corn seedlings in Example 7. Detailed Implementation

[0021] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but these should not be construed as limiting the scope of protection of the present invention. The strains used in the following embodiments were all provided by Guangxi University for Nationalities. Among them, *Pseudomonas aeruginosa* GXun-2 has been disclosed in CN202110583659.4, with the number GDMCC No: 61615. The wet crayfish shells were purchased from Hubei Province and transported via cold chain, in three batches; the average moisture content of the wet crayfish shells was 80%.

[0022] Example 1: Screening of crayfish shell deproteinization strains

[0023] Sixteen bacterial strains capable of secreting protease, previously screened by the inventors, were plated onto casein plates and incubated at 37°C for 2 days. Ten strains showed distinct clear zones. The colony diameter and clear zone diameter were measured, and the ratio of clear zone diameter to colony diameter (diameter-to-clear zone ratio) was calculated. The results are as follows: Figure 1 As shown in Table 1, GXun-1, GXun-2, Gxun-7, Gxun-3, Gxun-4, and Gxun-1 exhibit high protease production activity.

[0024] Table 1. Results of colony diameter and clear zone diameter of different strains on casein plates.

[0025] Six strains with a diameter-to-circle ratio greater than 2.3 were inoculated into a sterilized (121℃ moist heat sterilization for 20 min) culture medium (composed of water and wet crayfish shells; 66.7 g of wet crayfish shells were added to 33.3 mL of distilled water, pH natural (approximately 7.8)). Fermentation was carried out at 35℃ and 180 rpm for 48 h on a shaker. The fermentation broth was centrifuged at 10000 rpm for 5 min, and the supernatant was used as the crude enzyme solution. The strains were further screened by measuring the protease activity in the crude enzyme solution and observing the degradation effect on crayfish shells. The results are as follows: Figure 2As shown, *Pseudomonas aeruginosa* GXun-2 showed the best deproteinization effect on crayfish shells, with a protease activity of 1219.36 U / mL in the crude enzyme solution. Although *Pseudomonas aeruginosa* Gxun-7 also showed good degradation effect in the early stage of degrading marine shrimp (Song Chaodong et al., Food and Fermentation Industries, 2024), its effect on degrading crayfish shells was poor, with a protease activity of only 724.18 U / mL. This may be related to the different protein composition of crayfish shells compared to marine shrimp, and the higher mineral content in crayfish shells. The protease activities of the other strains were as follows: *Pseudomonas aeruginosa* Gxun-1, 557.36 U / mL; *Aureobacterium chrysogenum* GXun-1, 816.57 U / mL; *Bacillus cereus* Gxun-3, 539.47 U / mL; and *Bacillus belyssus* Gxun-4, 367.44 U / mL. Therefore, Pseudomonas aeruginosa GXun-2 was selected as the deproteinizing strain for crayfish shells.

[0026] Protease activity assay: Dilute the supernatant 100-fold, take 200 μL and preheat in a 50°C water bath for 2 min. Then add 300 μL of the same preheated 2% casein solution (m / v, pH 7.5), mix well, and react in a 50°C water bath for 10 min. Immediately add 500 μL of 4 mol / L trichloroacetic acid (TCA) solution to terminate the reaction. Centrifuge and take 200 μL of the supernatant. Add 1 mL of 0.5 mol / L Na₂CO₃ solution and 200 μL of Folin-Ciocalteu solution sequentially, and react at 50°C for 10 min. Use the supernatant inactivated by trichloroacetic acid as a control. After the reaction, measure the absorbance at 660 nm. One unit of enzyme activity is defined as the amount of enzyme required to hydrolyze casein to produce 1 μg of tyrosine per minute at a reaction temperature of 50°C. Each experiment was repeated 3 times, and the average value was taken.

[0027] Example 2

[0028] In this embodiment, *Pseudomonas aeruginosa* GXun-2 was used as the crayfish shell deproteinization strain to study the effects of initial pH (6, 6.5, 7, 7.5, 8, 8.5, 9), nitrogen source (peptone, yeast extract powder, corn steep liquor, NaNO3, (NH4)2SO4), inorganic salts (MnSO4, MgSO4, CuSO4, FeSO4, K2SO4, CaCl2), and fermentation temperature (25, 30, 35, 40, 45℃) on the deproteinization effect of crayfish shells.

[0029] Preparation of Pseudomonas aeruginosa GXun-2 seed culture: Pseudomonas aeruginosa GXun-2 was activated into a primary strain using LB solid slant culture. The activated strain was then inoculated into LB liquid medium and cultured at 35℃ and 180 rpm until the bacterial concentration reached 1 billion cells / mL to obtain the seed culture.

[0030] 1. Study on the effect of initial pH on deproteinization effect

[0031] Fermentation medium was prepared by combining wet crayfish shells and water, with a mass-to-volume ratio of 2g:1mL for wet crayfish shells to water. After adjusting the pH, the medium was sterilized by moist heat at 121℃ for 20min.

[0032] Inoculation with Pseudomonas aeruginosa GXun-2: Inoculate the seed culture of Pseudomonas aeruginosa GXun-2 into the fermentation medium at an inoculation rate of 2% (v / v), ferment at 35℃ and 180 rpm for 48 h, centrifuge at 1200 rpm for 1 min, and take the supernatant to determine the protease activity.

[0033] 2. Study on the effect of nitrogen source type on deproteinization effect

[0034] Fermentation medium was prepared from wet crayfish shells, a nitrogen source, and water, with a mass-to-volume ratio of 2 g crayfish shells to 1 mL water. The nitrogen source concentration was 10 g / L. After adjusting the pH to 7.5, the medium was sterilized by moist heat at 121℃ for 20 min. Fermentation medium without added nitrogen source served as the control (CK).

[0035] Inoculation with Pseudomonas aeruginosa GXun-2: Inoculate the seed culture of Pseudomonas aeruginosa GXun-2 into the fermentation medium at an inoculation rate of 1% (v / v), ferment at 35℃ and 180 rpm for 48 h, centrifuge at 1200 rpm for 1 min, and take the supernatant to determine the protease activity.

[0036] 3. Research on the Influence of Inorganic Salt Types on Deproteinization Efficiency

[0037] Fermentation medium was prepared by combining wet crayfish shells, inorganic salts, and water, with a mass-to-volume ratio of 2 g crayfish shells to 1 mL water and an inorganic salt concentration of 1 g / L. After adjusting the pH to 7.5, the medium was sterilized by moist heat at 121℃ for 20 min. Fermentation medium without added inorganic salts served as the control (CK).

[0038] Inoculation with Pseudomonas aeruginosa GXun-2: Inoculate the seed culture of Pseudomonas aeruginosa GXun-2 into the fermentation medium at an inoculation rate of 5% (v / v), ferment at 35℃ and 180 rpm for 48 h, centrifuge at 1200 rpm for 1 min, and take the supernatant to determine the protease activity.

[0039] 4. Study on the effect of fermentation temperature on deproteinization effect

[0040] Fermentation medium was prepared by combining wet crayfish shells and water, with a mass-to-volume ratio of 2g:1mL for wet crayfish shells to water. After adjusting the pH, the medium was sterilized by moist heat at 121℃ for 20min.

[0041] Inoculation with Pseudomonas aeruginosa GXun-2: Inoculate the seed culture of Pseudomonas aeruginosa GXun-2 into the fermentation medium at an inoculation rate of 5% (v / v), ferment at 180 rpm for 48 h, centrifuge at 1200 rpm for 1 min, and take the supernatant to determine the protease activity.

[0042] The method for determining protease activity is the same as in Example 1.

[0043] The results of Pseudomonas aeruginosa GXun-2 demineralizing crayfish shell proteins under different conditions are as follows: Figure 3 As shown, CK is the blank control. It can be seen that the initial pH of the fermentation medium at 6.5–7.5 has no significant effect on enzyme activity (P>0.05). Figure 3 (A) Fermentation broths with different initial pH values ​​were measured with a pH meter, and the pH values ​​remained stable at around 9.5, which may be related to the fact that crayfish shells contain a large amount of CaCO3, which can act as a pH buffer for fermentation. External nitrogen sources have a certain inhibitory effect on GXun-2 enzyme production. Figure 3 (In case B), it's possible that the crayfish shell itself is rich in protein. Although adding a nitrogen source met the growth requirements of the strain (the number of viable bacteria in the fermentation broth increased slightly after adding nitrogen source compared to the control, but the results are not shown), it inhibited the substrate's induction of the enzyme. Therefore, no additional nitrogen source is needed during the deproteinization process of crayfish shells using GXun-2. Fermentation temperature has a significant impact on enzyme activity. When the fermentation temperature is between 25℃ and 35℃, enzyme activity increases with increasing fermentation temperature. However, when the temperature exceeds 35℃, enzyme activity decreases rapidly with increasing fermentation temperature. Figure 3 The optimal fermentation temperature (C) is likely related to the optimal growth temperature of GXun-2 and the optimal temperature for the protease; therefore, the optimal fermentation temperature is 35℃. Exogenous addition of inorganic salts, especially Cu, has a certain inhibitory effect on GXun-2 enzyme production. 2+ It significantly inhibits enzyme production by the strain. Figure 3 Therefore, GXun-2 does not require the addition of additional inorganic salts during the deproteinization process of crayfish shells (D).

[0044] Example 3

[0045] This embodiment uses *Pseudomonas aeruginosa* GXun-2 as the crayfish shell deproteinization strain to study the effect of crayfish shell concentration in the fermentation medium on the deproteinization effect. The specific methods are as follows: Fermentation medium was prepared by mixing wet crayfish shells with water at a mass-to-volume ratio (m / v, g / mL) of 1:1, 2:1, 3:1, and 1:0 (i.e., whole shells). The Pseudomonas aeruginosa GXun-2 seed culture prepared by the method in Example 2 was inoculated at a 2% (v / v) inoculation rate. Fermentation was carried out at 35℃ and 180 rpm for 48 h. After centrifugation at 1200 rpm for 1 min, the supernatant was collected to determine the protease activity, amino acid content, and protein content.

[0046] The method for determining protease activity is the same as in Example 1.

[0047] Amino acid content determination: The amino acid content was determined using a fully automated amino acid analyzer.

[0048] Protein content determination: The protein content of crayfish shells before and after fermentation was determined by the Kjeldahl method in GB 5009.5—2016 "Determination of Protein in Food", and the deproteinization rate was calculated.

[0049]

[0050] Where: 0.05 — concentration of hydrochloric acid standard titration solution, in mol / L; 0.014 — mass of nitrogen equivalent to 1.0 mL of hydrochloric acid standard titration solution, in g; 6.25 — coefficient for nitrogen to protein conversion; 30.04 — average percentage content of protein in the shrimp shell sample used.

[0051] Figure 4 Table 1 shows the state of wet crayfish shells at different fermentation times (CK is the blank control without bacterial inoculation). Table 2 shows the deproteinization effect measured after 48 hours of fermentation. Figure 4 As shown in Table 2, when the ratio of wet shrimp shells to water was 3:1, the amino acid content was highest after 48 hours of fermentation, reaching 3148.37±5.31 mg / L, and the protein removal rate reached 95.15%. When the ratio of wet shrimp shells to water was 1:1, the protein removal rate was highest, reaching 98.36%, and the amino acid content also reached 2587.36±4.15 mg / L. However, when fermentation was carried out using all wet shrimp shells, the degradation effect of shrimp shells was poor, with obvious large particles present, and the protein removal effect was poor, only 89%. This may be because the concentration of wet shrimp shells was too high, resulting in too little water content in the culture medium, increasing osmotic pressure and affecting the growth of the strain. In the CK group, some shrimp shells were also observed to be broken. This was because the shrimp shells underwent some physical damage after treatment at 35℃ and 180rpm on a shaker (the shrimp shells in the CK group treated with all shrimp shells were more severely broken, possibly because the low water content in the culture medium caused the shrimp shells to impact the bottle wall more forcefully, resulting in more severe physical damage). Therefore, taking into account both the deproteinization rate and amino acid concentration, the optimal wet shrimp shell to water ratio for deproteinization of strain GXun-2 is 3:1.

[0052] Table 2. Effect of crayfish shell concentration on the deproteinization effect of GXun-2.

[0053] Example 4

[0054] This embodiment uses *Pseudomonas aeruginosa* GXun-2 as the crayfish shell deproteinization strain to study the effect of fermentation time on the deproteinization effect of crayfish shells. The specific methods are as follows: The wet shells of crayfish were mixed with water at a mass-to-volume ratio of 3g:1mL to obtain a fermentation medium. The Pseudomonas aeruginosa GXun-2 seed culture prepared by the method in Example 2 was inoculated at a 2% (v / v) inoculation rate. Fermentation was carried out at 35℃ and 180rpm. Samples were taken after 12, 24, 36 and 48h of fermentation, respectively. The samples were centrifuged at 1200rpm for 1min and the supernatant was taken to determine the protease activity, amino acid and protein content (the protease activity determination method is the same as in Example 1, and the amino acid and protein content determination method is the same as in Example 3).

[0055] The results are as follows Figure 5 As shown in Table 3, fermentation time significantly affects the deproteinization rate, protease activity, and amino acid content of crayfish. During fermentation of 12-24 hours, enzyme activity increases with increasing fermentation time; at 36 hours, enzyme activity decreases slightly but not dramatically, and the amino acid content is highest at this time; however, at 48 hours, the amino acid content decreases, possibly due to the consumption of amino acids by other microorganisms over time. In the control group (CK) without added fermentation bacteria, some shell degradation was also observed, possibly due to physical damage to the shells after treatment on a shaker at 35℃ and 180rpm. Therefore, the optimal fermentation time is 36 hours.

[0056] Table 3. Effect of fermentation time on the deproteinization effect of GXun-2

[0057] The above results also indicate that although Pseudomonas aeruginosa GXun-2 was previously used for feather degradation (A strain of Pseudomonas aeruginosa GXun-2 and its application, CN202110583659.4), its degradation process in crayfish shells is completely different from that of feather degradation. The protease activity and amino acid concentration of the strain are much higher than those of feather degradation.

[0058] Example 5

[0059] A method for preparing deproteinized crayfish shells using Pseudomonas aeruginosa GXun-2, the steps of which are as follows: (1) Pseudomonas aeruginosa GXun-2 was activated into a primary strain by LB solid slant culture. The activated strain was inoculated into LB liquid medium and cultured at 35℃ and 180rpm until the bacterial concentration reached 1 billion cells / mL to obtain the seed culture.

[0060] (2) Mix the wet crayfish shells with water at a mass-volume ratio of 3g:1mL and adjust the pH to 6.5 to obtain the fermentation culture medium.

[0061] (3) The seed liquid obtained in step (1) was inoculated into the fermentation medium obtained in step (2) at an inoculation rate of 2% (v / v), fermented at 35℃ and 180 rpm for 36 h, centrifuged at 1200 rpm for 1 min, and the supernatant was collected to obtain the deproteinized crayfish shell liquid.

[0062] The crayfish shell deproteinization solution prepared above was used to determine the types and contents of amino acids using an automatic amino acid analyzer. The results are shown in Table 4. It can be seen that 16 amino acids were detected in the crayfish shell deproteinization solution, with a total content of 43.45 g / L. Histidine, phenylalanine, lysine, aspartic acid, and threonine were present in relatively high amounts, indicating potential for further development into amino acid fertilizers and feeds.

[0063] Table 4. Amino acid composition and content in deproteinized crayfish shell solution

[0064] Furthermore, the amino acid content of crayfish shells degraded by Pseudomonas aeruginosa GXun-2 (43.45 g / L) was much higher than that of Pseudomonas aeruginosa Gxun-7 in the early stage of degrading shrimp (1.86 g / L) (Song Chaodong et al., Food and Fermentation Industries, 2024).

[0065] Example 6: Wheat Growth Promotion Experiment

[0066] Wheat seedling cultivation: Wheat seeds (Jimai 22) were surface-sterilized (75% alcohol for 30 seconds, sterile water washing 3 times, 1% sodium hypochlorite disinfection for 10 minutes, sterile water washing 5 times), soaked in sterile water for 2 hours, and then kept moist with gauze in the dark for 12 hours. They were then cultivated under light conditions until germination. A sterile substrate was prepared by mixing nutrient soil (purchased from Guangxi Baomei Horticulture Co., Ltd.) and vermiculite at a mass ratio of 2:1 and sterilizing it. 400g of this substrate was placed in plastic pots. Wheat seedlings with uniform growth and sprouts approximately 1cm in length were selected and transplanted into plastic pots containing the sterile substrate, with 6 seedlings per pot. The pots were then placed in an artificial climate chamber with the following cultivation conditions: 26℃ / 18℃ (day / night), photoperiod 14h / 10h (day / night), and relative humidity 80%.

[0067] Preparation of amino acid liquid fertilizer: The crayfish shell deproteinization liquid prepared in Example 5 was diluted with water by 20 times, 60 times, 80 times and 100 times respectively to prepare amino acid liquid fertilizer.

[0068] Growth-promoting experiment: The prepared amino acid liquid fertilizer was applied to the roots of wheat seedlings at a rate of 4 mL / pot, once every 10 days. A treatment with an equal volume of sterile water was used as a control. Each treatment was replicated three times in a randomized block design. After 40 days, the plant height of wheat seedlings in each treatment group was measured, and the average value was taken. The growth status and plant height measurement results of wheat seedlings are as follows: Figure 6 As shown, when the crayfish shell deproteinizing solution was diluted 20, 60, and 80 times, the wheat seedlings showed significantly better growth than the control group and the treatment group with a crayfish shell deproteinizing solution diluted 100 times. This indicates that the crayfish shell deproteinizing solution has a significant growth-promoting effect on wheat when diluted 20 to 80 times, with the growth-promoting effect reaching 39.47% when diluted 80 times.

[0069] Example 7: Maize Growth Promotion Experiment

[0070] Cultivation of corn seedlings: Corn seeds (Guidan 162) were surface-sterilized (75% alcohol for 30 seconds, sterile water for 3 washes, 1% sodium hypochlorite for 10 minutes, sterile water for 5 washes), and then soaked in sterile water for 2 hours. Wheat seeds were then placed in the dark with gauze to maintain moisture for 12 hours, followed by cultivation under light conditions until germination. Nutrient soil (purchased from Guangxi Baomei Horticulture Co., Ltd.) and vermiculite were mixed at a mass ratio of 2:1 and sterilized to obtain a sterilized substrate, which was then filled into plastic pots at a rate of 400g / pot. Two corn seedlings with uniform growth and a sprout length of approximately 2cm were transplanted into each plastic pot containing the sterilized substrate. The pots were then placed in an artificial climate chamber with the following cultivation conditions: 26℃ / 18℃ (day / night), photoperiod 14h / 10h (day / night), and relative humidity 80%.

[0071] Preparation of amino acid liquid fertilizer: The crayfish shell deproteinization liquid prepared in Example 5 was diluted with water by 20 times, 60 times, 80 times and 100 times respectively to prepare amino acid liquid fertilizer.

[0072] Growth-promoting experiment: The prepared amino acid liquid fertilizer was applied to the roots of maize seedlings at a rate of 4 mL / pot, once every 10 days. A treatment with an equal volume of sterile water was used as a control. Each treatment was replicated three times in a randomized block design. After 40 days, the plant height of maize seedlings in each treatment group was measured, and the average value was taken. The results of maize growth status and plant height measurements are as follows: Figure 7 As shown, when the crayfish shell deproteinizing solution was diluted 20, 60, and 80 times, the corn growth was significantly better than the control group and the treatment group with the crayfish shell deproteinizing solution diluted 100 times. This indicates that the crayfish shell deproteinizing solution has a significant growth-promoting effect on corn when diluted 20 to 80 times, with the growth-promoting effect reaching 41.01% when diluted 80 times.

[0073] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of Pseudomonas aeruginosa GXun-2 in deproteinization of crayfish shells.

2. The application according to claim 1, characterized in that, The crayfish shells include by-products generated during crayfish processing; the by-products include crayfish shells, crayfish heads, and crayfish tails.

3. A method for preparing deproteinized crayfish shell products using Pseudomonas aeruginosa GXun-2, characterized in that, Includes the following steps: (1) Preparation of seed culture containing Pseudomonas aeruginosa GXun-2; (2) Prepare a fermentation medium containing crayfish shells; (3) The seed liquid obtained in step (1) is inoculated into the fermentation medium obtained in step (2) for culture. After centrifugation, the liquid part is taken to obtain the crayfish shell deproteinization liquid.

4. The method according to claim 3, characterized in that, The concentration of Pseudomonas aeruginosa GXun-2 in the seed solution in step (1) is 0.5 × 10⁻⁶. 9 cfu / mL ~1.5×10 9 cfu / mL.

5. The method according to claim 3, characterized in that, The fermentation medium in step (2) uses water as a solvent and contains wet or dried crayfish shells; the pH of the fermentation medium is 6.5 to 8.

5. When the fermentation medium contains wet crayfish shells, the mass-to-volume ratio of the wet crayfish shells to water is 1-3 g: 0.8-1.2 mL, and the water content of the wet crayfish shells is 70-85%. When the fermentation medium contains dried crayfish shells, the mass-to-volume ratio of the dried crayfish shells to water is 0.2~0.6g:0.8~1.2mL.

6. The method according to claim 3, characterized in that, In step (3), the seed liquid is inoculated into the fermentation medium at a volume ratio of 0.5 to 5.0%; the culture temperature is 25 to 42°C, the rate is 100 to 300 rpm, and the time is 1 to 5 days.

7. A deproteinized crayfish shell product prepared by the method according to any one of claims 3 to 6.

8. The application of the deproteinized crayfish shell product according to claim 7 in promoting plant growth.

9. The application according to claim 8, characterized in that, The plants mentioned include wheat and corn.

10. An amino acid fertilizer, characterized in that, Including the deproteinized crayfish shell product as described in claim 7.

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