Shrimp shell fermentation liquor and preparation method thereof

Abstract

The invention relates to the field of coastal agriculture, in particular to shrimp shell fermentation liquor and a preparation method thereof. The preparation method comprises the following steps: drying fresh shrimp shells to obtain dried shrimp shells; based on the total volume of the culture medium, inoculating trichoderma fermentation strains into the fermentation culture medium for culturing; fermentation seed liquid is obtained; and mixing the dried shrimp shells without sterilization with the fermentation seed solution, sealing, and then fermenting. According to the technical scheme, the trichoderma harzianum is used for fermenting the shrimp shells, and the shrimp shell fermentation liquor is prepared from the unsterilized shrimp shells and multiple strains of the trichoderma harzianum in a synergistic manner, so that the degradation rate of the shrimp shells is optimal. When the shrimp shell fermentation liquor is applied to rice, the contents of rapidly available phosphorus, rapidly available potassium and organic matters in rice soil are increased, and the content of alkali-hydrolyzable nitrogen is remarkably increased, so that the growth of plant height, stem diameter, stem leaves and root systems of the rice is promoted.

Description

Technical Field

[0001] This application relates to the field of coastal agriculture, and more specifically, to a shrimp shell fermentation liquid and its preparation method. Background Technology

[0002] Shrimp, a type of aquatic product, is one of the most important international fishery trade commodities due to its significant economic value. However, the shrimp shell waste generated during processing pollutes the environment. Traditional chemical methods for processing shrimp shells are costly and cause serious environmental pollution.

[0003] Therefore, it is of great significance to find better ways to utilize shrimp shell waste. Summary of the Invention

[0004] The purpose of this application is to provide a shrimp shell fermentation liquid and its preparation method.

[0005] In a first aspect, this application provides a method for preparing shrimp shell fermentation broth, comprising: Fresh shrimp shells are dried to obtain dried shrimp shells; Fermentation strains of the genus Trichoderma were inoculated into a fermentation medium and cultured to obtain a fermentation seed liquid; The dried shrimp shells are not sterilized and are mixed with the fermentation seed liquid and then sealed for fermentation.

[0006] The above-mentioned technical solution utilizes *Trichoderma harzianum* to ferment shrimp shells. The shrimp shell fermentation broth, prepared using a combination of non-sterilized shrimp shells and multiple *Trichoderma harzianum* strains, exhibits the best shrimp shell degradation rate. Applying this shrimp shell fermentation broth to rice increases the content of available phosphorus, available potassium, and organic matter in the paddy soil, and significantly improves the content of alkaline nitrogen, thereby promoting the growth of rice plants in terms of height, stem diameter, stems, leaves, and roots.

[0007] Traditional chemical methods for treating shrimp shells are costly and cause serious environmental pollution, while microbial fermentation of shrimp shells offers advantages such as high controllability, mild reaction conditions, and environmental friendliness. The aforementioned technical solution, which uses microbial fermentation of shrimp shells to produce organic fertilizer from shrimp shell waste, is more environmentally friendly than traditional chemical methods. Furthermore, the shrimp shell fermentation broth produced using unsterilized shrimp shells and *Trichoderma harzianum* shows the best results in terms of shrimp shell degradation rate and rice growth.

[0008] In other embodiments of this application, based on the total volume of the culture medium, 5-10% of the Trichoderma fermentation strain is inoculated into the fermentation culture medium by volume percentage, and cultured at 150-200 r / min and 25-30 ℃ for 2-5 days to obtain the fermentation seed liquid; In other embodiments of this application, dried shrimp shells are mixed with fermentation seed liquid without sterilization and sealed, and then fermented at 25~30 ℃ and 110~140 r / min for 45~65 days.

[0009] In other embodiments of this application, the above-mentioned mixing of dried shrimp shells with fermented seed liquid includes: Mix the dried shrimp shells with the fermentation seed liquid according to the ratio of dried shrimp shells to fermentation culture medium of (15~25 g): (100~300 mL).

[0010] In other embodiments of this application, the fermentation medium comprises, by weight, 150-250 parts potato, 15-25 parts glucose and 900-1100 parts water.

[0011] In other embodiments of this application, the Trichoderma fermentation strain is Trichoderma harzianum.

[0012] In other embodiments of this application, the enzyme activity of the Trichoderma fermentation strains is 0.056~0.088 U / mL.

[0013] In other embodiments of this application, the preparation of the fermentation seed liquid includes: Wash the fresh shrimp shells and bury them in paddy soil. Dig up and collect the remaining shrimp shells and soil samples to obtain soil samples. Weigh a soil sample and place it in sterile water to obtain a diluted soil solution. Spread the soil solution onto chitin selection medium and incubate at 25-30 ℃ for 2-5 days to obtain a culture. Isolate colonies from the culture and inoculate the colonies into PDA medium for purification culture to obtain pure colonies. Select strains from the pure colonies and inoculate them into LB medium for culture to obtain cultured bacterial strains. Inoculate the cultured bacterial strains into chitin fermentation medium for scale-up culture.

[0014] In other embodiments of this application, the chitin screening culture medium includes: colloidal chitin, Na2HPO4, KH2PO4, NaCl, NH4Cl, MgSO4•7H2O, agar, and water.

[0015] In other embodiments of this application, the LB culture medium includes: yeast extract, NaCl, peptone, and agar.

[0016] In other embodiments of this application, the PDA culture medium includes: potato, agar, glucose, and water.

[0017] Secondly, this application provides a shrimp shell fermentation liquid, which is prepared by any of the preparation methods provided in the first aspect above. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is the standard curve for N-acetylglucosamine; Figure 2 The transparent zone formed by strain ZR on a chitin plate; Figure 3 This is a molecular biological identification diagram of ZR; Figure 4 The value represents the degradation rate of shrimp shells after fermentation. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Obviously, the described embodiments are some embodiments of this application, but not all embodiments.

[0021] Therefore, the following detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments of the application. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

[0022] Shrimp production and consumption are increasing year by year, but the slow degradation rate of shrimp shells leads to their long-term accumulation, posing a serious threat to the ecological environment. Shrimp shell waste is rich in a large number of useful components and is a highly valuable biological resource. Using modern biochemical methods to efficiently decompose shrimp shell waste can not only reduce its environmental pollution but also turn waste into treasure, generating more high-value-added products and providing a new approach to the resource utilization of aquatic product waste.

[0023] Further research revealed that processing shrimp shell waste into organic fertilizer can improve the nutrients in paddy soil and promote rice growth. Applying fermented shrimp shell liquid increased the content of available phosphorus, available potassium, and organic matter in paddy soil, and significantly increased the content of alkaline nitrogen, thereby promoting the growth of rice plants in terms of height, stem diameter, stems, leaves, and roots.

[0024] Based on the above research, this application provides a method for preparing shrimp shell fermentation liquid, comprising: Fresh shrimp shells are dried to obtain dried shrimp shells; Fermentation strains of the genus Trichoderma were inoculated into a fermentation medium and cultured to obtain a fermentation seed liquid; The dried shrimp shells are not sterilized and are mixed with the fermentation seed liquid and then sealed for fermentation.

[0025] Traditional chemical methods for treating shrimp shells are costly and cause serious environmental pollution, while microbial fermentation of shrimp shells offers advantages such as high controllability, mild reaction conditions, and environmental friendliness. The aforementioned technical solution uses microbial fermentation of shrimp shells to produce organic fertilizer, which can be used to improve rice growth. Analysis of the physicochemical properties of paddy soil and rice growth indicators shows that unsterilized shrimp shells treated with *Trichoderma harzianum* exhibit the most significant degradation rate and growth-promoting effect on rice.

[0026] Furthermore, in some embodiments of this application, based on the total volume of the culture medium, 5% of the Trichoderma fermentation strain is inoculated into the fermentation culture medium by volume percentage, and cultured at 150-200 r / min and 25-30 ℃ for 2-5 days to obtain the fermentation seed liquid.

[0027] Furthermore, in some embodiments of this application, the dried shrimp shells are mixed with the fermentation seed liquid without sterilization and sealed, and then fermented at 25~30 ℃ and 110~140 r / min for 45~65 days.

[0028] Furthermore, in some embodiments of this application, mixing the dried shrimp shells with the fermented seed liquid includes: Mix the dried shrimp shells with the fermentation seed liquid according to the ratio of dried shrimp shells to fermentation culture medium of (15~25 g): (100~300 mL).

[0029] For example, in some embodiments of this application, the dried shrimp shells are mixed with the fermentation seed liquid according to the following ratios: (15 g, 16 g, 17 g, 18 g, 19 g, 20 g, 21 g, 22 g, 23 g, 24 g, 25 g or any two of the aforementioned values) (100 mL, 120 mL, 150 mL, 180 mL, 200 mL, 210 mL, 220 mL, 230 mL, 240 mL, 250 mL, 260 mL, 280 mL, 300 mL or any two of the aforementioned values).

[0030] Furthermore, in some embodiments of this application, the fermentation culture medium comprises, by mass parts, 150-250 parts potato, 15-25 parts glucose, and 900-1100 parts water.

[0031] For example, in some embodiments of this application, the fermentation culture medium comprises, by mass parts, 150, 160, 180, 150, 150, 250 parts of potato or any two of the aforementioned values; 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 25 parts of glucose or any two of the aforementioned values; and 900, 920, 950, 980, 1000, 1100 parts of water or any two of the aforementioned values.

[0032] For example, in some embodiments of this application, the fermentation medium (PDB) is: 200 g of potato, 2% glucose, 1000 mL of distilled water, natural pH, sterilized at 121 °C for 20 min.

[0033] Furthermore, in some embodiments of this application, the Trichoderma fermentation strain is Trichoderma harzianum.

[0034] Furthermore, in some embodiments of this application, the enzyme activity of the Trichoderma fermentation strain is 0.056~0.088 U / mL.

[0035] For example, the enzyme activity of Trichoderma fermentation strains is 0.056 U / mL, 0.058 U / mL, 0.060 U / mL, 0.062 U / mL, 0.065 U / mL, 0.070 U / mL, 0.075 U / mL, 0.080 U / mL, 0.085 U / mL, 0.088 U / mL, or any two of the aforementioned values.

[0036] Furthermore, in some embodiments of this application, the preparation of the fermentation seed liquid includes: Wash the fresh shrimp shells and bury them in paddy soil. Dig up and collect the remaining shrimp shells and soil samples to obtain soil samples. Weigh a soil sample and place it in sterile water to obtain a diluted soil solution. Spread the soil solution onto chitin selection medium and incubate at 25-30 ℃ for 2-5 days to obtain a culture. Isolate colonies from the culture and inoculate the colonies into PDA medium for purification culture to obtain pure colonies. Select strains from the pure colonies and inoculate them into LB medium for culture to obtain cultured bacterial strains. Inoculate the cultured bacterial strains into chitin fermentation medium for scale-up culture.

[0037] Many pests and pathogenic microorganisms contain chitin; therefore, chitinases are of great importance in pest control. Most chitinase-producing bacteria possess strong antifungal activity, and most can inhibit pests in vitro. Because they contain chitin in both reproduction and dispersal, chitinases can be used to control Staphylococcus aureus and Brucella. In the field of biological control, the bactericidal mechanism of microorganisms such as Trichoderma harzianum and Bacillus is related to the chitinases they secrete, and utilizing biocontrol microorganisms (such as those that promote lignin synthesis) can enhance their antibacterial activity.

[0038] By using coastal saline soil to screen out bacteria that can degrade chitin, shrimp shells are fermented to produce shrimp shell fermentation liquid, which is then used to prepare organic fertilizer, thus improving the available nutrients in coastal soil.

[0039] Chitin, also known as chitosan, is the second most abundant natural polysaccharide after cellulose, and also the natural organic compound with the highest nitrogen content on Earth besides proteins. Chitin is widely distributed in the shells of crustaceans, mollusks, and arthropods, such as shrimp, crabs, and locusts, and also exists in the cell walls of higher plants and fungi. It is a key substance and energy source for marine ecosystems, and also one of the major sources of organic pollution along my country's coast.

[0040] Chitin is a white or gray, odorless, amorphous, translucent natural mucopolysaccharide crystal, a polymer of N-acetyl-D-glucosamine. Due to its regular spatial structure and strong rigidity, chitin readily forms strong hydrogen bonds between its molecules. These hydrogen bonds between the polysaccharide macromolecules endow chitin with excellent chemical and physical stability, making it insoluble in water, dilute acids, dilute alkalis, and most organic solvents. It can only dissolve in concentrated acids, concentrated alkalis, and certain aqueous solutions of hexafluoroacetone.

[0041] The structural formula of chitin (a polymer of N-acetyl-D-glucosamine) is as follows:

[0042] The enzymatic degradation of chitin involves relatively mild reaction conditions, high controllability, high efficiency, and is environmentally friendly, aligning with the contemporary concept of green development. Furthermore, it preserves the structure of chitin, allowing for the production of different polymerization products by controlling various enzymes and reaction conditions, resulting in products with higher purity, antibacterial properties, and acetylation levels.

[0043] The broad concept of chitinase includes all enzymes involved in chitin degradation, including lysozyme (EC 3.2.1.17), chitosan disaccharide deacetylase (EC 3.5.1.105), chitinase (EC 3.2.1.14), exochitinase (EC 3.2.1.200), V-cath endopeptidase (EC 3.4.22.50), N,N'-diacetylchitobiose non-reducing terminal deacetylase (EC 3.5.1.136), and endochitinase (EC 3.2.1.202). In a narrower sense, chitinase refers to enzymes that catalyze the hydrolysis of glycosidic bonds containing at least one N-acetylglucosamine group. Chitinases include endochitinases and exochitinases. Endochitinases randomly break glycosidic bonds within the sugar chain, while exochitinases hydrolyze the sugar chain sequentially from the non-reducing ends, unit by unit, in chitobioses. Furthermore, chitinases exhibit diversity, induction, and secretion. The types and properties of chitinases produced by different microorganisms vary greatly. In the presence of an inducer, most microorganisms can secrete chitinase extracellular bodies, thus making it an inducible enzyme.

[0044] Furthermore, in some embodiments of this application, the chitin screening culture medium includes: colloidal chitin, Na2HPO4, KH2PO4, NaCl, NH4Cl, MgSO4•7H2O, agar, and water.

[0045] Furthermore, in some embodiments of this application, the LB culture medium includes: yeast extract, NaCl, peptone, and agar.

[0046] For example, in some embodiments of this application, the LB culture medium comprises: 0.5% yeast extract, 1% NaCl, 1% peptone, 2% agar, pH 7.0, sterilized at 121°C for 20 min.

[0047] Furthermore, in some embodiments of this application, the PDA culture medium includes: potato, agar, glucose, and water.

[0048] For example, in some embodiments of this application, the PDA culture medium comprises: 200 g of potato, 2% agar, 2% glucose, 1000 mL of distilled water, natural pH, sterilized at 121 °C for 20 min.

[0049] Some embodiments of this application provide a shrimp shell fermentation liquid, which is prepared using the preparation method provided in any of the foregoing embodiments.

[0050] Applying shrimp shell fermentation liquid increases the content of available phosphorus, available potassium, and organic matter in paddy soil, and significantly increases the content of alkaline nitrogen, thereby increasing the nutrient content in paddy soil, providing nutrients for rice, and promoting the growth of rice plant height, stem thickness, stems, leaves, and roots.

[0051] The features and performance of this application will be further described in detail below with reference to embodiments: Example 1 A method for preparing shrimp shell fermentation broth is provided, comprising the following steps: 1. Soil samples Wash the fresh shrimp shells and bury them 15 cm deep in paddy soil. After 15 days, dig out and collect the remaining shrimp shells and soil samples.

[0052] 2. Preparation of chitin Preparation of colloidal chitin: Weigh 25 g of finely powdered chitin and add 250 cm³ of water. 3 85% phosphoric acid was swollen at 4°C for 24 hours. Distilled water was added and stirred until the colloid precipitated. After chitin was precipitated, the mixture was centrifuged and washed with distilled water until neutral. The final concentration was adjusted to 2% with PBS at pH 6.0 and stored at 4°C.

[0053] 3. Culture medium (1) Screening medium: 200 mL of 2% colloidal chitin, 2 g of Na2HPO4, 1 g of KH2PO4, 0.5 g of NaCl, 1 g of NH4Cl, 0.5 g of MgSO4•7H2O, 12 g of agar, bring the volume to 1 L, and sterilize at 121 °C for 20 min.

[0054] (2) Seed culture medium: 4 g glucose, 0.7 g KH2PO4, 0.3 g K2HPO4, 0.5 g MgSO4•7H2O, 2 g yeast powder, 2 g peptone, pH 7.0, sterilized at 121 ℃ for 20 min.

[0055] (3) Potato culture medium (PDA): 200 g potato, 2% agar, 2% glucose, 1000 mL distilled water, natural pH, sterilized at 121 ℃ for 20 min.

[0056] Potato culture medium (PDB): 200 g potatoes, 2% glucose, 1000 mL distilled water, natural pH, sterilized at 121 ℃ for 20 min.

[0057] (4) LB medium: yeast extract 0.5%, NaCl 1%, peptone 1%, agar 2%, pH 7.0, sterilized at 121 ℃ for 20 min.

[0058] (5) Preservation of culture medium: Sterilize PDA culture medium at 121 ℃ for 20 min, and cool it to about 60 ℃ for use on slant.

[0059] (6) Fermentation medium (PDB): 200 g potato, 2% glucose, 1000 mL distilled water, natural pH, sterilized at 121 °C for 20 min.

[0060] 4. Isolation and purification of chitinase-producing strains (1) Separation Weigh 0.1 g of soil sample and place it in a centrifuge tube containing 0.9 mL of sterile water to prepare 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 10 -8 Soil solutions of different dilutions. Take 0.1 mL of the solution and spread it onto chitin selection medium. After spreading, open the plate lid, blow off the surface moisture, seal the plate, invert it, and incubate at 28 ℃ for 3 days.

[0061] (2) Separation, purification and screening Purification: Colonies that produce a clear zone are picked from the first diluted plate and inoculated onto PDA medium for four-zone streak purification. The plates are then incubated at 30 °C for 72 h to obtain pure colony plates.

[0062] Secondary screening: Use an inoculation needle to pick up and inoculate onto chitin medium. Select strains with a larger clear zone diameter than colony diameter from the pure colony plates of the second screening and inoculate them into 50 mL of LB seed medium. After culturing for 12 h, inoculate them into 100 mL of chitin fermentation medium and scale up the culture at 30 ℃.

[0063] (3) Observation of the transparent zone The plate clear zone method is a highly efficient method for screening chitinous bacteria. It involves adding poorly soluble nutrients that can be utilized by microorganisms to a solid culture medium, creating a turbid and opaque background. A clear zone will then form near the colony to be screened, and the size of the clear zone reflects the colony's ability to degrade this substance.

[0064] 5. Chitinase activity assay Chitinase hydrolyzes the β-1,4-glycosidic bonds of chitin to generate reducing sugars. Chitinase activity was determined using the 3,5-dinitrosalicylic acid (DNS) method, with N-acetyl-D-glucosamine as the substrate. Absorbance was measured at 530 nm to establish a standard curve. Several strains with relatively large diameters were selected, and their enzyme activity was determined using the DNS method with colloidal chitin as the liquid culture medium. The enzyme activities were compared, and strains with higher enzyme activity were further selected to investigate their ability to degrade shrimp shells.

[0065] (1) Preparation of DNS (3,5-dinitrosalicylic acid) reagent Dissolve 45.5 g of potassium sodium tartrate in 125 mL of hot distilled water. Then add 1.575 g of 3,5-dinitrosalicylic acid, 4.2 g of sodium hydroxide, 1.25 g of phenol, and 1.25 g of sodium sulfite in sequence. Stir until completely dissolved (heating can be done to below 50°C). After cooling, dilute to 250 mL with distilled water. Store in the dark and let stand at room temperature for one week before use.

[0066] (2) Determination of NAG (N-acetyl-α-D-glucosamine) standard curve The principle of the 3,5-dinitrosalicylic acid (DNS) method for determining reducing sugars: 3,5-dinitrosalicylic acid reacts with reducing sugars to form a brownish-red amino compound. This compound exhibits a brownish-red color, and within a certain range, the degree of color development is directly proportional to the content of reducing sugars. The absorbance is measured using a spectrophotometer. The content of reducing sugars in this system was measured using the DNS method, with the chitin monomer NAG as a standard, and the absorbance was measured at 530 nm. A standard solution of N-acetyl-α-D-glucosamine was prepared with a concentration of 5 μmol / mL. N-acetyl-α-D-glucosamine was first dried to constant weight at 90 °C, then 5 mmol was weighed, dissolved in an appropriate amount of distilled water, and transferred to a 100 mL volumetric flask for dilution. The solution was stored at 4 °C.

[0067] Table 1. Preparation of the standard curve for N-acetyl-α-D-glucosamine

[0068] (3) Methods for determining chitinase activity The isolated and purified strains were inoculated into seed culture medium. Each strain's seed culture was inoculated into 250 mL of chitin liquid culture medium at a 5% inoculation rate and cultured on a shaker at 180 r / min and 28 ℃ for 4 days. Chitinase activity was then measured.

[0069] Take 5 mL of culture medium from a shake flask and centrifuge at 6000 r / min at 4 ℃ for 10 min. Take 0.5 mL of the supernatant and add 1 mL of PBS buffer and 0.5 mL of 2% colloidal chitin. Incubate at 37 ℃ with shaking for 30 min, then in a boiling water bath for 10 min. Take 1 mL of the mixture and centrifuge at 4000 r / min for 5 min. Add 0.5 mL of DNS reagent to terminate the reaction. Incubate in a boiling water bath for 5 min and immediately cool. Make up to 25 mL with distilled water. Use distilled water as a blank control and measure the color at 530 nm.

[0070] (4) N-acetyl-α-D-glucosamine standard curve The standard curve for N-acetyl-α-D-glucosamine is shown in the figure. Figure 1 .

[0071] The chitinase activity unit is defined as the amount of enzyme required to convert colloidal chitin into 1 μmol of NAG per minute at 37 °C in a phosphate buffer environment at pH 7.0. It is represented by U.

[0072] Chitinase activity (U / mL) = (OD530 nm - 0.0268) × 5 / 0.0183 × 30 × 5 In the formula: 5 is the NAG concentration, 30 is the reaction time, and 0.5 is the volume of enzyme solution participating in the reaction (mL).

[0073] This application screened a fungus capable of degrading chitin, with an enzyme activity as high as 0.088 U / mL. It was identified as *Harzianum sp.*

[0074] (5) Results of strain screening: Table 2

[0075] Three colonies with clear zones were obtained from the initial screening. The enzyme activity was determined by the DNS method, and the strain with the highest enzyme activity, 4-9, was selected and named ZR for further observation.

[0076] The clear zone formed by strain ZR on chitin plates is shown in the instruction manual. Figure 2 .

[0077] By observing the spore morphology of fungus ZR, it was confirmed that it belongs to the Trichoderma genus.

[0078] (5) Molecular biological identification of chitin-degrading bacteria Using ZR as a template, and ITS1 and ITS4, EF-2 and EF-728F, and RPB2-4F and RPB2-7cR as primers, the PCR system was configured in 40 μL. PCR amplification was performed according to Table 3, with a total of 38 cycles. The specific program is shown in Table 4. The products were then subjected to agarose gel electrophoresis. The PCR products containing the target fragment were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were uploaded to the BLAST database of the National Center for Biotechnology Information (NCBI) for 18S rDNA sequence analysis to determine the phylogenetic relationship of strain ZR.

[0079] Table 3

[0080] Table 4

[0081] The results of the identification are as follows: Based on the 18S rDNA sequence of strain ZR, a phylogenetic tree was constructed by comparing the sequencing results using BLAST on NCBI. The results are as follows: Figure 3 As shown, ZR and Trichoderma harzianum had the highest matching rate, with a homology of over 97%, and the identified strain ZR was identified as Trichoderma harzianum sp.

[0082] 6. Chitin-degrading bacteria ferment shrimp shells The steps for preparing shrimp shell fermentation broth are as follows: Fresh shrimp shells were purchased from Dongtou Fish Farm. The shrimp shells were dried to constant weight and then used for later use. A portion of the shells were sterilized at 121 ℃ for 20 min.

[0083] The specific steps for processing shrimp shells are as follows: Unsterilized shrimp shells with *Trichoderma harzianum*: The purified strain ZR was inoculated at a 5% inoculum into 200 mL of PDB medium and cultured on a shaker at 180 r / min and 28 ℃ for 3 days. 20 g of unsterilized shrimp shells were weighed and aseptically mixed with the fermentation broth and sealed. The resulting fermentation broth was then placed in a constant-temperature shaker and fermented at 120 r / min and 28 ℃ for 60 days. After fermentation, the solid residue was filtered through gauze, dried, and stored at 4 ℃.

[0084] Comparative Example 1 The only difference from Example 1 is the step in preparing the shrimp shell fermentation liquid, as follows: Shrimp shell sterilization and Trichoderma harzianum inoculation: The purified strain ZR was inoculated at a rate of 5% into 200 mL of PDB medium and cultured on a shaker at 180 r / min and 28 ℃ for 3 days. 20 g of sterilized shrimp shells were weighed and aseptically mixed with the fermentation broth and sealed. The resulting fermentation broth was then placed in a constant-temperature shaker and fermented at 120 r / min and 28 ℃ for 60 days. After fermentation, the solid residue was filtered through gauze, dried, and stored at 4 ℃.

[0085] Comparative Example 2 The only difference from Example 1 is the step in preparing the shrimp shell fermentation liquid, as follows: Sterilized shrimp shells without Trichoderma harzianum: Weigh 20 g of sterilized shrimp shells, and under aseptic conditions, mix 200 mL of PDB medium with 20 g of sterilized shrimp shells and seal. Place the resulting fermentation broth in a constant temperature shaker and ferment at 120 r / min and 28 ℃ for 60 days. After fermentation, filter the solid residue with gauze, dry it for later use, and collect the fermentation broth for storage at 4 ℃.

[0086] Comparative Example 3 The only difference from Example 1 is the step in preparing the shrimp shell fermentation liquid, as follows: Unsterilized shrimp shells without Trichoderma harzianum: Weigh 20 g of sterilized shrimp shells, and under aseptic conditions, mix 200 mL of PDB medium with 20 g of unsterilized shrimp shells and seal. Place the resulting fermentation broth in a constant temperature shaker at 120 r / min and 28 ℃ for 60 days. After fermentation, filter the solid residue with gauze, dry it for later use, and collect the fermentation broth for storage at 4 ℃.

[0087] The shrimp shell fermentation broths prepared in the above embodiments and comparative examples were tested: 1. Determination of shrimp shell degradation rate Take 0.2 g of fermented shrimp shells, dry them, weigh them, and record the weight of the dried shrimp shells. Calculate the degradation rate of the fermented shrimp shells using the following formula: X(%) = (m0 - m) / m0 × 100% In the formula: X is the shrimp shell degradation rate, m0 is the mass of dried shrimp shells without fermentation, and m is the mass of dried shrimp shells after fermentation.

[0088] Test results are available Figure 4 .

[0089] according to Figure 4 The degradation rate of shrimp shell fermentation broth can be determined as follows: Comparative Example 2 (shrimp shells sterilized without Trichoderma harzianum) showed almost no degradation. Comparative Example 1 (shrimp shells sterilized with Trichoderma harzianum) showed partial decomposition of the shrimp shells after 60 days of fermentation. Example 1 (shrimp shells unsterilized with Trichoderma harzianum) and Comparative Example 3 (shrimp shells unsterilized without Trichoderma harzianum) showed almost complete decomposition of the shrimp shells after 60 days of fermentation, leaving only solid powder, indicating a high degradation efficiency. This is because a single microbial species can only act on one enzyme cleavage site, while there are seven known chitin cleavage sites. Comparing Comparative Example 1 and Example 1, the degradation efficiency of adding Trichoderma harzianum alone is clearly inferior to the synergistic effect of multiple microbial species. However, comparing Example 1 and Comparative Example 3 shows that adding Trichoderma harzianum increases the shrimp shell degradation rate, demonstrating a significant synergistic effect of multiple strains.

[0090] Therefore, Example 1 (unsterilized shrimp shells with Trichoderma harzianum) showed the best shrimp shell degradation rate. The treatment group with unsterilized shrimp shells and Trichoderma harzianum exhibited the highest shrimp shell degradation rate, reaching 48%.

[0091] 2. Composition analysis of shrimp shell fermentation liquid The nitrogen, phosphorus, and potassium contents of the fermentation broth were simultaneously determined using the sulfuric acid-perchloric acid digestion method, and the results are shown in the table below.

[0092] Table 5

[0093] The experimental data in Table 5 above show that: Example 1 (shrimp shells unsterilized with Trichoderma harzianum) had the highest organic matter and nitrogen, phosphorus and potassium content, followed by Comparative Example 3 (shrimp shells unsterilized without Trichoderma harzianum), Comparative Example 1 (shrimp shells sterilized with Trichoderma harzianum), and Comparative Example 2 (shrimp shells sterilized without Trichoderma harzianum). Based on the above results, the treatment with higher degradation efficiency also had higher organic matter and nitrogen, phosphorus and potassium content.

[0094] Therefore, Example 1 (unsterilized shrimp shells with Trichoderma harzianum) also had the highest nutrient content, with organic matter measured at 185 g / L and N+P2O5+K2O at 28 g / L.

[0095] 3. Effects of shrimp shell fermentation liquid on rice growth (1) Experimental design and methods: After the rice seeds sprouted, they were sown in seedling trays. Seedlings were transplanted when they reached the four-leaf stage, and seedlings with similar growth were selected for transplanting. Pot experiments were conducted in a greenhouse at Guangdong Ocean University (temperature 28±3 ℃, relative humidity 18.2%). Each pot contained 1 kg of air-dried and sieved soil, and harvesting was carried out on day 30. Shrimp shell fermentation liquid was applied near the rice roots, 4 mL every five days, with five parallel trials per treatment group. Basic physicochemical properties of the soil were determined using conventional analysis methods.

[0096] The physical and chemical properties of the test soil are as follows: Table 6 Basic physicochemical properties of the tested soils

[0097] Five treatment groups were set up using the prepared shrimp shell fermentation broth, and five parallel experiments were set up in each treatment group. The treatment groups were CK (distilled water), Comparative Example 1 (shrimp shell sterilization with Trichoderma harzianum), Comparative Example 2 (shrimp shell sterilization without Trichoderma harzianum), Example 1 (shrimp shell non-sterilized with Trichoderma harzianum), and Comparative Example 3 (shrimp shell non-sterilized without Trichoderma harzianum).

[0098] Experimental data were processed using Microsoft Excel 2021 software. Duncan's method in SPSS 21.0 statistical software was used to perform significance analysis between treatment groups at the P < 0.05 level, and the results are expressed using different letters. The experimental results are shown in Tables 7 and 8.

[0099] (2) The effects of shrimp shell fermentation liquid on rice growth are shown in Table 7.

[0100] Table 7 Effects of different treatments on rice growth indicators

[0101] Note: Different lowercase letters after the data in the same column indicate significant differences between different groups (P < 0.05). Table 7 shows the effects of different treatments on plant height, stem diameter, aboveground fresh weight, and underground fresh weight of rice as follows: Plant height comparison: Example 1 > Comparative Example 3 > Comparative Example 1 > Comparative Example 2 > CK. The plant height of the Example 1 and Comparative Example 3 treatment groups was significantly higher than the control group (CK), increasing by 3.71 to 4 times. Stem diameter comparison: Example 1 > Comparative Example 3 > Comparative Example 1 > Comparative Example 2 > CK. The stem diameter of the Example 1 treatment group was the largest, significantly higher than the other treatment groups, indicating that the Example 1 treatment group had the strongest promoting effect on stem diameter. Aboveground fresh weight comparison: Example 1 > Comparative Example 1 > Comparative Example 3 > Comparative Example 2 > CK, indicating that the Example 1 and Comparative Example 1 treatment groups had a significant promoting effect on rice root growth. In summary, the treatment in Example 1 (unsterilized shrimp shells plus Trichoderma harzianum) showed the best growth effect in all four indicators: plant height, stem diameter, above-ground fresh weight, and below-ground fresh weight, indicating that the treatment in Example 1 (unsterilized shrimp shells plus Trichoderma harzianum) had the most significant growth-promoting effect on rice.

[0102] (3) The physical and chemical properties of paddy soil after applying shrimp shell fermentation liquid are shown in Table 8.

[0103] Table 8. Physicochemical properties of paddy soil

[0104] Table 8 shows that, compared with the control group (CK), the available phosphorus content was in the order of Example 1 > Comparative Example 3 > Comparative Example 1 > Comparative Example 2 > CK; the available potassium content was in the order of Example 1 > Comparative Example 3 > Comparative Example 1 > Comparative Example 2 > CK; the organic matter content was in the order of Example 1 > Comparative Example 2 > CK; and the alkaline nitrogen content was in the order of Example 1 > Comparative Example 3 > Comparative Example 1 > Comparative Example 2 > CK. Compared with the control group, the shrimp shell fermentation liquid treatment group increased the content of available phosphorus, available potassium, and organic matter in the soil, and significantly increased the content of alkaline nitrogen, thus increasing the nutrient content in the soil.

[0105] In summary, Example 1 (unsterilized shrimp shells plus Trichoderma harzianum) showed the best effect on rice growth. Applying this shrimp shell fermentation liquid promoted rice growth and provided nutrients. It promoted plant height, stem diameter, stem and leaf growth, and root development. Furthermore, it increased the content of available phosphorus, available potassium, and organic matter in the soil, and significantly improved the content of alkaline nitrogen, thus increasing the overall nutrient content of the soil.

[0106] In summary, compared with the various comparative examples, the treatment group of Example 1 (unsterilized shrimp shells plus Trichoderma harzianum) showed the best results in terms of shrimp shell degradation rate and rice growth.

[0107] The alkaline nitrogen content of the original coastal saline soil is low, indicating that the application of shrimp shell fermentation liquid promoted nitrogen absorption by rice and thus promoted rice growth. This is likely because the nitrogen in the shrimp shell fermentation liquid mainly originates from organic nitrogen compounds such as ammonia and amino acids generated after protein decomposition. These nitrogens usually exist in the form of ammonia nitrogen, which is easily absorbed by plants. Compared to most commercially available organic fertilizers, the nitrogen mineralization process in the shrimp shell fermentation liquid of Example 1 is more rapid, which may result in a less lasting effect on increasing soil nitrogen content and difficulty in effectively accumulating nitrogen nutrients in the soil. The organic matter in organic fertilizers accelerates the release of organic phosphorus during decomposition, effectively increasing the phosphorus content in the soil.

[0108] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for preparing shrimp shell fermentation liquid, characterized in that, include: Fresh shrimp shells are dried to obtain dried shrimp shells; Fermentation strains of the genus Trichoderma were inoculated into the fermentation medium and cultured. The fermented seed liquid was obtained; The dried shrimp shells are not sterilized and are mixed with the fermentation seed liquid and then sealed for fermentation.

2. The method for preparing shrimp shell fermentation liquid according to claim 1, characterized in that, Based on the total volume of the culture medium, 5-10% of the Trichoderma fermentation strains were inoculated into the fermentation medium by volume percentage and cultured at 150-200 r / min and 25-30 ℃ for 2-5 days to obtain the fermentation seed liquid; Optionally, the dried shrimp shells are mixed with the fermented seed liquid without sterilization and sealed, and then fermented at 25~30℃ and 110~140 r / min for 45~65 days; Optionally, mixing the dried shrimp shells with the fermented seed liquid includes: The dried shrimp shells and the fermentation culture medium are mixed in a ratio of (15~25 g):(100~300 mL).

3. The method for preparing shrimp shell fermentation liquid according to claim 1, characterized in that, The fermentation medium comprises, by weight, 150-250 parts potato, 15-25 parts glucose and 900-1100 parts water.

4. The method for preparing shrimp shell fermentation liquid according to claim 1, characterized in that, The fermentation strain of the Trichoderma genus is Trichoderma harzianum.

5. The method for preparing shrimp shell fermentation liquid according to claim 1, characterized in that, The enzyme activity of the Trichoderma fermentation strains is 0.056~0.088 U / mL.

6. The method for preparing shrimp shell fermentation liquid according to any one of claims 1-5, characterized in that, The preparation of the fermentation seed liquid includes: Wash the fresh shrimp shells and bury them in paddy soil. Dig up and collect the remaining shrimp shells and soil samples to obtain soil samples. Weigh the soil sample and place it in sterile water to obtain a diluted soil solution; spread the soil solution onto chitin selection medium and incubate at 25-30 ℃ for 2-5 days to obtain a culture; isolate the colonies from the culture and inoculate the colonies into PDA medium for purification culture to obtain pure colonies; select strains from the pure colonies and inoculate them into LB medium for culture to obtain cultured bacterial strains; inoculate the cultured bacterial strains into chitin fermentation medium for large-scale culture.

7. The method for preparing shrimp shell fermentation liquid according to claim 6, characterized in that, The chitin screening medium comprises: colloidal chitin, Na2HPO4, KH2PO4, NaCl, NH4Cl, MgSO4•7H2O, agar, and water.

8. The method for preparing shrimp shell fermentation liquid according to claim 6, characterized in that, The LB medium comprises: yeast extract, NaCl, peptone, and agar.

9. The method for preparing shrimp shell fermentation liquid according to claim 6, characterized in that, The PDA culture medium includes: potato, agar, glucose, and water.

10. A shrimp shell fermentation liquid, characterized in that, It is prepared by the preparation method according to any one of claims 1-9.

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