Method for detecting content of chitin in hermetia illucens sand, detection kit and application

Abstract

The invention belongs to the technical field of biological substance detection, and discloses a method for detecting the content of chitin in hermetia illucens sand, which comprises the following steps: (1) adding hermetia illucens sand dry powder to be detected into a buffer solution containing a surfactant, and crushing to obtain a sample suspension to be detected; (2) adding a working solution containing a fluorescent probe capable of specifically binding with chitin into the suspension of the sample to be detected, and incubating in a dark place; (3) after the incubation is finished, detecting the fluorescence intensity value of the reaction system under the excitation wavelength and emission wavelength of the fluorescent probe; and (4) calculating the content of chitin in the sample according to the fluorescence intensity-chitin concentration standard working curve. According to the detection method, the time is shortened from traditional 1-2 days to within 30 minutes, the detection efficiency is greatly improved, and the method is suitable for rapid screening of large-batch samples.

Description

Technical Field

[0001] This invention belongs to the field of biological material detection technology, and in particular relates to a method, a detection kit, and applications for detecting chitin content in black soldier fly sand. Background Technology

[0002] Black soldier fly larvae, as resource-efficient insects, demonstrate significant potential in processing organic waste. They transform organic waste into high-value insect protein and frass. Frass is rich in organic matter, nitrogen, phosphorus, potassium, and chitin, an important bioactive substance. Chitin and its derivatives can be used as biostimulants in agriculture, inducing disease resistance in plants, promoting plant growth, and improving soil. Therefore, accurate and rapid determination of chitin content in black soldier fly frass is crucial for quality control, grading, and the development of functional products.

[0003] The chitin in the insect sand mainly originates from the molted residue of black soldier fly larvae during their growth process. This chitin has a natural high affinity for the chitin-binding proteins of the black soldier fly itself. Based on bioinformatics analysis, the black soldier fly genome contains multiple chitin-binding module (CBM) gene families, especially the CBM14 family. These domains are specifically responsible for recognizing and binding chitin molecules.

[0004] Currently, the conventional methods for determining chitin content are mainly acid hydrolysis-gravimetric methods or alkali hydrolysis-gravimetric methods. The main principle of these methods is to use strong acids or bases to hydrolyze and remove other components in the sample (such as proteins, fats, ash, etc.), leaving chitin as the residue, which is then quantified by weighing. These methods have significant drawbacks: 1) They are time-consuming, typically requiring 24-48 hours for the entire process; 2) They are cumbersome, involving multiple high-temperature and strong acid / alkali treatments, posing risks to operators and the environment; 3) They are highly destructive to samples, making them unsuitable for in-situ or non-destructive testing; 4) They have low sensitivity and precision, especially with larger errors at low sample concentrations; 5) They are susceptible to interference factors, as components such as proteins and cellulose in the sample may affect the accuracy of the results.

[0005] In recent years, methods for detecting chitin based on spectrophotometry and high-performance liquid chromatography (HPLC) have also been reported. However, these methods either require complex sample pretreatment or expensive equipment and professional technicians, making them difficult to promote and apply in the production line.

[0006] Therefore, there is an urgent need to develop a new method for the quantitative detection of chitin in black soldier fly larvae that is easy to operate, fast to detect, highly sensitive, and low in cost. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to overcome the deficiencies and defects mentioned in the background art above, and to provide a method, test kit and application for detecting chitin content in black soldier fly sand.

[0008] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows: A method for detecting chitin content in black soldier fly sand includes the following steps: (1) Add the dried black soldier fly sand powder to be tested into a buffer solution containing surfactant, and crush it to obtain a suspension of the sample to be tested; (2) Add a working solution containing a fluorescent probe that can specifically bind to chitin to the sample suspension to be tested, and incubate in the dark; (3) After incubation, the fluorescence intensity of the reaction system was detected at the excitation and emission wavelengths of the fluorescent probe; (4) Calculate the chitin content in the sample based on the standard working curve of fluorescence intensity-chitin concentration.

[0009] The preferred method for detecting chitin content in black soldier fly sand, as described above, includes the following steps in step (4): using a series of chitin standards with known concentrations, employing the same processing steps as steps (1) to (3), measuring their fluorescence intensity, plotting a curve with fluorescence intensity as the ordinate and chitin concentration as the abscissa, and a linear correlation coefficient R² ≥ 0.995.

[0010] In the above method for detecting chitin content in black soldier fly sand, preferably, in step (1), the surfactant is at least one of sodium dodecyl sulfate (PBS), Triton X-100 or Tween-20, and the mass-volume concentration of the sample to be tested is 0.1%-2%.

[0011] In the above method for detecting chitin content in black soldier fly sand, preferably, in step (1), the crushing treatment is ultrasonic treatment, the ultrasonic power is 100-300W, the treatment time is 3-10 minutes, and the temperature is 4-25℃.

[0012] In the above-described method for detecting chitin content in black soldier fly sand, preferably, in step (2), the fluorescent probe is a chitin binding domain (CBD) labeled with a fluorescent group, and the fluorescent group is one of green fluorescent protein (GFP), fluorescein isothiocyanate (FITC), rhodamine, or Alexa Fluor series dyes.

[0013] In the above-described method for detecting chitin content in black soldier fly sand, preferably, in step (2), the chitin binding domain (CBD) labeled with a fluorescent group is prepared by fusing the CBM14 chitin binding domain derived from the black soldier fly's own genome with a fluorescent protein (such as GFP) to form a recombinant fluorescent probe (CBD-GFP). Compared with traditional general probes such as Calcofluor White, this self-derived probe has high specificity and affinity for chitin in black soldier fly sand. The probe specifically binds to the N-acetylglucosamine polymer chain of chitin via non-covalent bonds, and is incubated in the dark at a preset temperature and pH to allow the probe to fully bind with chitin.

[0014] In the above-described method for detecting chitin content in black soldier fly sand, preferably, the amino acid sequence of the chitin-binding domain of CBM14 is as shown in any one of SEQ ID NO:1-3, or a variant sequence that has at least 80% similarity to these sequences and maintains chitin-binding activity.

[0015] In the above-described method for detecting chitin content in black soldier fly sand, preferably, the chitin-binding protein comprises a fusion protein containing a single, double, or triple CBM14 domain, and multiple CBM14 domains are linked together by a flexible linker peptide to enhance binding affinity.

[0016] In the above-mentioned method for detecting chitin content in black soldier fly sand, preferably, in step (2), the conditions for incubation in the dark include: temperature 20-37℃, pH value 6.0-8.0, and incubation time 5-30 minutes.

[0017] In the above-mentioned method for detecting chitin content in black soldier fly sand, preferably, in step (3), the device for detecting the fluorescence intensity value of the reaction system includes any one of a fluorescence microplate reader, a fluorescence microscope, a flow cytometer, or a portable fluorometer.

[0018] In the above-described method for detecting chitin content in black soldier fly sand, preferably, unbound free probes are removed by centrifugation and washing after incubation to reduce background signal.

[0019] The method for detecting chitin content in black soldier fly sand described above preferably includes a quality control step: verifying the accuracy and reliability of the detection method by adding a chitin standard of known concentration as a positive control and a chitin-free matrix as a negative control.

[0020] Based on a general inventive concept, the present invention also provides a detection kit for the above-described method of detecting chitin content in black soldier fly sand, comprising the following components: Component A: Chitin concentration gradient standards; Component B: A fluorescent probe solution that can specifically bind to chitin; Component C: Sample pretreatment buffer containing surfactant; Component D: Positive control and negative control.

[0021] In the above-mentioned detection kit, preferably, the concentration of the fluorescent probe solution in component B is 1-20 μg / mL.

[0022] Preferably, the above-mentioned test kit also includes a detailed instruction manual, which describes the preparation method of the standard working curve, sample processing steps, detection condition settings, and result calculation methods.

[0023] Based on a general inventive concept, the present invention also provides a method for detecting chitin content in black soldier fly sand as described above, or an application of the detection kit as described above in the quality control of fly sand products, the development of biostimulants, or the evaluation of organic fertilizers.

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention uses the CBM14 chitin-binding domain derived from the black soldier fly's own genome and fuses it with a fluorescent protein (such as GFP) to prepare a recombinant fluorescent probe (CBD-GFP). This self-derived probe has high specificity and affinity for chitin in black soldier fly sand. The probe specifically binds to the N-acetylglucosamine polymer chain of chitin through non-covalent bonds, realizing a rapid and efficient detection process, shortening the traditional 1-2 days to less than 30 minutes, greatly improving detection efficiency, and is suitable for rapid screening of large batches of samples.

[0025] (2) The method for detecting chitin content in black soldier fly sand of the present invention is simple to operate, does not require dangerous operations such as high temperature, strong acid and strong alkali, has a simple process, low requirements for personnel and equipment, and is easy to promote and apply in the production line.

[0026] (3) The method of the present invention for detecting chitin content in black soldier fly sand has extremely high sensitivity and the detection limit can reach the microgram level, which is far superior to the traditional gravimetric method.

[0027] (4) The method of detecting chitin content in black soldier fly sand of the present invention uses a fluorescent probe that can specifically bind to chitin, effectively eliminating the interference of other organic matter in the fly sand.

[0028] (5) The method for detecting chitin content in black soldier fly sand of the present invention is easy to standardize and commercialize. The developed standardized reagent kit is easy to promote and apply, and provides a technical basis for establishing industry quality standards for fly sand products.

[0029] (6) The method of the present invention for detecting chitin content in black soldier fly sand is low in cost and suitable for large-scale application compared with expensive methods such as HPLC.

[0030] (7) The method of the present invention for detecting chitin content in black soldier fly sand can process multiple samples at the same time, which is suitable for industrial quality control needs.

[0031] (8) The method and kit for detecting chitin content in black soldier fly sand of the present invention can be used to rapidly determine the chitin content in commercial fly sand products, providing technical support for product grading, pricing and quality certification; it can also be used to monitor changes in chitin content in biostimulant products made from fly sand, guiding product formulation optimization and process improvement; and it can also be used for organic fertilizer evaluation. Detailed Implementation

[0032] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0033] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0034] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0035] The method for detecting chitin content in black soldier fly larvae sand according to the present invention includes the following steps: (1) Sample pretreatment: Take dried black soldier fly sand powder, add buffer solution containing surfactant, and break it up by physical means such as ultrasound. The purpose of this step is to break the complex structure of the fly sand, fully expose the chitin encapsulated inside, and at the same time allow some soluble impurities to enter the solution, which is convenient for subsequent cleaning.

[0036] (2) Fluorescent labeling: A fluorescently labeled chitin-binding protein (CBD-GFP) that specifically binds to chitin is added to the treated sample. This type of probe can specifically bind to the N-acetylglucosamine polymer chain of chitin via non-covalent bonds. Then, the sample is incubated in the dark at a preset temperature and pH to allow the probe to fully bind to chitin.

[0037] (3) Signal detection: After incubation, unbound free probes were removed by centrifugation and washing to reduce background signal. Then, the fluorescence intensity of the reaction system was detected using a fluorescence microplate reader or portable fluorometer at the optimal excitation and emission wavelengths of the probe.

[0038] (4) Content Calculation: Before sample testing, a series of commercial chitin standards with known concentrations are used, and the same pretreatment steps as for the sample are followed to measure their fluorescence intensity and plot a standard working curve of fluorescence intensity versus chitin concentration. The mass fraction of chitin in the sample can be calculated by substituting the fluorescence intensity value of the sample into the regression equation of the standard curve.

[0039] Example 1: Preparation process of recombinant fluorescent probe (CBD-GFP) This embodiment aims to produce a highly specific fluorescent probe derived from black soldier flies (… Hermeticism illuminating It is formed by fusing the chitin-binding domain (CBD) of GFP with green fluorescent protein (GFP), and the specific preparation process includes: 1. Gene design and cloning Determination of target gene sequence: Using bioinformatics methods, the CBM14 (Carbohydrate-Binding Module family 14) chitin-binding domain gene with high affinity was screened from the black soldier fly genome. Its typical amino acid sequence is shown in SEQ ID NO:1-SEQ ID NO:3.

[0040] SEQ ID NO:1 CGTSLNGLFADPSNCRKYIYCIDGVAHSMTCLSGFYFNPLTRICGPNIPSGC SEQ ID NO:2 CATSMNGVFADTTNCQKYFTCMDGTAYTMTCPNNYYFNPLTRLCGSNI SEQ ID NO:3 CPTGVNTMLPDPNGDCQRYVLCLNGQASYQTCPNGFYFQPTTGYCGPNMPNSC.

[0041] Domain design: Single domain: Directly select sequences such as SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0042] Multiple domains: By tandemly connecting 2-3 sequences such as those in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 via a flexible linker, the binding affinity of the probe to chitin is enhanced.

[0043] In this embodiment, the sequence of SEQ ID NO:1 is used to form a single structural domain design.

[0044] Construction of expression carriers: The synthesized CBM14 gene sequence was ligated in vitro with the green fluorescent protein (GFP) gene sequence.

[0045] The ligated fusion gene (CBD-GFP) was cloned into the high-efficiency protein expression vector pET-28a(+). This vector typically adds a His tag to the fusion protein to facilitate subsequent purification.

[0046] 2. Transformation and Expression Host transformation: The constructed recombinant expression plasmid (e.g., pET-28a-CBD-GFP) is transformed into a suitable protein expression host bacterium, *Escherichia coli*. E. coli In BL21(DE3) strain.

[0047] Induced expression: The transformed engineered bacteria were inoculated into a suitable culture medium (LB medium containing kanamycin) and cultured at 37°C with shaking until the logarithmic growth phase (OD600 ≈ 0.6-0.8).

[0048] Add the inducer IPTG (isopropyl-β-D-thiogalactoside) to a final concentration of 0.1-0.5 mM.

[0049] Continue culturing at a lower temperature (16-25℃) for 12-16 hours to promote proper folding and soluble expression of the fusion protein.

[0050] 3. Protein purification and identification Cell disruption: Bacterial cells were collected by centrifugation, resuspended in buffer, and then lysed by sonication to release intracellular fusion proteins.

[0051] Affinity chromatography purification: Centrifuge the cell lysate at high speed and collect the supernatant.

[0052] Purification was performed using immobilized metal ion affinity chromatography (IMAC). The supernatant was passed through a buffer pre-treated with nickel ions (Ni... 2 + A balanced chromatography column allows the CBD-GFP protein to bind specifically to the column.

[0053] After removing impurities with washing buffer, the target protein is eluted with a high concentration of imidazole solution, and the eluent is collected.

[0054] Purity and concentration determination: The purity and molecular weight of the purified protein were determined by SDS-PAGE gel electrophoresis. The expected molecular weight of the single CBM14-GFP fusion protein was approximately 34.2 kDa. The concentration of the purified protein was determined using the BCA method or the Bradford method.

[0055] 4. Freeze-drying and storage Buffer replacement: The purified high-concentration protein solution is replaced with a buffer suitable for long-term storage by dialysis or ultrafiltration.

[0056] Freeze-drying: The protein solution is freeze-dried to produce freeze-dried powder.

[0057] Storage: The lyophilized powder can be stored stably at -20°C for extended periods. Before use, dissolve the powder in a buffer solution (such as PBS) to the working concentration (e.g., 1-10 μM or 1-20 μg / mL), and store at 4°C after dissolution.

[0058] By following the above steps, a high-purity, high-activity recombinant fluorescent probe (CBD-GFP) can be prepared for rapid and specific detection of chitin content in black soldier fly sand.

[0059] Example 2: Detection of chitin content in black soldier fly based on recombinant fluorescent probe (CBD-GFP) A method for detecting chitin content in black soldier fly sand according to the present invention includes the following steps: (1) Preparation of standard curve: a) Accurately weigh 10.0 mg of commercially pure chitin standard (derived from fungi, degree of deacetylation >90%), dissolve it in 5% (w / v) lithium chloride-N,N-dimethylacetamide (LiCl / DMAc) solution, and dilute to 10 mL to prepare a stock solution of 1.0 mg / mL. b) Dilute the stock solution with buffer (1% PBS) to prepare a series of standard solutions with concentrations of 100, 50, 25, 12.5, 6.25, and 0 μg / mL; the 0 μg / mL standard solutions are pure buffer solutions and do not introduce chitin. c) Preparation of CBD-GFP working solution: Take the CBD-GFP lyophilized powder prepared in Example 1, dissolve and dilute it with PBS buffer to a working concentration of 5 μM.

[0060] d) Take 100 μL of standard series solutions of different concentrations, add 100 μL of LBD-GFP working solution (pH=7.0) to each solution, and react at room temperature in the dark for 10 minutes.

[0061] e) Using a fluorescence microplate reader, the fluorescence intensity (RFU) was measured at an excitation wavelength of 488 nm and an emission wavelength of 509 nm. Specific data are shown in Table 1.

[0062] f) A standard curve was plotted with fluorescence intensity as the ordinate (Y) and chitin concentration as the abscissa (X). Linear regression was performed based on the data in Table 1, yielding the regression equation Y = 80.15X + 98.7, with a correlation coefficient R² = 0.9998. The results indicate a good linear relationship between chitin concentration and fluorescence intensity within the concentration range of 0-100 μg / mL.

[0063] Table 1: Data from the determination of chitin standard curve

[0064] (2) Sample testing: a) Accurately weigh 1.0 g of dried, pulverized, and sieved black soldier fly sand sample.

[0065] b) Add the black soldier fly sand sample to 10 mL of PBS buffer containing 1% SDS, and treat it with a probe-type ultrasonic cell disruptor for 5 minutes. The ultrasonic power is 200W, the temperature is 22℃, the operation time is 3 seconds, and the pause time is 3 seconds to obtain the sample suspension.

[0066] c) Centrifuge the treated suspension at 4000 rpm for 5 minutes, discard the supernatant, and wash the precipitate twice with 1% PBS buffer to remove soluble impurities and some pigments.

[0067] d) Resuspend the washed precipitate in 10 mL of 1% PBS buffer to obtain the sample test solution.

[0068] e) Take 100 μL of sample solution, add 100 μL of CBD-GFP working solution (5 μM), pH=7.0, and react at room temperature in the dark for 10 minutes.

[0069] f) Using a fluorescence microplate reader, the fluorescence intensity (RFU) was measured at an excitation wavelength of 488 nm and an emission wavelength of 509 nm. The measured RFU was 2295.1. Substituting the measured RFU value into the standard curve equation: 2295.1 = 80.15X + 98.7, we obtained X = 27.40 μg / mL. Then, we calculated the chitin content in the insect sand: (27.40 μg / mL * 10 mL) / (1.0 g * 1000 mg / g) = 0.0274% (w / w), which is 2.74 mg / g.

[0070] Example 3: Methodological Validation 1. Precision testing: Black soldier fly sand samples from the same batch as in Example 2 were taken and measured six times using the method described in Example 2. The results are shown in Table 2.

[0071] The results showed that the RSD of the six parallel determinations was 2.01%, which is less than 5%, indicating that the detection method of the present invention has good precision and repeatability.

[0072] Table 2 Precision Test Results

[0073] 2. Accuracy test (spike recovery) Three samples of insect excrement with known chitin content (background value 2.73 mg / g) were weighed, each 1.0 g. Chitin standards of low (1.00 mg), medium (2.50 mg), and high (5.00 mg) concentrations were added respectively. The chitin content was determined according to the method in Example 1, and the recovery rate was calculated. The results are shown in Table 3.

[0074] The results showed that the spiked recovery rate ranged from 95.0% to 103.2%, with an average recovery rate of 98.6%, indicating that the method of the present invention has high accuracy and the sample matrix has little interference with the determination results.

[0075] Table 3 Results of Spiked Recovery Test

[0076] 3. Comparison with the traditional gravimetric method Three samples of insect excrement from different sources (labeled A, B, and C, respectively) were taken. Each sample was divided into two groups, and the fluorescent labeling method of Example 1 of this invention and the traditional acid hydrolysis-gravimetric method were used for detection, respectively. The results are shown in Table 4. The traditional acid hydrolysis-gravimetric method specifically includes the following steps: (1) Sample pretreatment: The black soldier fly sand samples were dried at 70°C to constant weight, then crushed using a pulverizer and passed through a standard sieve (80 mesh) to ensure the homogeneity of the samples.

[0077] (2) Deproteinization: Accurately weigh 2.0 g of dried insect sand powder and place it in an Erlenmeyer flask.

[0078] Add an alkaline solution, 1 M (mol / L) sodium hydroxide (NaOH) solution, at a solid-liquid ratio of 1:30 (i.e., 2 g of sample added to 60 mL of alkaline solution), to obtain a mixture.

[0079] The mixture was heated and stirred in a water bath at 90°C for 3 hours. The purpose of this step was to hydrolyze the protein into water-soluble amino acids or peptides.

[0080] After the reaction is complete, allow it to cool, then repeatedly centrifuge and wash the precipitate with deionized water until the pH of the supernatant becomes neutral (pH≈7).

[0081] (3) Demineralization: Transfer the deproteinized precipitate obtained in the previous step to a new beaker, add an acid solution, 1 M hydrochloric acid (HCl), and treat it at a solid-liquid ratio of about 1:30.

[0082] Then stir at room temperature for 1.5 hours. The purpose of this step is to dissolve inorganic minerals such as calcium carbonate and phosphate in the sample.

[0083] After the reaction was complete, the precipitate was repeatedly centrifuged and washed with deionized water until the pH of the supernatant returned to neutral.

[0084] (4) Decolorization and degreasing: To remove pigments and residual lipids from the sample, the precipitate obtained in the previous step was washed with 95% ethanol.

[0085] Stir at room temperature for 30 minutes, then centrifuge and discard the colored supernatant. Repeat this step twice until the precipitate turns grayish-white.

[0086] (5) Drying and weighing: The final precipitate (i.e., crude chitin) is transferred to a pre-weighed weighing dish.

[0087] Dry the residue in an oven at 105°C until constant weight is reached (i.e., the weight change is less than 0.2 mg in two consecutive weighings), and accurately weigh the final weight of the residue after drying (m_chitin).

[0088] (6) Calculate: Chitin content (%) = (m_chitin / m_sample) × 100%, where m_chitin is the weight of the final residue and m_sample is the dry weight of the starting sample.

[0089] The results show that the test method of the present invention has no significant difference from the traditional gravimetric method, with relative deviations within ±5%. However, the test time is drastically reduced from about 48 hours to at least 30 minutes, and the efficiency is improved by more than 95%, which is extremely significant.

[0090] Table 4 Comparison of detection results between the method of Embodiment 1 of the present invention and the traditional method

[0091] 4. Method stability test The stability of the method was tested under different experimental conditions (different temperatures, pH values, and reaction times). The results showed that the method was stable and reliable within the range of temperature 20-25℃, pH 6.8-7.6, and reaction time 8-15 minutes.

[0092] 5. Limit of detection and limit of quantitation testing By measuring the standard deviation of blank samples, the limit of detection (LOD) of the method was calculated to be 0.5 μg / mL and the limit of quantitation (LOQ) to be 1.5 μg / mL, which meets the quality control requirements for insect sand products.

[0093] Example 4: CBD-GFP-based detection kit This embodiment provides a detection kit for detecting chitin content in black soldier fly sand, comprising: Component A: Chitin standard (1.0 mg / mL, 1 mL).

[0094] Component B: Recombinant chitin-binding protein-green fluorescent protein fusion protein (CBD-GFP) lyophilized powder (10 μg). This fusion protein contains the CBM14 domain (sequence shown in SEQ ID NO:1) derived from the black soldier fly genome, exhibiting high specific chitin-binding activity. It should be dissolved in buffer before use. Technical characteristics of the fusion protein: molecular weight: 34.2 kDa, CBM14 domain score: 46.5 (E-value: 8.40e-15), working concentration: 1-10 μM, storage conditions: lyophilized at -20℃, thawed at 4℃ before use.

[0095] Component C: 10x sample processing buffer (containing 10% SDS, 50 mL).

[0096] Component D: Positive control sample (standard insect sand with known chitin content, 5 g).

[0097] Component E: Negative control sample (chitin-free matrix, 5 g).

[0098] Instructions: This manual details the operating steps, preparation of the standard curve, calculation methods for results, and excitation / emission wavelengths (488 nm / 509 nm). The specific instructions are the same as those for the method of detecting chitin content in black soldier fly sand, and will not be repeated here.

[0099] Users can easily and quickly complete the test by following the instructions.

[0100] Example 5: Industrial Application Verification A three-month industrial application verification was conducted at a large-scale insect sand production enterprise: 20-30 batches of insect sand samples were tested daily, and the results were compared with traditional methods. The testing time, cost, accuracy and other indicators were recorded.

[0101] The results show that the detection time is reduced by more than 95%; the detection cost is reduced by 60%; and the accuracy is comparable to traditional methods (relative deviation <5%). It is evident that the detection method of this invention greatly improves quality control efficiency and saves enterprises a lot of manpower and resources.

[0102] Example 6: Applicability verification of different types of insect sand samples The applicability of the method of the present invention to insect sand samples from different raw material sources (vegetable waste, kitchen waste, livestock and poultry manure, etc.) and different processing techniques was tested. The results showed that the method has good versatility.

[0103] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting chitin content in black soldier fly sand, characterized in that, Includes the following steps: (1) Add the dried black soldier fly sand powder to be tested into a buffer solution containing surfactant, and crush it to obtain a suspension of the sample to be tested; (2) Add a working solution containing a fluorescent probe that can specifically bind to chitin to the sample suspension to be tested, and incubate in the dark; (3) After incubation, the fluorescence intensity of the reaction system was detected at the excitation and emission wavelengths of the fluorescent probe; (4) Calculate the chitin content in the sample based on the standard working curve of fluorescence intensity-chitin concentration.

2. The method for detecting chitin content in black soldier fly sand as described in claim 1, characterized in that, In step (4), the process of drawing the standard working curve of fluorescence intensity-chitin concentration includes: using a series of chitin standards with known concentrations, adopting the same processing steps as steps (1) to (3), measuring their fluorescence intensity, drawing a curve with fluorescence intensity as the vertical axis and chitin concentration as the horizontal axis, and the linear correlation coefficient R²≥0.

995.

3. The method for detecting chitin content in black soldier fly sand as described in claim 1, characterized in that, In step (1), the surfactant is at least one of sodium dodecyl sulfate, Triton X-100 or Tween-20, and the mass-volume concentration of the sample to be tested is 0.1%-2%.

4. The method for detecting chitin content in black soldier fly sand as described in claim 1, characterized in that, In step (1), the crushing process is ultrasonic treatment, with an ultrasonic power of 100-300W, a treatment time of 3-10 minutes, and a temperature of 4-25℃.

5. The method for detecting chitin content in black soldier fly sand as described in claim 1, characterized in that, In step (2), the fluorescent probe includes a chitin-binding protein labeled with a fluorescent group, wherein the fluorescent group is one of green fluorescent protein, fluorescein isothiocyanate, rhodamine, or Alexa Fluor series dyes.

6. The method for detecting chitin content in black soldier fly sand as described in claim 1, characterized in that, In step (2), the conditions for incubation in the dark include: temperature 20-37℃, pH value 6.0-8.0, and incubation time 5-30 minutes.

7. The method for detecting chitin content in black soldier fly sand as described in any one of claims 1 to 6, characterized in that, It also includes quality control steps: verifying the accuracy and reliability of the detection method by adding chitin standards of known concentrations as positive controls and a chitin-free matrix as a negative control.

8. A test kit for the method of detecting chitin content in black soldier fly sand according to any one of claims 1 to 7, characterized in that, It contains the following components: Component A: Chitin concentration gradient standards; Component B: A fluorescent probe solution that can specifically bind to chitin; Component C: Sample pretreatment buffer containing surfactant; Component D: Positive control and negative control.

9. The detection kit as described in claim 8, characterized in that, The concentration of the fluorescent probe solution in component B is 1-20 μg / mL.

10. The application of a method for detecting chitin content in black soldier fly sand as described in any one of claims 1 to 6, or the detection kit as described in claim 8 or 9, in the quality control of fly sand products, the development of biostimulants, or the evaluation of organic fertilizers.