Shrimp-derived chitin and preparation method thereof

Abstract

The invention relates to the technical field of chitin preparation, and particularly discloses shrimp-derived chitin and a preparation method thereof. The invention provides a method for preparing chitin through segmented fermentation in combination with biomagnetism, protease generated by bacillus subtilis and organic acid prawn shells generated by lactic acid bacteria are used for deproteinization and decalcification, and green and efficient extraction of chitin in waste prawn shells is achieved. The deproteinization rate of the shrimp shells is 97.2%-98.4%, and the decalcification rate of the shrimp shells is 96.5%-98.8%. The chitin prepared by the method has high structural similarity with commercial shrimp-derived chitin, the crystallization index is 60.2-64.8%, and the chitin has wide application potential.

Description

Technical Field

[0001] The present invention relates to the technical field of chitin preparation, and specifically discloses a shrimp-derived chitin and a preparation method thereof. Background Art

[0002] Chitin is polymerized by N-acetylglucosamine through β-glycosidic bonds. Its chemical properties are very stable, insoluble in water, dilute acids, dilute alkalis and general organic solvents, and it is very difficult to degrade under natural conditions. As the second largest natural polysaccharide in nature, the output of chitin is second only to cellulose, and it is widely distributed in the cell walls of fungi, the exoskeletons of invertebrates and crustaceans. In recent years, with the increasing production and consumption of crustaceans such as shrimps and crabs, a large amount of by-products such as shrimp and crab shells have been generated. Due to current market and technological limitations, a large amount of shrimp and crab shells are directly treated as garbage, which not only seriously pollutes the environment, but also greatly wastes chitin resources. Therefore, the development and utilization of chitin in shrimp and crab shell waste has important economic and environmental benefits.

[0003] The extraction methods of chitin from shrimp and crab shells mainly include chemical methods (such as acid-base methods), biological methods (such as enzymatic hydrolysis and microbial fermentation methods) and physical methods. Among them, the microbial fermentation method has attracted increasing attention due to its advantages such as simple and mild fermentation conditions, reducible usage of strong acids and strong alkalis, no need for subsequent wastewater treatment, and greatly reduced production costs. The microbial fermentation method mainly includes single-strain fermentation and mixed-strain fermentation. In the process of single-strain fermentation, it is difficult to balance the two factors of deproteinization and deashing, resulting in low chitin purity and extraction rate. Mixed-strain fermentation has great uncertainty and may be ineffective. Summary of the Invention

[0004] Aiming at the technical problems such as long fermentation cycle, low chitin purity and extraction rate, and great uncertainty in mixed-strain fermentation during the process of microbial fermentation of chitin, the present invention provides a method for preparing chitin by segmented fermentation combining biomagnetics by optimizing strains and improving the fermentation process.

[0005] To solve the above technical problems, the present invention adopts the following technical solutions: In the first aspect, the present invention provides a method for preparing chitin, and the treatment method includes the following steps: Step 1: After inoculating a Bacillus subtilis seed solution into a shrimp shell culture medium, it is placed in a magnetic field environment for aerobic fermentation for 12 h to 18 h, and then in a non-magnetic field environment for aerobic fermentation for 18 h to 24 h to obtain a Bacillus fermentation broth; Step 2: Sterilize the Bacillus fermentation broth, inoculate a compound lactic acid bacteria seed solution, oscillate and culture for 6 h to 12 h, and then perform anaerobic fermentation for 36 h to 48 h to obtain a compound bacteria fermentation broth; Step 3: Decolorize the compound bacteria fermentation broth to obtain chitin; Among them, the compound lactic acid bacteria include Lactococcus lactis and Lactobacillus plantarum.

[0006] The present invention provides a method for preparing chitin by segmented fermentation combining biomagnetics. The protease produced by Bacillus subtilis and the organic acid produced by lactic acid bacteria are used to deproteinize and decalcify shrimp shells, realizing the green and efficient extraction of chitin from waste shrimp shells.

[0007] Among them, the present invention combines biomagnetics with traditional industrial fermentation. The magnetic field may improve the ability of Bacillus subtilis to secrete protease by improving metabolism, membrane permeability, gene expression and other ways, and accelerate the decomposition of proteins in shrimp shells. The reason for the segmented magnetic field fermentation of Bacillus subtilis is as follows: in the early stage of fermentation (12h - 18h), the magnetic field environment can stimulate the activity of the bacterial cells; in the later non-magnetic field environment (18h - 24h), it maintains the stability of the bacterial cells, avoiding the premature formation of spores induced by long-term exposure to the magnetic field and reducing the protease production. This design conforms to the "activation-stability" fermentation kinetics.

[0008] The present invention uses mixed bacteria fermentation for decalcification. In the process of mixed bacteria fermentation, various factors such as the interaction between strains, nutritional competition, environmental condition control and the influence of metabolites need to be fully considered. For example, there is an antagonistic relationship between strains, the metabolite of one strain will inhibit the growth of another strain, and the optimal fermentation conditions are significantly different. Therefore, the simultaneous fermentation of many mixed bacteria is ineffective. The present invention uses the co-fermentation of Lactococcus lactis and Lactobacillus plantarum to jointly remove the minerals in shrimp shells. Based on the time-space segmentation of aerobic-anaerobic and the matching of the metabolic requirements of the two bacteria, the time of oscillatory culture and anaerobic fermentation in the decalcification stage is reasonably set to achieve the relay cooperation of "Lactobacillus plantarum enzymatically degrades to build channels and produces acid in small amounts, and Lactococcus lactis produces acid to dissolve minerals", which not only avoids the competition for oxygen between the two bacteria, but also activates their respective dominant metabolic pathways dynamically using the change of pH, realizing the efficient removal of minerals in shrimp shells.

[0009] Among them, the oscillatory culture stage promotes the colonization of Lactobacillus plantarum on the surface of shrimp shells, and the protease produced further removes the remaining proteins during the previous deproteinization step. Organic acids are produced in the later stage of oscillatory culture, which reduces the pH of the system to be suitable for the growth of Lactococcus lactis and activates its acid production pathway at the same time. The oscillatory culture stage can be regarded as the "energy reserve period" of Lactococcus lactis. In the anaerobic stage, Lactococcus lactis produces lactic acid efficiently, and the pH drops to 3.8 - 4.2, directly dissolving the calcium carbonate in shrimp shells; in addition, the accumulation of lactic acid inhibits the excessive acid production of Lactobacillus plantarum, forming a pH steady state, which is conducive to the dissolution of minerals rather than the degradation of chitin. In the anaerobic stage, through the concentrated acid production of Lactococcus lactis, while dissolving minerals, it protects the chitin structure. The design of 36h - 48h is a triple balance of kinetics, economy and product quality.

[0010] Furthermore, the magnetic field environment is an alternating magnetic field.

[0011] Further, the intensity of the magnetic field is 0.2 mT to 0.6 mT, and its magnetic field frequency is 50 Hz.

[0012] Further, in step one, the viable count of the Bacillus subtilis seed solution is 5.0×10 8 CFU / mL to 1.0×10 9 CFU / mL, and the inoculation amount is 3% to 5%. In the present invention, the Bacillus subtilis is Bacillus subtilis subsp. subtilis ( Bacillus subtilis subsp. Subtilis ), and the strain number is BNCC132861.

[0013] Further, the temperature of the aerobic fermentation is 35°C to 38°C, and the rotation speed is 180 rpm to 220 rpm.

[0014] Further, in step two, in the compound lactic acid bacteria seed solution, the viable count of Lactobacillus plantarum is 1.0×10 8 CFU / mL to 5.0×10 8 CFU / mL, and the viable count of Lactococcus lactis is 5.0×10 8 CFU / mL to 1.5×10 9 CFU / mL.

[0015] Further, the inoculation amount of the compound lactic acid bacteria seed solution is 2% to 5%.

[0016] The Lactobacillus plantarum selected in the present invention ( Lactobacillus plantarum ) has the strain number of BNCC132861; the Lactococcus lactis selected in the present invention is Lactococcus lactis subsp. lactis ( Lactococcus lactis subsp. Lactis ), and its strain number is BNCC336437; Further, the temperature of the anaerobic fermentation is 32°C to 35°C.

[0017] Further, sterilize the Bacillus fermentation broth, add sterilized carbon source and buffer salts, adjust the pH to obtain a lactic acid bacteria culture medium; inoculate the compound lactic acid bacteria seed solution into the lactic acid bacteria culture medium, shake and culture for 6 h to 12 h, and then perform anaerobic fermentation for 36 h to 48 h to obtain a compound bacteria fermentation broth.

[0018] Further, the carbon source includes glucose or lactose, and the addition amount is 3% to 5% of the volume of the Bacillus fermentation broth.

[0019] Further, the buffer salt includes a phosphate-citrate composite buffer system.

[0020] The components of the phosphate-citrate composite buffer system consist of dipotassium hydrogen phosphate (1 g / L - 3 g / L), potassium dihydrogen phosphate (1 g / L - 2 g / L), citric acid (0.5 g / L - 1 g / L), and sodium citrate (3 g / L - 5 g / L). The total buffer concentration is 100 mM - 150 mM. After adding the composite buffer system, the pH of the medium is adjusted to 5.5 - 5.9.

[0021] During the fermentation process, lactic acid bacteria produce a large amount of acid, resulting in a rapid decrease in pH and inhibiting their own growth. This buffer system neutralizes H + , slows down the pH fluctuation, and prolongs the logarithmic growth phase of the bacteria. In addition, phosphate in the buffer system provides phosphorus element, which participates in the synthesis of bacterial ATP and the construction of cell membrane; citrate serves as a carbon source supplement to support energy metabolism. Compared with the phosphate buffer system, the phosphate-citrate composite buffer system provided by the present invention can significantly improve the fermentation efficiency and product quality of lactic acid bacteria.

[0022] Furthermore, the shrimp shell medium includes shrimp shell powder after microwave treatment, glucose, peptone, and ammonium sulfate.

[0023] Among them, the shrimp shell powder after microwave treatment refers to the shrimp shell powder that has been microwave-treated at a power of 200 W - 260 W for 10 min - 15 min. The preparation steps of the shrimp shell powder are as follows: The cleaned and dried shrimp shells are coarsely crushed by a hammer crusher and finely crushed by an ultrafine grinder, and then sieved through a 40-mesh - 80-mesh sieve to obtain.

[0024] Microbial fermentation is used to remove protein and demineralize. It requires effective contact between microorganisms and shrimp shells and decomposition of target components. Microwave treatment can break the structure, increase the specific surface area of shrimp shells, making it easier for microorganisms to attach and decompose; microwave pretreatment can unfold protein molecules, expose more enzymatic hydrolysis sites, and shorten the enzymatic hydrolysis time of proteins; in addition, microwave treatment can also make the shrimp shell structure looser, facilitating the dissolution of calcium ions in the shrimp shell.

[0025] In the second aspect, the present invention provides a shrimp-derived chitin, which is prepared by the chitin preparation method provided in the first aspect.

[0026] The chitin obtained in the present invention is prepared by deproteinizing and demineralizing shrimp shell powder. The deproteinization rate of the shrimp shell is 97.2% - 98.4%, and the demineralization rate is 96.5 - 98.8%. The obtained chitin has a high structural similarity with commercial shrimp-derived chitin, and the crystallization index is 60.2% - 64.8%, having broad application potential. The present invention is of great significance for making full use of waste shrimp shell resources, reducing environmental pollution, and increasing product added value. Description of the Drawings

[0027] To more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the accompanying drawings required in the embodiments. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0028] Figure 1 is the process flow chart of the preparation method of chitin provided in Embodiment 1 of the present invention; Figure 2 is the Fourier transform infrared spectrum of the chitin prepared in Embodiment 1 of the present invention and commercial shrimp-derived chitin. Detailed implementation manners

[0029] In order to make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following further details the present invention in combination with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] The present invention selects the shrimp shells of Litopenaeus vannamei.

[0031] Embodiment 1 The embodiment of the present invention provides a preparation method of chitin. The process flow chart of the preparation method is as Figure 1 shown, and specifically includes: Step 1: After inoculating the Bacillus subtilis seed liquid into the shrimp shell culture medium, it is placed in a magnetic field environment for aerobic fermentation and then in a non-magnetic field environment for aerobic fermentation to obtain a Bacillus fermentation liquid. Specifically, it includes: S11. Preparation of the seed liquid The Bacillus subtilis BNCC132861 is activated in the LB culture medium and passaged 3 times. It is inoculated into the LB modified culture medium (calculated based on 1 L, including 20 g of glucose, 1 g of beef extract, 5 g of peptone, and 5 g of NaCl) at an inoculation amount of 3% and placed under the conditions of 37 °C and 200 rpm for scale-up culture to obtain the Bacillus subtilis BNCC132861 seed liquid. After measurement, the viable count of the seed liquid is 8.0×10 8 CFU / mL; The Lactobacillus plantarum BNCC132861 is activated in the MRS liquid culture medium and inoculated into the modified MRS liquid culture medium [on the basis of the MRS liquid culture medium, adding 2% (w / v) fructose and 2.5% (w / v) yeast extract] for scale-up culture at 37 °C to obtain the Lactobacillus plantarum BNCC132861 seed liquid with a viable count of 5.0×10 8 CFU / mL; Lactococcus lactis BNCC336437 was activated in M17 medium, passaged 3 times, and inoculated into MRS broth medium. After two rounds of subculturing under anaerobic conditions at 37 °C, a Lactococcus lactis BNCC336437 seed solution with a viable cell count of 3.0×10 9 CFU / mL was obtained; The seed solution of Lactobacillus plantarum BNCC132861 and the seed solution of Lactococcus lactis BNCC336437 were mixed in equal volumes to obtain a compound lactic acid bacteria seed solution; S12. Preparation of shrimp shell medium Shrimp shells were taken and washed, dried at 60 °C, coarsely crushed by a hammer crusher and finely crushed by an ultrafine grinder, and then passed through a 60-mesh sieve to obtain shrimp shell powder; The microwave power of the microwave oven was adjusted to 200 W, and the above-mentioned shrimp shell powder was placed in the microwave oven and treated with microwave for 15 min to obtain microwave-pretreated shrimp shell powder; According to the ratio of 6% shrimp shell powder, 6% glucose, 0.4% peptone, and 0.1% ammonium sulfate (the balance is water), shrimp shell medium was prepared and sterilized at 121 °C for 15 min by moist heat to obtain sterilized shrimp shell medium; S13. Fermentation stage of Bacillus subtilis The above-mentioned Bacillus subtilis BNCC132861 seed solution was inoculated into the sterilized shrimp shell medium at an inoculation amount of 5%, placed in a magnetic field incubator, and cultured with shaking at 37 °C and 200 rpm for 15 h, and then cultured with shaking at 37 °C and 200 rpm for 18 h in a non-magnetic field environment to obtain Bacillus fermentation broth; Wherein the magnetic field is an alternating magnetic field, the magnetic field frequency is 50 Hz, and the magnetic field intensity is 0.5 mT.

[0032] In step two, the Bacillus fermentation broth was sterilized at 121 °C for 15 min by moist heat, glucose sterilized at 115 °C for 20 min was added, and the pH of the system was adjusted to 5.7 by adding a sterilized phosphate-citrate composite buffer system to obtain lactic acid bacteria medium; The above-mentioned compound lactic acid bacteria seed solution was inoculated at an inoculation amount of 3%, cultured with shaking at 37 °C and 200 rpm for 10 h, and then anaerobically cultured at 33 °C for 42 h to obtain a compound bacteria fermentation broth; Wherein the addition amount of glucose is 4% of the Bacillus fermentation broth.

[0033] In step three, the compound bacteria fermentation broth was centrifuged and washed with deionized water until neutral, added to a 10% H2O2 solution, decolorized at 80 °C, the precipitate was collected after decolorization was completed, and dried in an oven at 70 °C to obtain chitin.

[0034] Example 2 An embodiment of the present invention provides a preparation method of chitin, and the preparation method specifically includes: Step 1: After inoculating the Bacillus subtilis seed liquid into the shrimp shell culture medium, it is placed in a magnetic field environment for aerobic fermentation, and then in a non-magnetic field environment for aerobic fermentation to obtain a Bacillus fermentation liquid; specifically including: S11. Preparation of seed liquid The Bacillus subtilis BNCC132861 is activated in LB medium, passaged 2 times, inoculated into the improved LB medium at an inoculation amount of 5%, and placed in an incubator at 37°C and 180 rpm for enlarged culture to obtain the Bacillus subtilis BNCC132861 seed liquid. After measurement, the viable count of the seed liquid is 5.0×10 8 CFU / mL; The Lactobacillus plantarum BNCC132861 is activated in MRS liquid medium, and after activation, it is inoculated into the improved MRS liquid medium for enlarged culture at 37°C to obtain a Lactobacillus plantarum BNCC132861 seed liquid with a viable count of 2.0×10 8 CFU / mL; The Lactococcus lactis BNCC336437 is activated in M17 medium, passaged 3 times, and inoculated into MRS broth medium at 37°C under anaerobic conditions to obtain a Lactococcus lactis BNCC336437 seed liquid with a viable count of 1.0×10 9 CFU / mL; The Lactobacillus plantarum BNCC132861 seed liquid and the Lactococcus lactis BNCC336437 seed liquid are mixed in equal volume to obtain a compound lactic acid bacteria seed liquid; S12. Preparation of shrimp shell culture medium Take the shrimp shell for cleaning, dry the shell at 60°C, coarsely crush it with a hammer crusher and finely crush it with an ultramicro grinder, and then pass through a 40-mesh sieve to obtain shrimp shell powder; Adjust the microwave power of the microwave oven to 260W, place the above shrimp shell powder in the microwave oven, and perform microwave treatment for 10 min to obtain microwave-pretreated shrimp shell powder; According to the ratio of 4% shrimp shell powder, 5% glucose, 0.2% peptone, and 0.12% ammonium sulfate (the balance is water), prepare the shrimp shell culture medium, and sterilize it at 121°C for 15 min by moist heat to obtain a sterilized shrimp shell culture medium; S13. Fermentation stage of Bacillus subtilis Inoculate the above-mentioned Bacillus subtilis BNCC132861 seed liquid into the sterilized shrimp shell culture medium at an inoculation amount of 3%, place it in a magnetic field incubator, and oscillate and culture it at 35°C and 180 rpm for 18 h, and then place it in a non-magnetic field environment and oscillate and culture it at 35°C and 180 rpm for 20 h to obtain a Bacillus fermentation liquid; Wherein the magnetic field is an alternating magnetic field, the magnetic field frequency is 50 Hz, and the magnetic field intensity is 0.2 mT.

[0035] Step 2: Sterilize the Bacillus fermentation broth at 121°C for 15 min by moist heat, add lactose sterilized at 115°C for 20 min, and adjust the pH of the system to 5.5 by adding a sterilized phosphate-citrate composite buffer system to obtain a lactic acid bacteria culture medium; Inoculate the above-mentioned compound lactic acid bacteria seed liquid at an inoculation amount of 5%, culture it by shaking at 37°C and 200 rpm for 6 h, and then culture it anaerobically at 32°C for 48 h to obtain a compound bacteria fermentation broth; Among them, the addition amount of lactose is 3% of the Bacillus fermentation broth.

[0036] Step 3: Centrifuge the compound bacteria fermentation broth and wash it with deionized water until it is neutral, add it to a 10% H2O2 solution, decolorize it at 80°C, collect the precipitate after decolorization is completed, and dry it in an oven at 70°C to obtain chitin.

[0037] Example 3 The embodiment of the present invention provides a preparation method of chitin, and the preparation method specifically includes: Step 1: After inoculating a Bacillus subtilis seed liquid into a shrimp shell culture medium, place it in a magnetic field environment for aerobic fermentation, and then perform aerobic fermentation in a non-magnetic field environment to obtain a Bacillus fermentation broth; specifically including: S11. Preparation of seed liquid Activate Bacillus subtilis BNCC132861 in LB culture medium, passage it 3 times, inoculate it into LB modified culture medium at an inoculation amount of 4%, and expand the culture at 37°C and 220 rpm to obtain a Bacillus subtilis BNCC132861 seed liquid. After measurement, the viable bacteria count of the seed liquid is 1.0×10 9 CFU / mL; Activate Lactobacillus plantarum BNCC132861 in MRS liquid culture medium, and after activation, inoculate it into modified MRS liquid culture medium and expand the culture at 36°C to obtain a Lactobacillus plantarum BNCC132861 seed liquid with a viable bacteria count of 1.0×10 9 CFU / mL; Activate Lactococcus lactis BNCC336437 in M17 culture medium, passage it 3 times, inoculate it into MRS broth culture medium, and perform two-stage expansion culture at 36.5°C under anaerobic conditions to obtain a Lactococcus lactis BNCC336437 seed liquid with a viable bacteria count of 2.0×10 9 CFU / mL; Mix the Lactobacillus plantarum BNCC132861 seed liquid and the Lactococcus lactis BNCC336437 seed liquid in equal volume to obtain a compound lactic acid bacteria seed liquid; S12. Preparation of shrimp shell culture medium Wash the shrimp shells, dry the shells at 60 °C, coarsely crush them with a hammer crusher and finely crush them with an ultrafine grinder, and then pass through an 80-mesh sieve to obtain shrimp shell powder; Adjust the microwave power of the microwave oven to 240 W, place the above shrimp shell powder in the microwave oven, and perform microwave treatment for 12 min to obtain microwave-pretreated shrimp shell powder; Prepare a shrimp shell medium according to the ratio of 8% shrimp shell powder, 8% glucose, 0.5% peptone, and 0.08% ammonium sulfate (the balance is water), and sterilize it at 121 °C for 15 min by moist heat to obtain a sterilized shrimp shell medium; S13. Bacillus subtilis fermentation stage Inoculate the above Bacillus subtilis BNCC132861 seed liquid into the sterilized shrimp shell medium according to an inoculation amount of 4%, place it in a magnetic field incubator, and oscillate and culture it at 38 °C and 220 rpm for 12 h, and then place it in a non-magnetic field environment and oscillate and culture it at 38 °C and 220 rpm for 24 h to obtain a Bacillus fermentation broth; Wherein the magnetic field is an alternating magnetic field, the magnetic field frequency is 50 Hz, and the magnetic field intensity is 0.6 mT.

[0038] Step 2: Sterilize the Bacillus fermentation broth at 121 °C for 15 min by moist heat, add glucose sterilized at 115 °C for 20 min, and adjust the pH of the system to 5.9 by adding a sterilized phosphate-citrate composite buffer system to obtain a lactic acid bacteria medium; Inoculate the above compound lactic acid bacteria seed liquid according to an inoculation amount of 2%, oscillate and culture it at 36 °C and 200 rpm for 12 h, and then anaerobically culture it at 35 °C for 36 h to obtain a compound bacteria fermentation broth; Wherein the addition amount of glucose is 5% of the Bacillus fermentation broth.

[0039] Step 3: Centrifuge the compound bacteria fermentation broth and wash it with deionized water until neutral, add it to a 10% H2O2 solution, decolorize it at 80 °C, collect the precipitate after decolorization is completed, and dry it in an oven at 70 °C to obtain chitin.

[0040] Comparative Example 1 This comparative example provides a method for preparing chitin. This preparation method is basically the same as that of Example 1, except that in step S13, in the Bacillus subtilis fermentation stage, "placed in a magnetic field incubator" is replaced with "placed in a conventional incubator", and the culture conditions and time remain unchanged, and the remaining steps are the same as those of Example 1 and will not be repeated. Finally, chitin is prepared.

[0041] Comparative Example 2 This comparative example provides a method for preparing chitin. This preparation method is basically the same as that of Example 1, except that in step S11, the seed solution of Lactobacillus plantarum BNCC132861 is not prepared; in step two, the sentence "Inoculate the above-mentioned compound lactic acid bacteria seed solution at an inoculation amount of 3%, shake and culture at 37°C and 200 rpm for 10 h, and then anaerobically culture at 33°C for 42 h to obtain a compound bacteria fermentation broth" is replaced with "Inoculate Lactococcus lactis BNCC336437 at an inoculation amount of 1.5%, and anaerobically culture at 33°C for 52 h to obtain a Lactococcus lactis fermentation broth"; the remaining steps are the same as those of Example 1 and will not be elaborated here. Finally, chitin is prepared.

[0042] Effect Example 1 In this effect example, the chitin prepared in Example 1 was used as a representative and commercial shrimp-derived chitin to obtain the intramolecular structure information by Fourier transform infrared spectroscopy (FTIR), where the infrared spectrum scanning wavelength range was 4000 cm -1 ~500 cm -1 . The characteristic peaks of chitin are the amide I region, amide II region, and amide III region.

[0043] It can be seen from Figure 2 that the spectral pattern of the chitin (MS) prepared in Example 1 is similar to that of the commercial shrimp-derived chitin (CC). The chitin (MS) prepared in the present invention shows characteristic peaks at 1654 and 1622 cm -1 , corresponding to the amide I region, and shows a signal at 1557 cm -1 , corresponding to the amide II region. When the shrimp shell powder has an absorption peak at 1544 cm -1 , the spectra of the chitin prepared in the present invention and the commercial chitin (CC) lack absorbance. Other characteristic peaks of chitin were observed in MS at 1377 cm -1 (C-H) and 950 cm -1 ~1200 cm -1 (C-O-C and C-O).

[0044] Effect Example 2 The main components of shrimp shells include protein, inorganic salts, and chitin. The protein content is determined by the Kjeldahl method, the ash content is determined by a muffle furnace, and the chitin content is determined by an acid-base method. Then, the decalcification rate and deproteinization rate of microorganisms are estimated based on the protein and ash contents.

[0045] (1) Decalcification rate D m The calculation formula 1 is as follows: 1 In the formula, M0 is the mass of the sample before fermentation, M F is the mass of the sample after fermentation, A0 is the ash content in the sample before fermentation, and A FIt is the ash content in the fermented sample.

[0046] (2) Deproteinization rate D p The calculation formula 2 is as follows: 2 Wherein, M0 is the mass of the sample before fermentation, M F is the mass of the sample after fermentation, P0 is the protein content in the sample before fermentation, and P F is the protein content in the sample after fermentation.

[0047] (3) Chitin content calculation Soften the shrimp shells with 1 mol / L HCl at a ratio of 1:10 (v / v) for 3 h. Then carry out deproteinization with 1 mol / L NaOH solution at a ratio of 1:10 (w / v) at 55 °C for 3 h, and wash with distilled water until the filtrate is neutral. Repeat the above operation 3 times, then wash and place in an oven at 60 °C for 12 h, and the obtained substance can be regarded as pure chitin.

[0048] (4) Crystallinity index (C rl ) is an important characteristic affecting indicators such as the solubility and biodegradability of chitin. The higher the crystallinity, the lower the solubility and biodegradability. C rl is calculated by the following formula 3: 3 Wherein, I 110 is the maximum intensity at 2θ = 20°, I am is the maximum intensity at 2θ = 16°.

[0049] It was determined that the main component contents in the raw material shrimp shells selected in the present invention are shown in Table 1 below.

[0050] Table 1

[0051] The determination results of the decalcification rate, deproteinization rate of the shrimp shells in Examples 1-3 and Comparative Examples 1-2, and the crystallinity index of the obtained chitin are shown in Table 2.

[0052] Table 2

[0053] It can be seen from the data analysis in Table 2 that in Comparative Example 1, without magnetic field cultivation, the ability of Bacillus subtilis to secrete protease is significantly weakened, and thus the deproteinization rate of the shrimp shells is significantly lower than that in Example 1. In Comparative Example 2, only Lactococcus lactis BNCC336437 was used in the decalcification stage of the shrimp shells, and its decalcification rate was significantly lower than that in Example 1.

[0054] The above are only the preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A preparation method of chitin, characterized in that, The processing method includes the following steps: Step 1: After inoculating the Bacillus subtilis seed liquid into the shrimp shell culture medium, it is placed in a magnetic field environment for aerobic fermentation for 12 h to 18 h, and then in a non-magnetic field environment for aerobic fermentation for 18 h to 24 h to obtain a Bacillus fermentation liquid; Step 2: Sterilize the Bacillus fermentation liquid, inoculate the compound lactic acid bacteria seed liquid, oscillate and culture for 6 h to 12 h, and then anaerobically ferment for 36 h to 48 h to obtain a compound bacteria fermentation liquid; Step 3: Decolorize the compound bacteria fermentation liquid to obtain chitin; Among them, the compound lactic acid bacteria include Lactococcus lactis and Lactobacillus plantarum.

2. The preparation method of chitin according to claim 1, characterized in that, The magnetic field environment is an alternating magnetic field.

3. The preparation method of chitin according to claim 1 or 2, characterized in that, The intensity of the magnetic field is 0.2 mT to 0.6 mT.

4. The preparation method of chitin according to claim 1, characterized in that, In Step 1, the viable count of the Bacillus subtilis seed liquid is 5.0×10 8 CFU / mL to 1.0×10 9 CFU / mL, and the inoculation amount is 3% to 5%; and / or The temperature of the aerobic fermentation is 35 °C to 38 °C, and the rotation speed is 180 rpm to 220 rpm.

5. The preparation method of chitin according to claim 1, characterized in that, In Step 2, in the compounded lactic acid bacteria seed liquid, the viable count of Lactobacillus plantarum is 1.0×10 8 CFU / mL to 5.0×10 8 CFU / mL, the viable count of Lactococcus lactis is 5.0×10 8 CFU / mL to 1.5×10 9 CFU / mL; and / or The inoculation amount of the compound lactic acid bacteria seed liquid is 2% to 5%; and / or The temperature of the anaerobic fermentation is 32 °C to 35 °C.

6. The preparation method of chitin according to claim 1, characterized in that, Step 2 includes the following steps: Sterilize the Bacillus fermentation liquid, add a sterilized carbon source and buffer salts, adjust the pH to obtain a lactic acid bacteria culture medium; inoculate the compound lactic acid bacteria seed liquid into the lactic acid bacteria culture medium, oscillate and culture for 6 h to 12 h, and then anaerobically ferment for 36 h to 48 h to obtain a compound bacteria fermentation liquid.

7. The preparation method of chitin according to claim 6, wherein, The carbon source includes glucose or lactose.

8. The preparation method of chitin according to claim 6, characterized in that, The buffer salts include a phosphate-citrate composite buffer system.

9. The preparation method of chitin according to claim 1, characterized in that, The shrimp shell culture medium includes shrimp shell powder after microwave treatment, glucose, peptone and ammonium sulfate.

10. A shrimp-derived chitin, characterized in that, Prepared by the preparation method of chitin according to any one of claims 1 to 9.

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