CN114672528A

CN114672528A - Preparation method of chitin

Info

Publication number: CN114672528A
Application number: CN202210212327.XA
Authority: CN
Inventors: 杨燊; 邢宇凡; 陈兴花; 张玉苍; 邓尚贵; 郑明静
Original assignee: Jimei University
Current assignee: Jimei University
Priority date: 2022-03-04
Filing date: 2022-03-04
Publication date: 2022-06-28

Abstract

The invention relates to the technical field of industrial production, in particular to a preparation method of chitin, which comprises the following steps: A. screening lactobacillus, determining single bacterium with strongest fermentation capacity, and selecting lactobacillus for fermentation; B. inoculating the fermented lactic acid bacteria into a culture medium, and culturing to obtain a bacterial liquid; C. placing the sterilized crab shells in sugar water to form a crab shell-sugar water mixture for later use; D. inoculating the bacterial liquid into the crab shell-sugar water mixture, and performing ultrasonic treatment fermentation to obtain primary fermentation liquid; E. and adding strong acid into the primary fermentation liquid for treatment, then adding strong base for treatment, and bleaching to obtain insoluble chitin. The invention solves the problems of high cost, environmental pollution and the like in chitin preparation in the prior art, the chitin is produced by combining biological fermentation with an acid-base method, the cost is reduced, the production efficiency is ensured, and the purity of the chitin is effectively improved to more than 90 percent, so that the chitin is an ideal industrial production method.

Description

Preparation method of chitin

Technical Field

The invention relates to the technical field of industrial production, in particular to a preparation method of chitin.

Background

Chitin, whose chemical name is beta- (1, 4) -2-acetamido-2-deoxy-D-glucopyranose, is a condensation product of N-acetylglucosamine with beta-1, 4 glycosidic bond, and chitin, etc., is a polysaccharide second to cellulose existing on earth, and is a nitrogen-containing natural organic high molecular compound with the largest amount except protein in nature. Chitin is widely present on the surfaces of crustaceans such as shrimps and crabs and various insects, the bones of mollusks such as cuttlefish and shellfish, and the cell walls of mushrooms and fungi. The crab shell contains abundant chitin, and the content of the chitin is up to one third of that of the crab shell.

The chitosan is a good antibacterial agent, can inhibit the growth of various bacteria and fungi, and can inhibit the growth of escherichia coli, salmonella, staphylococcus aureus, aeromonas, certain fungi and the like. Both chitin and chitosan have good biodegradability. Chitin can be degraded by lysozyme, acetylglucosaminidase and lipase, and chitosan can be biodegraded by chemical (acidolysis) or enzymatic methods. In addition, chitosan has good biocompatibility, good adaptability to animals and plants, no irritation to organisms, and small inflammatory reaction, and is approved for food in japan, italy, and finland, and also approved for wound dressings by FDA.

In the food industry, the crab wastes discarded every year around the world are about 600-800 million tons, most of the wastes are buried or burned, which not only causes a considerable problem of secondary environmental pollution, but also cannot fully utilize the value of the wastes. The crab shell is rich in organic matters such as chitin and protein, and inorganic matters such as calcium. The content of calcium carbonate in the crab shell is up to 20-50%, the content of protein is up to 20-40%, except calcium carbonate and protein, the most main component in the crab shell is chitin, and the content in the crab shell is 15-40%.

The method for extracting chitin from crab shell includes acid-base method, enzyme method and biological method. The most common extraction method of chitin is acid-base method, which removes inorganic salts such as calcium carbonate with acid, deproteinizes with alkali, and then decolors to obtain chitin. The traditional acid-base method is convenient to operate and wide in application, but a large amount of acid-base waste liquid is generated in the production process, so that the environment is greatly polluted, and the waste liquid needs to be treated, so that the production cost is increased. In contrast, the biological method is not only environment-friendly and does not need high cost, but also has a long production period and low fermentation efficiency, so that the method for improving the fermentation efficiency by finding a low-cost, convenient and efficient way has profound significance.

Lactic acid bacteria are probiotics which ferment carbohydrates into lactic acid and can be used for preparing chitin. Lactic acid produced during fermentation of lactic acid bacteria is produced by the breakdown of glucose, thereby creating a low pH condition that inhibits the growth of spoilage microorganisms. Calcium lactate precipitate formed by the reaction of lactic acid and calcium carbonate can be removed by washing; the deproteinization of the shrimp and crab wastes is hydrolyzed by proteolytic enzyme produced by the strain, so that the minerals and proteins in the crab shells can be removed. In addition, substances such as organic acids, special enzyme systems, acidomycins and the like generated by fermentation of lactic acid bacteria have special physiological functions. A large number of research data show that the lactobacillus can promote the growth of animals, regulate the normal flora of the gastrointestinal tract and maintain the microecological balance, thereby improving the gastrointestinal tract function, improving the food digestibility and the biological value, reducing the serum cholesterol, controlling the endotoxin, inhibiting the growth of putrefying bacteria in the intestinal tract, improving the immunity of the organism and the like, and is closely related to the health of people. Lactic acid bacteria are widely present in our lives, and many foods contain lactic acid bacteria, such as yogurt, active lactic acid bacteria beverages, kimchi, and the like. The lactobacillus can improve the flavor of the food, improve the nutritive value of the food, prolong the storage life of the food and endow the food with higher utilization value.

Disclosure of Invention

The invention aims to research and design a preparation method for fermenting chitin by a low-intensity ultrasonic-promoted biological method, and solve the problems of high cost, environmental pollution and the like of chitin preparation in the prior art.

In order to solve the technical problem, the invention adopts the following technical scheme:

a preparation method of chitin comprises the following steps:

A. screening lactobacillus, determining single bacterium with strongest fermentation capacity, and selecting lactobacillus for fermentation;

B. inoculating the fermented lactic acid bacteria into a culture medium, and culturing to obtain a bacterial liquid;

C. placing the sterilized crab shells in sugar water to form a crab shell-sugar water mixture for later use;

D. inoculating the bacterial liquid into the crab shell-sugar water mixture, and performing ultrasonic treatment fermentation to obtain primary fermentation liquid;

E. and adding strong acid into the primary fermentation liquid for treatment, then adding strong base for treatment, and bleaching to obtain insoluble chitin.

The invention fully utilizes the crab shell which is a waste in food industry to extract the chitin from the crab shell. The chitin is produced by combining biological fermentation with an acid-base method, and the biological fermentation method is adopted at the initial stage of reaction, so that the use amount of acid and base is greatly reduced (the reduction amount reaches 70-80%), the pollution is reduced, and the cost is reduced. In the later stage of the reaction, the biological fermentation tends to be slow, so the acid-base method is adopted to assist the complete extraction of the chitin in the last stage, the higher production efficiency is ensured, and the purity of the chitin produced by combining the biological fermentation with the acid-base method reaches 90 percent, so the method is an ideal industrial production method.

Preferably, the method comprises the following steps:

A. screening lactobacillus, determining single bacterium with strongest fermentation capacity, and selecting the single bacterium as the fermentation lactobacillus;

B. inoculating the fermented lactic acid bacteria into a liquid culture medium in an inoculation amount of 5-10%, and culturing at constant temperature for 8-12 h to obtain a bacterial liquid after culturing;

C. and (2) mixing the sterilized crab shell powder according to the mass volume ratio of 1 g: 2-6 ml of the crab shell-sugar water mixture is prepared in sugar water, wherein the mass concentration of sugar in the sugar water is 5-25%, and the crab shell-sugar water mixture is formed for later use

Inoculating the bacterial liquid into the crab shell-sugar water mixture in a bacterial liquid inoculation amount of 5-10%, performing ultrasonic treatment, and fermenting at constant temperature for 32-72 h to obtain primary fermentation liquid;

D. adding 1mol/L of primary fermentation liquor^-1Then adding 8% NaOH solution for treatment, and bleaching to obtain the insoluble chitin.

Preferably, in the step C, the ultrasonic treatment is single-time-point intermittent ultrasonic, specifically: performing ultrasonic treatment at an interval of 8h for 5-20 min after performing ultrasonic treatment for the first time for 5-20 min at the beginning of fermentation, wherein the ultrasonic power is 0.167W/cm²The ultrasonic frequency is 40KHz, and the ultrasonic temperature is 37 ℃.

Ultrasound is a sound wave with a frequency above the human threshold. The ultrasonic energy generating device is a vibration energy generated by an ultrasonic transducer, can generate a heating effect, a mechanical mass transfer effect and a cavitation effect, and the ultrasonic energy is used as a green and economic food processing technology, has good directionality and strong penetrating power, and also has a series of reaction effects, emulsification effects, thermal effects, mechanical effects and the like, so that the potential application of the ultrasonic energy generating device in a plurality of food processing applications is opened up by the non-thermal treatment means without harm and chemical addition. Ultrasound can be classified into high-intensity ultrasound and low-intensity ultrasound according to intensity, and ultrasonic waves of different intensities can affect microorganisms to different degrees. The cavitation effect generated by the low-intensity ultrasonic treatment has small damage to cells, and can improve the permeability of the membrane, thereby accelerating the transfer of substances, promoting the growth and the reproduction of the cells, and improving the yield of metabolites, so that the ultrasonic technology has wide application prospect in microbial fermentation engineering. In the research of the invention, the ultrasonic treatment, especially the single-time-point intermittent ultrasonic treatment, is adopted in the process of preparing the chitin by fermenting the crab shells, so that the reaction efficiency and the fermentation quality can be effectively improved.

Preferably, the first sonication is carried out 10min at the beginning of the fermentation, followed by 10min every 8 h.

Preferably, in the step C, the sterilized crab shells are mixed according to the mass volume ratio of 1 g: 3ml of the mixture is prepared in the sugar water; the sugar water is glucose water, and the mass concentration of sugar in the sugar water is 15%.

Preferably, in the step A, the fermentation lactic acid bacteria are lactobacillus paracasei, and the NCBI number of the fermentation lactic acid bacteria is NR _ 025880.1.

Preferably, the specific operation of screening lactobacillus in the step a is as follows: separating and purifying the compound lactobacillus, selecting a plurality of single bacterial colonies, respectively fermenting crab shells by the obtained single bacteria, determining the single bacteria with the strongest fermentation capacity, identifying the strains, and determining the fermented lactobacillus.

Preferably, the isolation and purification is performed by plate-streaking, and the identification of the strain is performed by 16S rDNA identification.

The screening of the lactobacillus for producing the chitin by fermenting the crab shells comprises the following steps:

step one, preparing a culture medium: weighing 1.08g of MRS nutrient broth powder on an electronic balance, pouring into a conical flask, adding 20mL of distilled water, shaking uniformly, covering the conical flask, and plugging the conical flask into an autoclave at 121 ℃ for 15min to obtain 20mL of MRS liquid culture medium. The MRS liquid culture medium is selected because the culture medium is used for selectively culturing the lactic acid bacteria and is suitable for culturing and separating the lactic acid bacteria.

Step two, inoculating and culturing: cooling the autoclaved MRS liquid culture medium to room temperature, taking out the composite lactic acid bacteria powder stored at the temperature of-20 ℃ from a refrigerator, inoculating the composite lactic acid bacteria powder with the inoculation amount of 4% into the MRS liquid culture medium, and culturing for 8 hours in a constant-temperature incubator at the temperature of 37 ℃ to obtain turbid composite lactic acid bacteria liquid.

Step three, carrying out plate-drawing culture: packaging the plate required for culture, preparing solid culture medium, and sterilizing in autoclave at 121 deg.C for 15 min. A total of 4 plates were extinguished, 100mL solid medium requiring 5.4g of MRS nutrient broth powder, 2g agar, 100mL distilled water. In a super clean bench, the sterilized solid culture medium is poured into the sterilized flat plates while the solid culture medium is still hot, about 20mL of the solid culture medium is needed for each flat plate, and the solid culture medium is condensed at room temperature for 20-40min until the culture medium is solidified.

In the superclean bench, each flat plate is divided into 4 regions. Burning the metal wire of the inoculating loop to be red by outer flame of an alcohol lamp, slightly inclining the inoculating loop, burning a metal rod, cooling, taking one-loop composite lactobacillus liquid by using the inoculating loop, carrying out plate lineation, wrapping the inoculated plate with a sealing film, and then culturing for 48 hours in a 37 ℃ constant-temperature incubator.

Step four, selecting bacteria: and (3) moving the plate from the incubator to a super clean bench, preparing a sterilized MRS liquid culture medium in advance, burning and cooling an inoculating loop, and selecting a large, full and single bacterial colony which is not overlapped with other bacterial colonies from the plate by using the inoculating loop to be placed in the liquid culture medium. And 7 single colonies are picked up and respectively placed in different test tubes, and then are moved into a constant-temperature incubator at 37 ℃ for static culture for 8 hours.

Step five, fermentation: the cultured bacterial solutions were separately reserved for seed, and the serial numbers were marked for storage at-20 ℃ in a refrigerator. And respectively fermenting crab shells by using the 7 bacteria, measuring the calcium content in the fermentation liquor by using an EDTA method after fermenting for 72 hours, calculating the decalcification rate, and selecting the single bacteria with the highest calcium content in the fermentation liquor.

Step six, strain identification: and (3) selecting the single bacterium with the strongest fermentation capacity through fermentation, and identifying the 16S rDNA strain of the single bacterium. The bacterium is identified as lactobacillus paracasei, and the NCBI number of the bacterium is NR _ 025880.1.

Preferably, in the step D, the bleaching operation is as follows: and (3) carrying out a normal-temperature reaction for 1h by adopting hydrogen peroxide with the mass concentration of 30% to complete the decolorization treatment.

Preferably, in step B, the culture medium is MRS liquid culture medium; in the step C, the crab shell sterilization is specifically performed in a sterilization pot at 121 ℃ for 10-30 min.

Compared with the prior art, the implementation of the invention has the following beneficial effects:

on one hand, the invention saves a large amount of strong acid and alkali and achieves the purpose of protecting the environment. On the other hand, the fermentation is promoted by using a single-time-point intermittent ultrasonic method, and the extraction of the chitin is completed by combining an acid-base method after the fermentation is carried out for 72 hours, so that the extraction efficiency of the chitin is improved on the premise of protecting the environment, the purity of the obtained chitin is improved, and the method is suitable for industrial large-scale production. The invention uses biological fermentation combined with acid-base method to produce chitin, reduces cost and ensures production efficiency, and effectively improves the purity of the chitin to more than 90 percent, thus being an ideal industrial production method.

Drawings

FIG. 1 is a schematic diagram of a strain evolutionary tree of bacteria obtained by 16S rDNA identification of bacteria obtained by separation and purification of the compound lactic acid bacteria.

FIG. 2 is a graph of the effect of different culture conditions of the invention on the calcium content of a fermentation broth, wherein: the influence of different A-glucose addition amount, B-strain inoculation amount, C-culture mode and D-solid-liquid ratio on the calcium content in the fermentation liquor.

FIG. 3 shows the effect of different ultrasonic intermittent durations (5min,10min,20min) and ultrasonic modes (single-time point ultrasonic intermittent and constant ultrasonic) on decalcification of the lactobacillus paracasei fermented crab shells.

FIG. 4 is a graph of the effect of low intensity ultrasound of the present invention on fermentation decalcification at various times.

FIG. 5 shows the effect of low intensity ultrasound on fermentation deproteinization under optimal fermentation conditions according to the present invention.

FIG. 6 shows the low intensity ultrasound of the present invention on Lactobacillus paracasei OD in different culture environments₆₀₀The influence of (c). Wherein: a is MRS culture medium, B is low intensity ultrasound to lactobacillus paracasei OD under 15% glucose₆₀₀The influence of (c).

FIG. 7 is a graph showing the effect of low intensity ultrasound of the present invention on the viable count of Lactobacillus paracasei.

FIG. 8 is the effect of low intensity ultrasound of the present invention on the pH of Lactobacillus paracasei.

Fig. 9 is a scanning electron micrograph of crab shells according to a different treatment mode of the present invention, wherein: a is crab shell, B is residue obtained by independently performing ultrasonic treatment on crab shell, C is residue obtained by fermenting crab shell under optimal fermentation condition, and D is 0.167W/cm²Scanning electron microscope image of residue obtained by intermittent ultrasonic for 10min in cooperation with fermentation.

FIG. 10 shows solid nuclear magnetism of the residue of crab shell fermentation according to the present inventionA resonance spectrum, wherein: a represents¹HNMR spectra, B stands for¹³C NMR spectrum, and gray and black represent the NMR spectrum of the residue obtained by fermenting crab shells under optimal fermentation conditions and the NMR spectrum of the residue obtained by intermittent treatment with low intensity ultrasound for 10min, respectively.

FIG. 11 is an infrared spectrum of crab shells according to different treatment modes of the present invention, wherein: a is crab shell, b is ultrasonic wave to crab shell, c is residue obtained by fermenting crab shell under optimum fermentation condition, d is 0.167W/cm²Performing intermittent ultrasonic treatment for 10min in cooperation with fermentation to obtain infrared spectrogram of residue.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, the present invention will be described in further detail below with reference to the accompanying drawings, but embodiments of the present invention are not limited thereto.

Example 1:

separating lactobacillus paracasei from compound lactobacillus

The invention cultures the compound lactobacillus in MRS solid culture medium by plate marking method, then picks single colony to be cultured in MRS liquid culture medium to obtain pure single bacterium, identifies the bacterium 16S rDNA, and the identification result is that the bacterium is lactobacillus paracasei, the NCBI number of the bacterium is NR _ 025880.1. The specific operation is as follows:

Step two, inoculating and culturing: after the MRS liquid culture medium after autoclaving is cooled to room temperature, the composite lactic acid bacteria powder stored at the temperature of minus 20 ℃ is taken out of a refrigerator, 0.8g of the composite lactic acid bacteria powder is inoculated into the MRS liquid culture medium by 4 percent of inoculation amount, and the MRS liquid culture medium is cultured for 8 hours in a constant temperature incubator at the temperature of 37 ℃ to obtain turbid composite lactic acid bacteria liquid.

Step three, carrying out plate-drawing culture: packaging the plate required for culture, preparing solid culture medium, and sterilizing in autoclave at 121 deg.C for 15 min. A total of 4 plates, 100mL solid medium, were plated, requiring 2% more agar in the solid medium configuration compared to the liquid medium, i.e., 5.4g MRS nutrient broth powder, 2g agar, 100mL distilled water for 100mL solid medium. In addition, the solid culture medium needs to be heated in an enamel jar to slightly boil and then poured into a conical flask for sterilization, and the culture medium needs to be stirred by a glass rod all the time in order to avoid uneven heating of the culture medium during heating. In a clean bench, the sterilized solid culture medium is poured into sterilized plates while the solid culture medium is hot, each plate needs about 20mL of the solid culture medium, and the solid culture medium is condensed at room temperature for 20-40min until the culture medium is solidified. It is worth noting that if the solid culture medium cannot be poured in time, the solid culture medium cannot be placed at room temperature for standby, because the culture medium can be condensed in the conical flask, and can be placed in an oven at 60 ℃ or a constant-temperature water bath for standby to avoid the solidification of the culture medium.

In the superclean bench, each flat plate is divided into 4 regions. Burning the metal wire of the inoculating loop to be red by the outer flame of an alcohol lamp, slightly inclining the inoculating loop, burning a metal rod, cooling, taking a loop of composite lactobacillus liquid by using the inoculating loop, and carrying out plate lineation. After each zone is marked, the inoculating loop is burnt, and the latter zone is required to be connected with the former zone end to end but cannot be lapped with other zones. As the lactic acid bacteria are facultative anaerobes, the inoculated flat plate is wrapped with a sealing film to facilitate the growth of the lactic acid bacteria, and then cultured in a constant temperature incubator at 37 ℃ for 48 hours.

Step four, selecting bacteria: and (3) moving the plate from the incubator to a super clean bench, preparing a sterilized MRS liquid culture medium in advance, burning the inoculating loop, cooling, and selecting a large, full and single bacterial colony which is not overlapped with other bacterial colonies from the plate by using the inoculating loop to be in the liquid culture medium. A total of 7 single colonies were picked, placed in different test tubes, and transferred to a 37 ℃ incubator for static culture for 8 hours.

Step five, fermentation: the cultured bacterial solutions were respectively reserved in sterilized 1.5mL centrifuge tubes, and the serial numbers were clearly indicated for storage at-20 ℃ in a refrigerator. And respectively fermenting crab shells by using the 7 bacteria, measuring the calcium content in the fermentation liquor by using an EDTA method after fermenting for 48 hours, calculating the decalcification rate, and selecting the single bacteria with the highest calcium content in the fermentation liquor. Because lactic acid bacteria are fermented to produce lactic acid, the lactic acid can react with calcium carbonate in crab shells to generate calcium salt dissolved in water, and the higher the calcium content in fermentation liquor is, the higher the decalcification rate of fermentation is, namely, the higher the produced lactic acid is, namely, the stronger the fermentation capacity of the bacteria is.

Step six, strain identification: and (3) selecting the single bacterium with the strongest fermentation capacity through fermentation, and identifying the 16S rDNA strain of the single bacterium. The 6S rDNA identification refers to species identification of bacteria by a method of sequencing a 16S rDNA sequence of the bacteria. The method comprises the steps of bacterial genome DNA extraction, 16S rDNA specific primer PCR amplification, amplification product purification, DNA sequencing, bacterial comparison and the like, a large amount of DNA sequence information is obtained, a conserved sequence region can reflect the genetic relationship among biological species, and a high-mutation sequence region can reflect the difference among the species. The 16S rDNA strain identification firstly needs to extract genome DAN, and mainly comprises the following steps: (1) the bacterial suspension was centrifuged at 4000 Xg for 10min at room temperature to precipitate the bacteria, and then TE Buffer was added to resuspend the bacteria. (2) Adding Lysozyme, and performing water bath at 37 ℃ for 10min to completely break the wall of the bacteria. (3) Adding BTL Buffer and protease K Solution, mixing by vortex, and carrying out water bath at 55 ℃ for 1h to completely lyse bacteria, wherein the vortex is carried out once every 20min during the water bath. (4) Adding RNase A, incubating for 5min, centrifuging to obtain supernatant, adding BDT Buffer, mixing, and incubating at 65 deg.C for 10 min. (5) The genomic DNA was eluted. Next, using 27F: 5' -AGAGAGTTTGATCCTGGCTCAG-3 and 27F: the 5' -AGAGAGTTTGATCCTGGCTCAG-3 primer is used for amplifying the sample DNA, and the amplification process has 35 cycles, wherein each cycle comprises: denaturation at 98 ℃ for 10s, annealing at 55 ℃ for 8s, and extension at 68 ℃ for 2 s. And verifying the amplification product by using 1% agarose gel electrophoresis, performing bidirectional primer sequencing on the PCR product by using a Sanger method, and splicing and processing a sequencing result by using MNAMAN software. The 16S rDNA sequence obtained by sequencing is subjected to Blas comparison on NCBI, so that a known sequence with higher homology with the sequence can be obtained, and a basis is provided for determining the taxonomic status of strains. As shown in FIG. 1, the bacterium was identified as Lactobacillus paracasei, and the NCBI number of the bacterium was NR _ 025880.1.

Example 2

Determination of optimal conditions for producing chitin by lactobacillus paracasei fermented crab shells

The crab shells are fermented by lactobacillus paracasei, the decalcification rate is obtained by measuring the calcium content in the fermentation liquor, the optimal fermentation condition is selected, and the calcium content is measured by using an EDTA method.

The method comprises the following steps:

step one, weighing: weighing 1.00g crab shell, 2-6ml distilled water and 5-25% sugar in a test tube for later use;

step two, sterilization: sterilizing weighed crab shells and the like, liquid culture medium and a wrapped 200 mu L of gun head in a sterilizing pot for 10-30min at high pressure and high temperature;

step three, culturing: cooling the liquid culture medium to room temperature, taking out lactobacillus paracasei (available from Beijing lanborlidide biotechnology, Inc.) with NCBI number NR _025880.1 screened in example 1 from a refrigerator, storing at-20 deg.C, inoculating the liquid culture medium with inoculum size of 5-10% in a super clean bench, and culturing in an incubator at constant temperature for 8-12 h;

step four, inoculating bacteria and fermenting: taking the cultured bacterial liquid out of the incubator, placing the bacterial liquid on a super clean bench, inoculating the bacterial liquid into the crab shells in an inoculation amount of 5-10%, and culturing the crab shells in the incubator for 32-72h at constant temperature.

Step five, an acid-base method: the obtained crude chitin also contains impurities such as calcium and protein, so that the residual calcium and protein are further removed by continuously adopting strong acid and alkali.

Step six, depigmentation: a common depigmentation method is bleaching treatment with strong oxidant such as potassium permanganate, hydrogen peroxide, etc. The invention adopts 30% hydrogen peroxide by mass concentration to react for 1 hour at normal temperature to complete decolorization treatment, and the insoluble chitin is obtained after cleaning and draining.

As shown in fig. 2, the decalcification rate gradually increased as the glucose content increased, and the highest decalcification rate, i.e., the highest fermentation capacity, was observed when the glucose content was 15%. Subsequent increases in glucose addition-15%, 20%, 20%, found that the glucose addition, which was still 15%, was the most fermentative, which was likely that too much sugar inhibited microbial growth. Solid-liquid ratio 1: 3 is that the lactobacillus paracasei has stronger fermentation capacity, and in addition, the inoculation amount is opposite to the fermentationThe effect of (3) is not so great, and 5% of the inoculum size is selected, and the lactobacillus paracasei is a facultative anaerobe, so that the standing fermentation is selected. Thus, the fermentation conditions were determined to be: glucose content 15%, solid-to-liquid ratio 1: 3, 5 percent of inoculation amount, and standing and fermenting for 72 hours at 37 ℃. And after the fermentation time reaches 72h, the fermentation speed is relatively slow, so that most of protein is removed by 72h fermentation, and in order to improve the extraction efficiency of the chitin, the chitin is further prepared by an acid-base method. Using 1.0mol/L ^-1The HCl was reacted at 50 ℃ for 1h to remove the remaining calcium and 8% NaOH was used at 90 ℃ for 1h to remove the remaining protein.

Example 3

The optimal ultrasonic condition for determining the lactobacillus paracasei (NCBI number NR _025880.1) fermenting crab shells is to ferment the crab shells under the determined optimal fermentation condition and carry out ultrasonic stimulation in different growth periods.

Firstly, ultrasonic power of 0.167W/cm is always applied to the fermentation²The ultrasonic treatment at the ultrasonic temperature of 37 ℃ and the ultrasonic frequency of 40KHz was carried out, and the decalcification efficiency of lactobacillus paracasei by the low-intensity ultrasonic treatment was improved by 28.75% for the fermentation of lactobacillus paracasei, taking the control group without the ultrasonic treatment. The experiment shows that the low-intensity ultrasound can promote the growth and metabolism of lactobacillus paracasei, so that more lactic acid is generated, the crab shells are fermented to prepare the chitin in an auxiliary manner, and the fermentation efficiency is improved. However, ultrasonic waves serving as an external physical field can generate certain stress action on microorganisms when acting on the microorganisms, the microorganisms can generate certain resistance to external stress, and certain promotion action can be generated when the conditions of sublethal state are reached, but the process is complex and variable. Therefore, suitable sonication conditions need to be screened. On one hand, the ultrasonic treatment easily causes over-strong ultrasonic action to generate inhibition on microorganisms, on the other hand, the energy is greatly wasted, and the method does not accord with the concept of energy conservation and environmental protection. Therefore, the method mostly adopts short-time and intermittent ultrasonic treatment, and has research significance for promoting the growth and metabolism of lactobacillus paracasei by means of low energy consumption so as to promote the fermentation of the lactobacillus paracasei.

Thus, single low intensity sonication is performed in the incubation, log phase, stationary phase5min, experiments show that single low-intensity ultrasound has no influence on fermentation, probably because the fermentation period is long, bacteria which act by single ultrasound are quickly updated and metabolized, and therefore intermittent ultrasound is selected to promote the fermentation of lactobacillus paracasei. Performing first ultrasonic treatment from the beginning of fermentation, and then performing ultrasonic treatment at an interval of 8h, wherein the ultrasonic power is 0.167W/cm²The ultrasonic temperature is 37 ℃, the ultrasonic frequency is 40KHz, and the ultrasonic time is 5min, 10min and 20min respectively. As can be seen from FIG. 3, compared with non-ultrasonic fermentation, ultrasonic fermentation can improve the decalcification efficiency, the fermentation speed is obviously accelerated, the fermentation capacity is obviously enhanced, ultrasonic treatment for 10min at an interval of 8h has the best promotion effect on fermentation, and the decalcification efficiency can be improved by 48.03%. While the decalcification efficiency can be improved by 28.75 percent by ultrasonic treatment all the time.

Experiments prove that the bacteria liquid is stimulated by low-intensity ultrasonic waves in the growth latency period of the lactobacillus paracasei to have the best promotion effect on the growth of the lactobacillus paracasei, the influence on the growth of the bacteria liquid is not large when the low-intensity ultrasonic treatment is applied to the lactobacillus paracasei in the logarithmic phase of growth, the growth capacity of the bacteria liquid in the logarithmic phase of growth is probably strong, the influence on the bacteria liquid is not large when the ultrasonic treatment is applied in the logarithmic phase, and the growth and reproduction capacity is not greatly improved. The reason for intermittent low-intensity stimulation every 8h is that lactobacillus paracasei enters a growth stabilization period after 8h, and obvious hypha precipitation begins to be generated at the time, which indicates that lactobacillus paracasei gradually enters a vigorous senescence period, so that the ultrasonic treatment is applied to enhance the growth of lactobacillus paracasei at the time.

It can be seen that compared with the common one-time ultrasonic fermentation promotion, the intermittent ultrasonic fermentation promotion method has the advantages that the energy required by a large amount of ultrasonic waves is saved (the one-time ultrasonic waves require 72h, and the total duration of the intermittent ultrasonic waves is only 90min), the fermentation promotion effect can be improved, the cavitation and mechanical effects generated by the ultrasonic effect can enable cell membranes to generate transient sonoporation, and the permeability change caused by the sonoporation is temporary and reversible, so that organisms can restore a normal physiological defense mechanism in a short time after the ultrasonic treatment, and the substances can be successfully transported without permanent cell damage, so that the ultrasonic fermentation has a promotion effect on the lactobacillus paracasei fermented crab shells, but the one-time ultrasonic treatment can cause permanent damage on the cells, and the fermentation promotion effect is inferior to that of the intermittent ultrasonic treatment.

Example 4

Extraction of chitin by combination of microbial fermentation and acid-base method

A total of 72 tubes of crab shells are taken and put in test tubes, and divided into two batches of 36 tubes, wherein one batch is subjected to single-time-point intermittent ultrasound for 5min for 8h, and the other batch is not subjected to ultrasound as a control. And taking 3 tubes out of the two batches at intervals of 8h, and measuring the calcium content in the fermentation liquor by an EDTA method so as to study the influence of the ultrasound on the fermentation at different periods. As can be seen from FIG. 4, in the early stage of fermentation, the low-intensity ultrasonic stimulation has no obvious promotion effect on the fermentation of the lactobacillus paracasei, and has weak inhibition effect, and the ultrasonic stimulation has no obvious promotion effect on the fermentation of the lactobacillus paracasei until the fermentation lasts for 24 hours. The cavitation phenomenon is generated by ultrasonic waves in the early stage of fermentation, the lactobacillus paracasei and bacterial membranes thereof can generate periodic expansion and contraction under the action of the ultrasonic waves, the bacteria cannot adapt to ultrasonic conditions, the bacterial cell membranes are damaged by low-intensity ultrasonic waves, so that the leakage cells of contents die, the bacteria adapt to the ultrasonic conditions gradually in the later stage, the metabolism of the lactobacillus paracasei is changed by the ultrasonic waves, the growth and the propagation are accelerated, and the fermentation rate is improved. It can also be seen from the figure that after the fermentation time reaches 72h, the fermentation speed of lactobacillus paracasei is greatly reduced, so that most of calcium and protein are removed by 72h fermentation.

In order to improve the extraction efficiency of the chitin, the chitin is further prepared by an acid-base method. Using 1.0mol/L^-1The HCl was reacted at 50 ℃ for 1h to remove the remaining calcium and 8% NaOH was used at 90 ℃ for 1h to remove the remaining protein.

Meanwhile, the removal of calcium and protein in the crab shells is completed by combining a microbial fermentation method, a single-time-point ultrasonic-assisted microbial fermentation method and a single-time-point ultrasonic-assisted microbial fermentation method with an acid-base method respectively, and the results are shown in table 1.

TABLE 1 duration required for different chitin extraction methods and its effect on chitin purity

As can be seen from table 1, the single time point ultrasonic-assisted microbial fermentation method can significantly reduce the fermentation time, but the acid-base method combined fermentation has higher chitin extraction efficiency, saves 43.94% of time compared with the single time point ultrasonic-assisted fermentation, saves 61.46% of time compared with the common fermentation, and greatly reduces the usage amount of strong acid and strong base compared with the acid-base method. In addition, compared with a fermentation method, the chitin obtained by combining the intermittent ultrasonic fermentation method with the acid-base method has higher chitin purity.

Example 5:

protein determination by Kjeldahl method

And (3) distilling and cleaning the crab shell residues after the fermentation is finished, performing solid-liquid separation in a centrifugal mode, discarding the supernatant, repeatedly cleaning for 3-4 times, and drying in an oven at 60 ℃ for 24 hours. And measuring the protein content in the fermented soybean by adopting a Kjeldahl method, and respectively calculating the deproteinization rate of ultrasonic fermentation and the deproteinization rate of non-ultrasonic fermentation. Putting a sample into a digestion tube, adding 0.4g of copper sulfate, 6g of potassium sulfate and 20mL of sulfuric acid into a graphite digestion instrument for digestion, continuing to digest for 1h when the temperature of the digestion tube reaches 420 ℃, cooling the liquid in the digestion tube to room temperature, realizing automatic liquid adding (adding sodium hydroxide solution, distilled water and boric acid solution containing a mixed indicator before use) on an automatic Kjeldahl azotometer after the volume is determined in a 50mL volumetric flask, titrating and recording data by using 0.01mol/L of HCl, and calculating the protein content, wherein the result can be shown in FIG. 5. As can be seen from FIG. 5, the removal rate of protein by intermittent fermentation is increased by 64.57% compared with that by non-ultrasonic fermentation, and the removal rate of protein by constant ultrasonic fermentation is increased by 54.78% compared with that by non-ultrasonic fermentation.

The amount of acid and base used, which can be reduced by promoting microbial fermentation with low-intensity ultrasound, can be calculated accordingly, and is shown in table 2.

TABLE 2 reduction of strong acid and base usage after fermentation

As can be seen from Table 2, the use of acid and alkali, particularly intermittent ultrasonic fermentation, in chitin produced by combining biological fermentation with an acid-base method can be greatly reduced, the reduction of strong acid is 84.97%, and the reduction of strong base is 70.6%, so that compared with common fermentation and ultrasonic fermentation, the cost can be reduced and the environment can be protected better.

Example 6:

effect of Low intensity ultrasound on growth of Lactobacillus paracasei

Inoculating lactobacillus paracasei into a sterilized liquid MRS culture medium, culturing for 12h in an incubator at 37 ℃, measuring the absorbance at 600nm every 2h, and drawing the growth curve of the strain according to the absorbance, thereby obtaining the incubation period and the logarithmic phase of the strain liquid. Meanwhile, the experimental group is set to stimulate the bacterial liquid with low-intensity ultrasound, and the influence of the low-intensity ultrasound on the growth of lactobacillus paracasei is researched.

In addition, to investigate the effect of low intensity ultrasound on the growth of Lactobacillus paracasei during fermentation, the study of the effect of low intensity ultrasound on Lactobacillus paracasei OD in a 15% glucose solution under fermentation conditions was specifically selected₆₀₀The influence of (c). Lactobacillus paracasei was inoculated into a sterilized 15% glucose solution, cultured in an incubator at 37 ℃ for 12 hours, and absorbance at 600nm was measured every 2 hours.

Because the lactobacillus paracasei can generate lactic acid, and the lactic acid reacts with calcium carbonate in the crab shells to complete the removal of calcium, the research on the influence of low-intensity ultrasound on the pH of the lactobacillus paracasei liquid can react to a certain extent to promote the lactobacillus paracasei.

As can be observed from FIG. 6, Lactobacillus paracasei is in the latent phase during the first 2h, in the logarithmic phase of growth during 2-8h, and gradually enters the stationary phase of growth later. Low intensity sonicated Lactobacillus paracasei OD₆₀₀Larger, more viable count (fig. 7), lower pH (fig. 8), indicating that low intensity ultrasound promoted growth and reproduction of lactobacillus paracasei.

Example 7:

scanning electron microscope

And (3) freeze-drying the raw crab shells which are not fermented and not subjected to ultrasonic treatment, the crab shells which are not fermented and only subjected to intermittent ultrasonic treatment, the lactobacillus paracasei fermentation residues which are not subjected to ultrasonic treatment and the lactobacillus paracasei fermentation residues subjected to ultrasonic stimulation for 24 hours in a freeze-drying machine, and observing the shape of the obtained dried sample under a scanning electron microscope. Because the powder is dry solid powder, the powder can be shot by directly sticking the powder on the electric conducting glue and spraying gold, the shot pictures are all magnified 10000 times, and the shooting result is shown in figure 9. As can be seen from fig. 9, a is the original crab shell form, B is the form of the crab shell after the crab shell is subjected to ultrasound, the two forms have no obvious difference, and the crab shell has a hollow structure, so that the form of the crab shell ultrasound crab shell is inferred to have no obvious change, and the ultrasound is considered to have no influence on the crab shell structure. C is the crab shell residue form obtained by fermentation, D is the crab shell residue form obtained by low-intensity ultrasound-promoted fermentation, and the obvious filamentous structure is shown in C, and the D picture is not shown, which is probably because the ultrasonic wave makes the crab shell fermented by the bacterial liquid to play a role, so that the filamentous structure of the crab shell is broken.

Example 8:

nuclear magnetic resonance

The chitin obtained by fermentation and the chitin obtained by promoting the fermentation through low-intensity ultrasonic treatment are subjected to high-resolution solid-state Nuclear Magnetic Resonance (NMR) spectra at Bruker 400M, the MAS spin rate is 10Khz, the recovery time is 4s, the pulse program used for acquisition is cp, the pre-scanning delay is 6.5 mus, and a rotor with the length of 4mm is adopted for carrying out solid-state nuclear magnetic resonance tests. Carbon Spectroscopy by CP MAS NMR (Cross-polarized magic Angle rotating Nuclear magnetic resonance) ((C))¹³C) Testing of hydrogen spectra using DDMAS NMR (dipole decoupled magic Angle rotating Nuclear magnetic resonance) ((ii))¹H)。

The solid nuclear magnetic resonance is used for characterizing the fermentation product, namely the chitin, and the result shows that the hydrogen spectrums of the fermentation residues of the fermentation group and the ultrasonic fermentation group are almost overlapped (figure 10A), and the anomeric proton (H-1) in the figure has a signal peak in the range of 4.5-5.5 ppm. Among them, 4.51ppm of the GlcN residue H-1 signal peak, 3.41ppm of the GlcN residue H-3 signal peak, 1.38ppm of the GlcN residue H-2 signal peak, which are characteristic hydrogen absorption peaks of chitin. The carbon spectra of the fermentation group and the ultrasonic fermentation group residues are almost the same (FIG. 10B)Wherein the absorption peaks at 172.84ppm and 22.69ppm are acetyl C ═ O and CH respectively₃Resonance absorption peak of carbon, and ¹³The C signal comprises 54.97ppm (C)₂)，73.46ppm(C₅)，75.60ppm(C₃)，83.06ppm(C₄)，103.7 9ppm(C₁) 128.30ppm (amide CN), these absorption spectra are also similar to those of chitin. The chitin configuration is not changed because the molecular chain arrangement mode of chitin can form a stable hydrogen bond structure, and the configuration of the chitin cannot be changed by ultrasonic waves. In addition, in the nuclear magnetic resonance spectrum, both alpha-chitin and gamma-chitin have two absorption peaks (MI-kyeon JANG) at about 73 ppm and 75ppm, so that the nuclear magnetic resonance spectrum cannot judge whether the fermentation product is chitin or gamma-chitin, and the infrared spectrum is required to further explore the structures of the chitin and the gamma-chitin.

Example 9:

infra-red spectrogram

The potassium bromide, sample were oven dried at 60 ℃ to constant weight, then the potassium bromide was mixed with the sample at a ratio of 100: 1 grinding in a mortar along one direction to avoid transgene of the sample. In addition, the grinding process does not need to breathe or speak as much as possible, so that CO generated is avoided₂And (3) entering a sample to generate an additional absorption peak, and grinding the mixed sample of the potassium bromide and the sample until no obvious particles are visible to naked eyes. Then, the sheet can be pressed, and the sheet is as flat and transparent as possible and placed under infrared light to take a spectrogram.

FIG. 11 is the infrared spectra of the crab shell, the intermittent ultrasonic crab shell, the fermented crab shell residue, and the intermittently ultrasonic fermented crab shell residue, respectively, and it can be seen from FIG. 12 that the chemical structure of chitin cannot be changed by low intensity ultrasound. The existence of chitin can be confirmed in the main absorption peak of the chitosan, which is 3443cm^-1And 3412cm^-1The absorption peak at the position may be caused by-OH or N-H stretching vibration in chitin, and the absorption peak at the position of 1121cm-1 may be caused by C-O-C stretching vibration in chitin, and the absorption peak at the position of 1616cm^-1The absorption peak at (b) may be caused by the C ═ O deformation shock of the amide I in chitin. The infrared spectrum of the ultrasonic-assisted lactobacillus paracasei after treatment is about 3497cm^-1And 3439cm^-1Two peaks corresponding to intramolecular hydrogen bonds of chitin O H (6) · O C and O H (3) · O-5, respectively, the strong absorption peak of carbonyl region is the typical characteristic of alpha-chitin, and the infrared absorption spectrum of alpha-chitin is 1662cm^-1And 1629cm^-1Two independent peaks are present, namely intermolecular hydrogen bond CO & HN and intramolecular hydrogen bond CO & HOCH₂. At 3261cm^-1The band caused by the amide NH belongs to the vibration of the bond NH group in the molecule, and the fermentation product is proved to be alpha-chitin. Research reports that chitin in crab shells is alpha-chitin, and the fact that the configuration of the chitin cannot be changed under ultrasonic-assisted lactobacillus paracasei treatment is proved.

The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the scope of the present invention, therefore, the present invention is not limited by the appended claims.

Claims

1. The preparation method of the chitin is characterized by comprising the following steps:

2. The method for preparing chitin according to claim 1, comprising the steps of:

B. inoculating the fermented lactic acid bacteria into a liquid culture medium in an inoculation amount of 5-10%, culturing at constant temperature for 8-12 h, and obtaining the bacterial liquid after culturing;

C. And (2) mixing the sterilized crab shell powder according to the mass volume ratio of 1 g: 2-6 ml of the crab shell-sugar water mixture is prepared in the sugar water, the mass concentration of sugar in the sugar water is 5-25%, and the crab shell-sugar water mixture is reserved

D. adding 1mol/L into the primary fermentation liquor^-1Then adding 8% NaOH solution for treatment, and bleaching to obtain the insoluble chitin.

3. The method for preparing chitin according to claim 1, wherein in step C, the ultrasonic treatment is single-time intermittent ultrasonic, specifically: performing ultrasonic treatment at an interval of 8h for 5-20 min after performing ultrasonic treatment for the first time for 5-20 min at the beginning of fermentation, wherein the ultrasonic power is 0.167W/cm²The ultrasonic frequency is 40KHz, and the ultrasonic temperature is 37 ℃.

4. The method for preparing chitin according to claim 3, wherein the first ultrasonic treatment is performed for 10min at the beginning of fermentation and then every 8h ultrasonic treatment is performed for 10 min.

5. The method for preparing chitin according to claim 1, wherein in step C, sterilized crab shells are mixed according to the mass volume ratio of 1 g: 3ml is prepared in the sugar water; the sugar water is glucose water, and the mass concentration of sugar in the sugar water is 15%.

6. The method of producing chitin according to claim 1, wherein in step A, the Lactobacillus paracasei is Lactobacillus paracasei having NCBI of NR _ 025880.1.

7. The method for preparing chitin according to claim 1, wherein the specific operation of screening lactobacillus in step a is: separating and purifying the compound lactobacillus, selecting a plurality of single bacterial colonies, respectively fermenting crab shells by the obtained single bacteria, determining the single bacteria with the strongest fermentation capacity, identifying the strains, and determining the fermented lactobacillus.

8. The method of claim 7, wherein the chitin is isolated and purified by plate streaking, and the species is identified by 16S rDNA identification.

9. The method for preparing chitin according to claim 1, wherein in step D, the specific operation of bleaching is as follows: and (3) carrying out a normal-temperature reaction for 1h by adopting hydrogen peroxide with the mass concentration of 30% to complete the decolorization treatment.

10. The method of claim 1, wherein in step B, the culture medium is MRS liquid medium; in the step C, the crab shell sterilization is specifically performed in a sterilization pot at 121 ℃ for 10-30 min.