Chitin-degrading strain and its application

Abstract

The present invention discloses a chitin degradation strain and its application, the strain is a luminous bacillus strain LYM-1, classified and named as luminous bacillus (Photobacterium sp.), and the deposit number is GDMCCNo.62616. The strain and budding short stalk mold are respectively degraded shrimp shells and straws for mixed fermentation of fermented liquid, a simple separation and purification method is used to obtain a degradable film polysaccharide component, and a biodegradable film with excellent performance is obtained by compounding with a cross-linking agent PVA and a plasticizer glycerol. Compared with the prior art, the raw material of the present invention is taken from agricultural by-products or waste, and the raw material processing process is green and pollution-free, and the obtained product degradable film is green and environmentally friendly. Therefore, the product and method provided by the present invention are not only green and environmentally friendly, but also simple in operation process, and high-value utilization of wastes such as straws and shrimp shells is realized at the same time.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to a chitin-degrading strain and application thereof. Background Art

[0002] Degradable film is a kind of degradable film mainly composed of cellulose, starch, chitosan and other natural polysaccharide substances, which is formed by adding plasticizers and film-forming agents. The final product of its degradation is CO 2 and H 2 O, will not cause secondary pollution to the environment. In recent years, more and more scientific researchers have prepared low-cost, environmentally friendly natural polymer compounds into biodegradable films, and developed environmentally friendly, degradable films by in-depth research on the modification technology and molding process of natural polymer materials. Cellulose-based degradable films are mainly mixed with polylactic acid (PLA), polyvinyl alcohol (PVA), modified starch, etc. to give them high mechanical strength, water resistance and other physical properties. Starch-based degradable films are mainly made by blending starch in different proportions with polymer materials such as polyethylene / polyvinyl chloride, polyvinyl alcohol, and protein, and then extruding and blowing them into films. The resulting films have strong mechanical properties. With the prevalence of environmental protection concepts, finding cheap raw materials for preparing degradable agricultural films and green and environmentally friendly film-making processes has become a major development trend. Summary of the invention

[0003] In view of the shortcomings of the prior art, the present invention provides a chitin-degrading strain and its application. The strain can use cheap by-products such as shrimp shells and straw as raw materials in a green and low-carbon way to prepare a degradable film with heat-insulating, moisture-keeping and waterproof properties that can be degraded by microorganisms and provide nutrition for plant growth.

[0004] The present invention is achieved through the following technical solutions:

[0005] A chitin degradation strain is a Photobacterium sp. strain LYM-1, which is classified and named as Photobacterium sp. and has a preservation number of GDMCC No.62616.

[0006] Preferably, the nucleotide sequence of 16s rDNA of the strain is as shown in SEQ ID NO:1.

[0007] Application of a chitin-degrading strain in degrading shrimp shells.

[0008] Application of a chitin-degrading strain in the preparation of degradable films.

[0009] A mixed bacterial fermentation method of chitin-degrading strains, characterized in that it comprises the following steps:

[0010] Step 1) inoculating the chitin-degrading strain into LB medium and placing it in an incubator at 37° C. for 12 hours for activation culture to prepare seed solution A;

[0011] Step 2) inoculating Aureobasidium pullulans into a YPD medium and placing it in an incubator at 28° C. for 72 hours of activation culture to prepare a seed solution B;

[0012] Step 3) seed solution A and seed solution B were inoculated into a container containing 100 mL of fermentation medium at a volume ratio of 1:1, with an inoculation amount of 5%, and cultured at 28° C. and 180 r / min for 7 days;

[0013] The preparation method of the fermentation medium is as follows: 15 g / L straw, 30 g / L shrimp shell, natural pH, and sterilization at 121° C. for 20 min.

[0014] The fermentation liquid obtained by the above mixed bacteria fermentation method.

[0015] Application of the fermentation broth in the preparation of degradable films.

[0016] A degradable film comprises the following raw materials in parts by weight: 0.1-10 parts of the chitin-degrading strain, 0.1-10 parts of budding short-stalked mold, 1-10 parts of straw powder, 1-10 parts of crayfish shell powder, 70 parts of water, and 1 part of PVA, wherein the budding short-stalked mold is purchased from the China General Microbiological Culture Collection Center with a collection number of CGMCC 3.3984.

[0017] Preferably, the mixture further comprises 0.2 parts of glycerol and 0.05 parts of Tween 20.

[0018] A method for preparing a degradable film comprises the following steps:

[0019] Step a) mixed fermentation: 0.1 to 10 parts of the chitin-degrading strain are inoculated into LB medium and placed in an incubator at 37° C. for 12 hours of activation culture to prepare seed solution A; 0.1 to 10 parts of budding Aureobasidium are inoculated into YPD medium and placed in an incubator at 28° C. for 72 hours of activation culture to prepare seed solution B; seed solution A and seed solution B are inoculated into a container containing 100 mL of fermentation medium at a ratio of 1:1, with an inoculation amount of 5%, and cultured at 28° C. and 180 r / min for 7 days to obtain fermentation solution;

[0020] The fermentation medium is prepared from 1 to 10 parts of straw powder and 1 to 10 parts of crayfish shell powder;

[0021] Step b) fermentation broth concentration: the fermentation broth obtained in step a) is centrifuged at 8000 r / min for 5 min to remove the fermentation residue, and then concentrated by a rotary evaporator to make the polysaccharide content reach 2 mg / mL. The concentrated fermentation broth is dialyzed to remove small molecular impurities therein to obtain a polysaccharide solution;

[0022] Step c) Preparation of a degradable film: dissolving 1 part of PVA in distilled water, heating and stirring to accelerate its dissolution, obtaining a uniform solution with a concentration of 2%, namely, a polyvinyl alcohol solution; taking equal amounts of the polysaccharide solution and the polyvinyl alcohol solution, stirring and mixing them, to obtain a film-forming solution;

[0023] 0.2 parts of glycerol and 0.05 parts of Tween 20 can be added to the film-forming solution;

[0024] Step d) Pour the film-forming liquid obtained in step c) into a polystyrene film mold, place it in an oven at 50° C. and dry it for 48 hours. After the film is formed, peel it off and place it in a dryer for 48 hours to obtain the film.

[0025] The beneficial effects of the present invention are as follows:

[0026] (1) The chitin-degrading strain of the present invention can effectively degrade chitin in shrimp and crab shells, which is beneficial to the efficient resource utilization of shrimp and crab shells.

[0027] (2) The present invention adopts a green and low-carbon fermentation method to achieve high-value utilization of low-value products, solving the problems of difficult treatment and environmental pollution of waste and agricultural products such as shrimp shells and straw.

[0028] (3) The present invention uses domestic waste and agricultural products such as shrimp shells and straw as raw materials to successfully prepare a degradable film that has heat preservation, moisture retention, and waterproof properties and can be degraded by microorganisms and provide nutrients for plant growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the phylogenetic tree of Photorhabditis elegans strain LYM-1;

[0030] Figure 2 This is a diagram showing the effect of photobacterium strain LYM-1 degrading chitin;

[0031] Figure 3 This is a graph showing the results of the strain compatibility test in Example 2;

[0032] Figure 4 The change curves of chitinase (a) and cellulase (b) activities of the experimental group and the standard group during the fermentation process in Example 2;

[0033] Figure 5 The curves of the content of reducing sugar (a), total sugar (b), and polysaccharide (c) in the experimental group and the standard group during the fermentation process in Example 2 are shown;

[0034] Figure 6 These are the morphological characteristics of the three degradable film samples in Example 4. DETAILED DESCRIPTION

[0035] The present invention is further explained below in conjunction with the accompanying drawings and specific embodiments. This specific implementation method is implemented based on the technical solution of the present invention. It should be understood that these solutions are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0036] The strain provided by the invention and the raw materials and reagents used in its application can all be purchased from the market.

[0037] Example 1 Screening and identification of bacterial strains

[0038] 1. Preparation of culture medium

[0039] Enrichment medium: MgSO 4 7H 2 O 0.5g / L, KH 2 PO 4 0.7g / L, K 2 HPO 4 0.3g / L, FeSO 4 7H 2 O0.01g / L, powdered chitin 4g / L.

[0040] Screening medium: colloidal chitin 4.0 g / L, (NH 4 ) 2 SO 4 2.0g / L, composite salt mother solution 10g / L, agar 15g / L, pH 7.0.

[0041] Preparation of colloidal chitin: Weigh 5g of chitin powder, slowly add it to 150mL of pre-cooled concentrated hydrochloric acid, mix and stir on ice overnight. Add the above mixture to 450mL of ice-cold 50% ethanol, stir rapidly and leave at room temperature (25°C) overnight. Centrifuge the precipitated colloidal chitin at 4°C, 5000g for 20min to collect the precipitate. Rinse the precipitate repeatedly with sterile distilled water until the pH is 7.0. Add 100mL of water or buffer to prepare a 5% colloidal chitin solution and store at 4°C. Sterilize the colloidal chitin solution separately from the other components (sterilize at 121°C for 20min), mix before pouring on the plate, adjust the pH to 7.2-7.4, and then pour on the plate.

[0042] 2. Screening of strains

[0043] The initial inoculum was mud from the Yellow Sea in Dafeng District, Yancheng City, Jiangsu Province. One gram of the collected mud sample was added to the sterilized enrichment medium and cultured at 30°C and 200 r / min for 48 hours. A sample of the enrichment culture fluid was taken and diluted with sterile water to make 10 1 ~10 6 times, then take 10 4 , 10 5 and 10 6 0.1 mL of each sample with three dilution multiples was spread on screening medium plates, cultured at 30°C for 72 hours, and observed. The strains that could produce transparent circles were selected and streaked on new screening medium plates until a single colony pure culture product was obtained. The strains were then further identified by molecular biology or stored at 4°C for later use.

[0044] 3. Molecular Biology Identification

[0045] (1)Universal primers were used. The primer names and sequences are as follows:

[0046] 27F (SEQ ID NO:2): 5'-AGAGTTTGATCCTGGCTCAG-3'

[0047] 1492R(SEQ ID NO:3):5'-TACGGCTACCTTGTTACGACTT-3'

[0048] (2) Extracting genomic DNA;

[0049] (3) synthesizing primers and amplifying 16S sequences;

[0050] Reaction system: 2×TaqPCR Master Mix 12.5μL, upstream and downstream primers 1μL each, DNA template 0.5μL, ddH 2 O 10 μL;

[0051] Reaction program: 95°C for 5 min; 94°C for 1 min, 55-58°C for 1 min, 72°C for 90 s, 30 cycles; 72°C for 10 min.

[0052] (4) PCR product purification: Ultra Clean PCR Clean-up kit;

[0053] (5) PCR product sequencing: The PCR product is sent to a sequencing company for 16S sequencing;

[0054] (6) BLAST sequencing results in the GenBank database; the sequencing results were analyzed using DNAStar software, and the 16s rDNA sequence of the strain was compared with the 16s rDNA sequences of other bacteria in GenBank. In the result with the highest matching degree, the 16s rDNA sequence measured by the strain had a homology of 99% with Photobacterium sp.LAM9072, confirming that the strain was a Photobacterium sp. and was named LYM-1.

[0055] Based on the measured 16s rDNA gene sequence of the isolated strain (as shown in SEQ ID NO: 1) and the 16s rDNA gene sequences of other bacteria with a high degree of matching in GenBank, a phylogenetic tree was constructed using MEGA11 software, as shown in FIG. Figure 1 As shown. Figure 1 It can be seen that the isolated strain belongs to the genus Photorhabdus of the order Vibrioales of the class Proteobacteria.

[0056] The isolated strain is a chitin-degrading strain, the Photobacterium strain LYM-1, classified and named Photobacterium sp., and was deposited in the Guangdong Provincial Microbial Culture Collection Center on July 11, 2022. The address of the collection center is No. 100, Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, and the collection number is GDMCC No. 62616.

[0057] The experiment showed that the selected luminescent bacteria strain LYM-1 had a good degradation effect on chitin. Figure 2 As shown, in the colloidal chitin culture dish, an obvious transparent circle appears around the colony. This is because the colloidal chitin is hydrolyzed into oligosaccharides or monosaccharides, resulting in the degradation of polysaccharides. The appearance of the transparent circle indicates that the chitinase produced by the metabolism of Photorhabdus luminescens can degrade chitin and use the hydrolysis products such as oligosaccharides as carbon sources for its own growth.

[0058] Example 2 Mixed Fermentation

[0059] 1. Strain compatibility test

[0060] Aureobasidium pullulans was purchased from China General Microbiological Culture Collection Center with the collection number of CGMCC 3.3984.

[0061] Pour the budding Aureobasidium into a YPD medium plate, inoculate the Photorhabdus species LYM-1 on it, and inoculate the budding Aureobasidium and Photorhabdus species LYM-1 separately on two other YPD medium plates as controls. Culture conditions: static culture at 28°C for 5 days.

[0062] Pour the budding Aureobasidium into an LB medium plate, inoculate the Photorhabdus species LYM-1 on it, and inoculate the budding Aureobasidium species and Photorhabdus species LYM-1 separately on two other LB medium plates as controls. Culture conditions: static culture at 28°C for 5 days.

[0063] Observe their growth and compare the growth of the two strains on the same plate. Figure 3 As shown, both strains can grow well. On the first day of culture, the two strains showed a clear and uniform distribution. On the second day of culture, the two strains showed fusion growth, and there was no inhibition or antagonistic growth phenomenon, indicating that the two strains can be cultured together without antagonism.

[0064] 2. Liquid fermentation

[0065] (1) Preparation of culture medium

[0066] LB medium: yeast extract powder 5 g / L, tryptone 10 g / L, sodium chloride 10 g / L, pH 7.0, sterilization at 121°C for 20 min.

[0067] YPD medium: 20 g / L tryptone, 10 g / L yeast extract powder, 20 g / L glucose, natural pH, sterilized at 121°C for 20 min.

[0068] Fermentation medium: straw 15g / L, shrimp shell 30g / L, natural pH, sterilization at 121℃ for 20min.

[0069] (2) Preparation of seed solution

[0070] The Photorhabditis elegans strain LYM-1 and Aureobasidium pullulans stored in a -80°C ultra-low temperature freezer were picked out respectively.

[0071] The Photorhabditis elegans strain LYM-1 was inoculated into LB medium and placed in an incubator at 37° C. for 12 h of activation culture to prepare seed solution A.

[0072] The budding Aureobasidium pullulans was inoculated into the YPD medium and placed in an incubator at 28°C for 72 hours for activation culture to prepare the seed solution B.

[0073] (3) Liquid fermentation culture

[0074] In the experimental group, seed solution A and seed solution B were inoculated into a 250 mL Erlenmeyer flask containing 100 mL of fermentation medium at a volume ratio of 1:1, with an inoculation amount of 5%, and cultured at 28°C and 180 r / min for 7 days.

[0075] In the standard group, seed solution A and seed solution B were separately inoculated into the fermentation medium, and other conditions were the same.

[0076] There were 3 parallels in each experimental group and standard group.

[0077] 3. Determination of chitinase, cellulase, reducing sugar, total sugar and polysaccharide content

[0078] (1) Chitinase activity determination: With colloidal chitin as substrate, the DNS colorimetric method (3,5-dinitrosalicylic acid) was used to measure reducing sugar. A reaction system consisting of 0.1 mL of 1% colloidal chitin and 50 μL of enzyme solution was added. The reaction was incubated at 37°C for 30 min, and then incubated in boiling water for 5 min to terminate the reaction. 300 μL of DNS reagent was added, and the reaction was incubated in boiling water for 5 min. The reaction was cooled to room temperature, and water was added to 1 mL. After centrifugation, the supernatant was collected and the absorbance was measured at a wavelength of 520 nm. An equal amount of inactivated enzyme solution was used as a blank control. Enzyme activity is defined as the amount of enzyme required to convert the substrate colloidal chitin to produce 1 μg of reducing sugar at 37°C for 1 min, which is defined as 1 activity unit (U).

[0079] Enzyme activity (U / mL) = ΔOD × V 总 ×n / Δt×V 酶 ×10 -6

[0080] In the above formula: ΔOD is the change value of absorbance OD; V 总 is the total volume of the reaction solution (mL); n is the enzyme solution dilution multiple; Δt is the reaction time; V 酶 is the volume of enzyme solution (mL).

[0081] (2) Cellulase activity determination: Carboxymethyl cellulose was used as the substrate, and the reducing sugar was measured by the DNS colorimetric method (3,5-dinitrosalicylic acid). A reaction system consisting of 0.1 mL of 1% carboxymethyl cellulose (dissolved in acetic acid-sodium acetate buffer at pH 4.8) and 50 μL of enzyme solution was added. The reaction was incubated at 50°C for 30 min, and then incubated in a boiling water bath for 5 min to terminate the reaction. 300 μL of DNS reagent was added, and the reaction was incubated in a boiling water bath for 5 min. The reaction was cooled to room temperature, and water was added to 1 mL. After centrifugation, the supernatant was collected and the absorbance was measured at a wavelength of 520 nm. An equal amount of inactivated enzyme solution was used as a blank control. Enzyme activity was defined as the amount of enzyme required to convert the substrate carboxymethyl cellulose to produce 1 μg of reducing sugar at 50°C for 1 min, which was defined as 1 activity unit (U).

[0082] Enzyme activity (U / mL) = ΔOD × V 总 ×n / Δt×V 酶 ×10 -6

[0083] In the above formula: ΔOD is the change value of absorbance OD; V 总 is the total volume of the reaction solution (mL); n is the enzyme solution dilution multiple; Δt is the reaction time; V 酶 is the volume of enzyme solution (mL).

[0084] (3) Determination of reducing sugar content: The standard curve method was used for determination. 2 mg of glucose standard was weighed and added to 2 mL of deionized water to prepare a 1 mg / mL glucose solution as the standard solution. Then, 0, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, and 0.2 mL of the glucose standard solution were respectively transferred into a 1.5 mL EP tube with a pipette. 300 μL of DNS reagent was added. The tube was placed in a boiling water bath for 5 min, cooled to room temperature, and filled with water to 1 mL. After centrifugation, the supernatant was collected and the absorbance was measured at a wavelength of 520 nm. The glucose concentration was used as the horizontal axis and the absorbance was used as the vertical axis to obtain a standard curve of the glucose solution.

[0085] After the absorbance of the fermentation broth is measured, the measured absorbance is substituted into the regression equation of the glucose standard curve to calculate the reducing sugar content in the fermentation broth.

[0086]

[0087] In the above formula: x is the reducing sugar content in the fermentation broth; y is the absorbance of the sample; a is the slope of the regression equation; b is the intercept of the regression equation on the y-axis; c is the dilution multiple of the sample.

[0088] (4) Determination of total sugar content: A standard curve was used for determination. 2 mg of glucose standard was weighed and added to 2 mL of deionized water to prepare a 1 mg / mL glucose solution as the standard solution. Then, 0, 0.02, 0.04, 0.06, 0.08, 0.1, 0.12, 0.14, 0.16, 0.18, and 0.2 mL of the glucose standard solution were respectively transferred into a 1.5 mL EP tube using a pipette. After adding distilled water to 0.2 mL, 0.2 mL of phenol (mass fraction: 5%) was added. Then, 1 mL of concentrated sulfuric acid was added. After mixing, the mixture was reacted at room temperature for 30 min. The absorbance of the solution was measured at the characteristic absorption peak of 490 nm. The standard curve of the glucose solution was obtained with the glucose concentration as the horizontal axis and the absorbance as the vertical axis.

[0089] Pipette 0.2 mL of filtered fermentation broth into a 1.5 mL EP tube. The remaining steps are the same as the determination method of the standard curve. After measuring the absorbance, substitute the measured absorbance into the regression equation of the glucose standard curve to calculate the total sugar content in the fermentation broth.

[0090]

[0091] In the above formula: x is the total sugar content in the fermentation broth; y is the absorbance of the sample; a is the slope of the regression equation; b is the intercept of the regression equation on the y-axis; c is the dilution multiple of the sample.

[0092] (5) The polysaccharide content in the fermentation broth was preliminarily estimated by the reducing sugar and total sugar contents (total sugar minus reducing sugar).

[0093] The experiment showed that there was no obvious incompatibility between the two strains used for fermentation, the growth rates of the two strains were similar, there was no inhibition zone between the two after 24 hours, and after 48 hours the two strains could fuse without obvious boundaries. Figure 4 (a) shows that the cellulase activity increased in the monoculture group (standard group) and coculture group (experimental group) of A. pullulans, while the Photorhabdus species LYM-1 did not have any cellulase activity. The highest activity of A. pullulans monoculture (standard group) at 7 days was 115.92 U / mL, showing good ability to degrade cellulose. For chitinase activity, Figure 4 As shown in (b), the maximum chitinase production observed in the single culture fermentation (standard group) using the Photorhabdus species LYM-1 was the same as that in the mixed culture fermentation, which was the highest at 5 days, with a value of 19.67 U / mL. Figure 5 As shown in (a), the reducing sugar in the fermentation broth of the monoculture of Aureobasidium pullulans (standard group) decreased in the first 4 days, indicating that Aureobasidium pullulans grew rapidly. Subsequently, a slight increase in the reducing sugar concentration at 5 days indicated a high yield of cellulase, which was consistent with the cellulase activity results. However, after 6 days, the reducing sugar tended to stagnate, indicating that the strain reached a balance in the utilization and production of reducing sugars. The monoculture of Photorhabdus species LYM-1 (standard group) maintained a similar reducing sugar concentration. The total sugar yield was Figure 5 (b) shows that there is not much difference between the monoculture (standard group) and the coculture (experimental group), both of which slowly decrease first and then increase, indicating that the strains first need them to obtain nutrients and then produce extracellular sugars. However, the polysaccharides required in the experiment were measured by subtracting reducing sugars from total sugars, such as Figure 5 As shown in (c), the polysaccharide concentration in the fermentation broth of the co-cultured (experimental group) decreased in the first 5 days, and then increased, reaching a maximum concentration of 1.17 mg / mL on day 7. The polysaccharide content in the fermentation broth of the single cultured (standard group) strain showed a similar trend.

[0094] Example 3 Fermentation broth treatment and film making

[0095] 1. Separation and purification of fermentation broth

[0096] The fermentation broth obtained by the liquid fermentation culture of the experimental group of Example 2 was filtered and centrifuged to obtain the supernatant. After the supernatant was concentrated by rotary evaporation at 55°C, 95% ethanol was added at a ratio of filtrate: ethanol = 1:4, and the mixture was allowed to stand at 4°C for 12 hours. After centrifugation at 4000r / min for 10 minutes, the precipitate was collected and dissolved in distilled water. The polysaccharide solution was mixed with Sevage reagent (chloroform: n-butanol = 4:1) at a volume ratio of 5:1, shaken for 30 minutes, and the aqueous phase was taken after centrifugation, and repeated until all proteins were removed. Three times the volume of anhydrous ethanol was added to the concentrated solution from which the protein had been removed, and the solution was allowed to stand at 4°C in a refrigerator for 24 hours, centrifuged at 8000r / min for 5 minutes, and the supernatant was discarded. Ultrapure water was added to the obtained polysaccharide precipitate to dissolve it to obtain a uniform solution, which was placed in a treated dialysis bag and dialyzed overnight.

[0097] 2. Fermentation liquid concentration and film making

[0098] The fermentation broth obtained by the liquid fermentation culture of the experimental group of Example 2 was centrifuged at 8000 r / min for 5 min to remove the fermentation residue, and then concentrated by a rotary evaporator to make the polysaccharide content reach 2 mg / mL. The concentrated fermentation broth was dialyzed to remove small molecular impurities therein to obtain a polysaccharide solution.

[0099] Weigh an appropriate amount of polyvinyl alcohol 17-99, dissolve it in distilled water, heat and stir to accelerate its dissolution, and obtain a uniform solution (polyvinyl alcohol solution) with a concentration of 2%. Take equal amounts of polysaccharide solution and polyvinyl alcohol solution and stir and mix them, pour the obtained film-forming liquid into a polystyrene film mold, put it in a 50°C oven to dry for 48 hours, and after it forms a film, peel it off and put it in a dryer to balance for 48 hours, so as to obtain a degradable film A.

[0100] 0.25% of the total volume of glycerol and 0.01% of the total volume of Tween 20 were added to the above membrane-forming solution to prepare a degradable membrane B.

[0101] Example 4 Performance Test of Degradable Film

[0102] The following performance tests were performed using polyvinyl alcohol (PVA) film as sample 1, degradable film A (PVA + fermentation broth) prepared in Example 3 as sample 2, and degradable film B (PVA + fermentation broth + glycerol + Tween 20) prepared in Example 3 as sample 3:

[0103] 1. Mechanical properties test

[0104] The mechanical properties (hardness, hardness deformation and hardness work) of each sample were measured at room temperature using a texture analyzer (Shanghai Baosheng Technology). The film samples were cut into square pieces (10×2 cm 2), clamped with a clamp and placed in the instrument. For this purpose, the compression test was analyzed at a target distance of 5.0 mm using a rod probe (TA39) with a diameter of 2 mm and applied at a test speed of 0.5 mm / s. The measurements were performed in triplicate and the average value with standard deviation is reported.

[0105] 2. Tensile performance test

[0106] The tensile tests were performed at room temperature using a texture analyzer (Shanghai Baosheng Technology) with a crosshead maximum strength capacity of 500 N. A fixed crosshead speed of 5 mm / min was used in all cases, and the results were taken as the average (standard deviation) of three tests. Rectangular film samples (10 × 2 cm 2 The significance of the mean values ​​of all parameters was tested by analysis of variance using SPSS software.

[0107] 3. Water absorption and water solubility test

[0108] The dried film samples were cut into squares (10 × 10 cm 2 ), weigh its mass W using an electronic balance 0 , placed in a conical flask containing 200 mL of distilled water, taken out after 24 hours, and the surface moisture of the film was absorbed with filter paper, and the weight was recorded as W 1 , and then dried at 50℃ to constant weight and weighed 2 .

[0109] The water absorption and solubility were calculated as follows:

[0110] Water absorption = (W 1 -W 0 ) / W 0 ×100%

[0111] Water solubility = (W 0 -W 2 ) / W 0 ×100%

[0112] The morphological characteristics of the three membrane samples are as follows: Figure 6 The performance test results are shown in Table 1 below:

[0113] Table 1 Performance test results

[0114]

[0115] From the data in Table 1, it can be seen that the performance of degradable membrane B (PVA+fermentation broth+glycerol+Tween 20) is improved compared with the single polyvinyl alcohol film. The strength is increased from 2.01±0.78MPa to 2.65±0.78MPa; the elongation at break is increased from 5.31±2.17% of the single membrane and 4.86±1.86% of the degradable membrane A (PVA+fermentation broth) to 118.18±36.01%; the water solubility is increased from 33.42±3.21% of the single membrane and 22.34±6.42% of the degradable membrane A to 18.12±4.23%; and the water absorption of the single membrane is 607.77±23.12%, the degradable membrane A is 659.11±33.42%, and the degradable membrane B is 619.21±45.02, and the difference between the three is not much.

[0116] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A chitin-degrading strain, It is characterized in that The strain is a Photorhabdus strain LYM-1, which is classified as Photorhabdus ( Photobacterium sp.), with the deposit number being GDMCC No.62616.

2. Use of a chitin-degrading strain as claimed in claim 1 in degrading shrimp shells.

3. Use of a chitin-degrading strain as claimed in claim 1 in the preparation of a degradable film.

4. A mixed fermentation method of chitin-degrading strains according to claim 1, It is characterized in that The following steps are involved: Step 1) inoculating the chitin-degrading strain as claimed in claim 1 into LB medium and placing it in an incubator at 37° C. for 12 hours for activation culture to prepare seed solution A; Step 2) inoculating budding Aureobasidium pullulans into a YPD medium and placing it in an incubator at 28° C. for 72 hours for activation culture to prepare a seed solution B; the budding Aureobasidium pullulans was purchased from the China General Microbiological Culture Collection Center with a collection number of CGMCC3.3984; Step 3) seed solution A and seed solution B were added to a container containing 100 mL of fermentation medium at a volume ratio of 1:1, with an inoculation amount of 5%, and cultured at 28°C and 180 r / min for 7 days; The preparation method of the fermentation medium is as follows: 15 g / L straw, 30 g / L shrimp shell, natural pH, and sterilization at 121° C. for 20 min.

5. The fermentation liquid obtained by the mixed bacteria fermentation method as claimed in claim 4.

6. Use of the fermentation broth as claimed in claim 5 in preparing a degradable film.

7. The use according to claim 3 or 6, It is characterized in that The preparation of the degradable film comprises the following steps: Step a) mixed fermentation: 0.1-10 parts of the chitin-degrading strain according to claim 1 are inoculated into LB medium and placed in an incubator at 37°C for 12 hours of activation culture to prepare seed solution A; 0.1-10 parts of budding Aureobasidium are inoculated into YPD medium and placed in an incubator at 28°C for 72 hours of activation culture to prepare seed solution B; seed solution A and seed solution B are inoculated into a container containing 100 mL of fermentation medium at a ratio of 1:1, with an inoculation amount of 5%, and cultured at 28°C and 180 r / min for 7 days to obtain a fermentation solution; The fermentation medium is prepared from 1 to 10 parts of straw powder and 1 to 10 parts of crayfish shell powder; The budding Aureobasidium pullulans was purchased from China General Microbiological Culture Collection Center with the collection number of CGMCC 3.3984; Step b) fermentation broth concentration: the fermentation broth obtained in step a) is centrifuged at 8000 r / min for 5 min to remove the fermentation residue, and then concentrated by rotary evaporator to make the polysaccharide content reach 2 mg / mL. The concentrated fermentation broth is dialyzed to remove small molecular impurities therein to obtain a polysaccharide solution; Step c) Preparation of a degradable film: dissolving 1 part of PVA in distilled water, heating and stirring to accelerate its dissolution, obtaining a uniform solution with a concentration of 2%, namely, a polyvinyl alcohol solution; taking equal amounts of the polysaccharide solution and the polyvinyl alcohol solution, stirring and mixing them, to obtain a film-forming solution; 0.2 parts of glycerol and 0.05 parts of Tween 20 can be added to the film-forming solution; Step d) Pour the film-forming liquid obtained in step c) into a polystyrene film mold, place it in an oven at 50°C and dry it for 48 hours. After the film is formed, peel it off and place it in a dryer for 48 hours to obtain the film.

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