Method for synthesizing pha by treating mildewed corn with a complex enzyme preparation and application thereof

Abstract

This invention discloses a method and application for treating moldy corn to synthesize polyhydroxy fatty acids (PHA) using a compound enzyme preparation, relating to the fields of fermentation engineering and waste resource utilization technology. The method for treating moldy corn to synthesize PHA using a compound enzyme preparation includes the following steps: 1) screening and culturing highly efficient halophilic bacteria as inoculum strains for open-enzyme PHA synthesis; 2) pretreating moldy corn with a compound enzyme preparation composed of α-amylase, β-amylase, and glucoamylase; 3) using the moldy corn hydrolysate as a fermentation carbon source, inoculating with PHA-producing halophilic bacteria for open-enzyme fermentation. This invention constructs a complete process system of specific halophilic bacteria screening – moldy corn enzymatic pretreatment – ​​open-enzyme fermentation to synthesize PHA, realizing the resource utilization and high-value utilization of moldy corn. Compared with existing technologies, it has significant economic, environmental, and industrial value, with outstanding comprehensive beneficial effects.

Description

Technical Field

[0001] This invention relates to the fields of fermentation engineering and waste resource utilization technology, and in particular to a method and application of treating moldy corn with a compound enzyme preparation to synthesize PHA. Background Technology

[0002] Polyhydroxyalkanoates (PHAs) are a class of natural high-molecular-weight polyesters synthesized by microorganisms. They possess excellent biodegradability, biocompatibility, and thermal processability, making them an ideal alternative to traditional petroleum-based plastics. They have broad application prospects in packaging, biomedicine, and agriculture, aligning with the global demand for green and environmentally friendly materials under the "dual carbon" goal. Their large-scale preparation technology has become a research hotspot in the field of biochemical engineering. Microbial fermentation is the main method for PHA preparation. Traditional processes often use purely chemical carbon sources such as glucose and fructose as fermentation substrates. While this achieves efficient PHA synthesis, the high cost of raw materials significantly limits the industrialization and market application of PHA. To reduce production costs, utilizing inexpensive biomass resources such as agricultural waste and industrial by-products as fermentation carbon sources for PHA preparation has become a research trend. Among these, corn, as a major food crop in my country, generates various corn-based wastes during processing and storage due to their high starch content and wide availability, making them a highly promising fermentation carbon source for PHA.

[0003] Corn is prone to mold growth during storage due to improper temperature and humidity control. Moldy corn not only suffers from varying degrees of degradation of its starch and other effective components, but may also produce mycotoxins such as aflatoxin and zearalenone. Under normal circumstances, it is mostly discarded or used only as low-value feed, resulting in a serious waste of agricultural resources and potential food safety and environmental pollution risks due to improper utilization. Currently, research on the resource utilization of corn-based waste to prepare PHA (property-based organic pollutants) focuses mainly on controllable industrial byproducts such as corn processing residues and corn cobs. For moldy corn, an agricultural waste with complex components and the risk of toxin inhibition, a mature and complete process system for high-value conversion has not yet been developed. Summary of the Invention

[0004] To address the problems of resource waste from moldy corn, high raw material costs for PHA preparation, and incomplete process systems in existing technologies, this invention uses moldy corn hydrolysate as the fermentation carbon source and PHA-producing halophilic bacteria strains as the fermentation strains to construct an open-loop process system for the synthesis of PHA from agricultural waste using fermentable sugars coupled with halophilic bacteria. The aim is to promote the efficient and low-cost conversion of moldy corn into PHA, ultimately achieving the resource utilization and high-value utilization of agricultural waste.

[0005] To achieve the above objectives, in a first aspect, the present invention provides a method for treating moldy corn with a compound enzyme preparation to synthesize PHA, comprising the following steps: (1) Screening of PHA-producing halophilic bacteria: The low-temperature preserved mud sample was inoculated into an inorganic salt liquid culture medium, shaken, and passaged to obtain a culture medium containing PHA-producing halophilic bacteria. The third-generation culture medium containing PHA-producing halophilic bacteria was diluted and spread on an inorganic salt solid culture medium plate containing Nile Red for further cultivation. Colonies that turned red under ultraviolet light were picked, centrifuged to collect the cell pellet, and strains were screened to obtain PHA-producing halophilic bacteria strains. (2) Enzymatic pretreatment of moldy corn: Moldy corn was pretreated with a compound enzyme preparation to obtain a hydrolysate of moldy corn. (3) Open-ended PHA synthesis: In an inorganic salt liquid culture medium, using moldy corn hydrolysate as the fermentation carbon source, a PHA-producing halophilic bacteria strain was inoculated and subjected to open fermentation to obtain PHA.

[0006] Preferably, in step (1), the mud sample is bottom mud from Yuncheng Salt Lake in Shanxi Province, and the mud sample is stored at a temperature of 0~4℃ for 2~4 days.

[0007] Preferably, in step (1), the inorganic salt in the inorganic salt liquid culture medium has a concentration of 25~35 g·L. -1 The NaCl solution was used, the shaking frequency was 150~250 rpm, the subculture temperature was 25~35℃, and the subculture time was 2~4 days.

[0008] Preferably, in step (1), the diluent for the third-generation culture medium containing PHA-producing halophilic bacteria is sterile water, and the dilution method is to dilute it sequentially at 10... -2 10 -3 10 -4 The inorganic salt was serially diluted by multiples, and the inorganic salt in the inorganic salt solid medium was NaCl. The culture temperature of the inorganic salt solid medium was 25~35℃ and the time was 12~36 h.

[0009] Preferably, in step (1), the screening method for PHA-producing halophilic bacteria strains is as follows: the selected colonies are fermented in an inorganic salt medium with glucose, fructose or glycerol as carbon sources, centrifuged at 5000~10000 rpm for 5~15 min, the supernatant is discarded, the cell pellet is collected, 10 mg of dried cell sample is placed in an esterification tube, 1 mL of methanol solution of concentrated sulfuric acid with a volume concentration of 15% (V / V) and 1 mL of chloroform solution are added, and the tube is sealed and placed in a constant temperature drying oven at 100℃ for 4 h of methyl esterification reaction. After the reaction was completed, the esterification tube was removed and cooled to room temperature. 1 mL of double-distilled water and 1 mL of chloroform solution containing 0.05% (w / v) methyl benzoate were added. The mixture was placed on a vortex mixer and shaken thoroughly until homogeneous. After standing and separating into layers, 1 mL of the lower organic phase solution was taken and the PHA yield was detected by gas chromatography. The PHA-producing halophilic bacteria strain with the highest PHA yield was selected as the inoculum strain for open PHA synthesis.

[0010] Preferably, in step (2), the compound enzyme preparation is one or more of α-amylase, β-amylase, and glucoamylase, and the amount of compound enzyme preparation added is 0.01 wt%~0.06 wt% of the moldy corn. The temperature of the enzymatic hydrolysis pretreatment is 40~60℃ and the time is 1~8 h.

[0011] Preferably, in step (3), the inorganic salt in the inorganic salt liquid culture medium is NaCl, the open fermentation temperature is 20~40℃, the time is 4~7d, and the pH is 6.0~8.0.

[0012] Secondly, an application of a method for treating moldy corn with a compound enzyme preparation to synthesize PHA is provided.

[0013] Preferably, a PHB-producing halophilic bacterium strain was obtained by screening according to the aforementioned screening method for PHA-producing halophilic bacteria strains. This strain belongs to the genus *Alkalophila* and is named... Halomonas sp. DYZ-4. Its 16S rRNA has the NCBI sequence number PV554778. The strain has been deposited at the China Center for Type Culture Collection and has been granted the accession number CCTCC M2026502.

[0014] Preferably, in an inorganic salt liquid culture medium, moldy corn hydrolysate is used as the fermentation carbon source, with Halomonas sp. DYZ-4 is a fermentation strain that synthesizes PHB using open fermentation.

[0015] Beneficial effects (1) This invention constructs a complete process system for screening specific halophilic bacteria, enzymatic hydrolysis pretreatment of moldy corn, and open fermentation synthesis of polyhydroxy fatty acids (PHA), realizing the resource utilization and high-value utilization of moldy corn; (2) This invention transforms moldy corn, which is usually discarded and treated as low-value, into high-value biodegradable plastic PHA, which not only greatly improves the utilization efficiency of agricultural waste and reduces the waste of corn resources, but also avoids the food safety and environmental pollution risks of improper disposal of moldy corn. (3) By optimizing the composition of the compound enzyme preparation and targeting the structure of moldy corn starch, the enzymatic hydrolysis parameters were optimized to achieve efficient starch hydrolysis and a reducing sugar yield of 0.36 g·g -1 Moldy corn, optimal yield 35.91 g·L -1 ; (4) Filtered results Halomonas strain DYZ-4 exhibits excellent compatibility and tolerance to moldy corn hydrolysate, achieving a PHB accumulation of 10.97 g·L⁻¹ in an open fermentation system over 72 h. -1 ; (5) The entire process is highly coordinated and compatible, with a wide range of raw material sources, readily available enzyme preparations, mild fermentation conditions, and low raw material and production costs, and has good industrialization prospects and promotion value.

[0016] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0017] Figure 1 It is a PHA-producing halophilic bacterium strain screened using Nile Red; Figure 2 This refers to the PHB production capacity of the six PHA-producing halophilic bacteria in Example 1 using different carbon sources; Figure 3 Results of DYZ-4 16S rRNA base sequence analysis; Figure 4 This is the dynamic monitoring result of substrate consumption and product accumulation in open fermentation for PHB synthesis using glucose as the carbon matrix; Figure 5 The changes in reducing sugar content under different enzymatic pretreatment conditions in Example 1, Comparative Example 1, and Comparative Example 4; Figure 6 The changes in reducing sugar content under different enzymatic pretreatment conditions in Example 1 and Comparative Examples 1-3; Figure 7 The changes in reducing sugar content under different enzymatic pretreatment conditions are compared in Examples 4-9. Figure 8 The changes in reducing sugar content under different enzymatic pretreatment conditions in Example 1 and Comparative Example 9; Figure 9 This refers to the change in reducing sugar content under different amounts of compound enzyme preparations added during enzymatic pretreatment. Detailed Implementation

[0018] The present invention will be further described below. It should be noted that this embodiment is based on the present technical solution and provides detailed implementation methods and specific operation processes, but the present invention is not limited to this embodiment.

[0019] Example 1 This embodiment provides a method for treating moldy corn with a compound enzyme preparation to synthesize PHB, including the following steps: (1) Screening of PHB-producing halophilic bacteria: Five g of mud sample collected from Yuncheng Salt Lake in Shanxi Province and stored at -20℃ for 3 days was inoculated into NaCl liquid culture medium (NaCl concentration of 30 g·L⁻¹). -1 The culture was passaged in a shaker at 30°C and 200 rpm for 3 days each time to obtain a culture medium containing PHB-producing halophilic bacteria. The third-generation culture medium containing PHB-producing halophilic bacteria was then diluted with sterile water sequentially at a ratio of 10... -2 10 -3 10 -4 After serial dilution with multiples of 1, the solution was coated onto a substrate containing 200 mg·L⁻¹. -1 Nile red was cultured on NaCl solid agar plates at 30°C for 24 h. Six parallel groups were cultured, and two colonies that turned red under ultraviolet light were picked from each group (see...). Figure 1 A total of 12 strains were selected and preliminarily identified as PHB-producing halophilic bacteria; The 12 selected bacterial colonies were each added to 20 g·L⁻¹ -1 Fermentation was carried out in NaCl liquid medium with glucose, fructose or glycerol as carbon source. After centrifugation at 8000 rpm for 10 min, the supernatant was discarded, the cell pellet was collected, and 10 mg of dried cell sample was placed in an esterification tube. 1 mL of methanol solution of 15% (V / V) concentrated sulfuric acid and 1 mL of chloroform solution were added. After sealing, the tube was placed in a constant temperature drying oven at 100℃ for 4 h for methyl esterification. After the reaction was completed, the esterification tube was removed and cooled to room temperature. 1 mL of double-distilled water and 1 mL of chloroform solution containing 0.05% (w / v) methyl benzoate were added. The solution was placed on a vortex mixer and shaken thoroughly until homogeneous. After standing and separating into layers, 1 mL of the lower organic phase solution was taken and the PHB yield was detected by gas chromatography. Six PHB-producing halophilic bacteria with PHB synthesis ability were identified and named DYZ-1, DYZ-2, DYZ-3, DYZ-4, DYZ-5, and DYZ-6, respectively. The PHB-producing halophilic bacteria strain with the highest PHB yield was selected as the inoculum strain for open PHB synthesis. (2) Enzymatic pretreatment of moldy corn: Moldy corn was pretreated with a compound enzyme preparation consisting of α-amylase, β-amylase and glucoamylase. The mass ratio of α-amylase, β-amylase and glucoamylase in the compound enzyme preparation was 1:1:1. The amount of compound enzyme preparation added was 0.05 wt% of the moldy corn. The enzymatic pretreatment temperature was 50℃ and the time was 8 h to obtain the moldy corn hydrolysate. (3) Open-ended PHB synthesis: In NaCl liquid medium, using moldy corn hydrolysate as the fermentation carbon source, a PHB-producing halophilic bacteria strain was inoculated and subjected to open fermentation at 30℃ for 5 days and pH 7.0 to obtain PHB.

[0020] Results Analysis In step (1) of Example 1, the PHB production capacity of the six PHB-producing halophilic bacteria DYZ-1, DYZ-2, DYZ-3, DYZ-4, DYZ-5, and DYZ-6 in different carbon sources is as follows: Figure 2 As shown in Table 1.

[0021] Table 1. PHB production capacity of 6 PHB-producing halophilic bacteria on different carbon sources.

[0022] Depend on Figure 1 As shown in Table 1, when glucose was used as the carbon source, DYZ-4 produced the highest PHB yield, at 3.78 g·L⁻¹. -1 Therefore, strain DYZ-4 was selected as the inoculum for open-source PHB synthesis. The 16S rRNA of DYZ-4 was sequenced, and the 16S rRNA base sequence of DYZ-4 is as follows: Analysis revealed that the NCBI sequence number of the DYZ-4 16S rRNA is PV554778, and the results are as follows: Figure 3 The results show that DYZ-4 and Halomonas alkaliphila The homology of 18bAG was over 99% and the similarity was the highest, proving that this strain belonged to the genus *Alkalophilus*, and was named... Halomonas sp. DYZ-4, the preservation information for this strain is as follows: 1) Preservation Institution (Name and Address): China Center for Type Culture Collection, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province 2) Applicant (Name / Company Name and Address): Henan University of Technology, No. 100 Lianhua Street, High-tech Industrial Development Zone, Zhengzhou, Henan Province 3) Accession number: CCTCC M2026502 4) Taxonomic name: Halomonas 5) Preservation date: March 25, 2026 Application Example 1 This application example provides a method for synthesizing PHB by treating moldy corn with a compound enzyme preparation, including the following steps: (1) The method in step (1) of Example 1 was used to screen for PHB-producing halophilic bacteria to determine the species that produce PHB. Halomona ssp. DYZ-4 as an inoculum strain for open-source PHB synthesis; (2) 5 g of moldy corn was pretreated by enzymatic hydrolysis using the method in step (2) of Example 1 to obtain 50 mL of moldy corn hydrolysate; (3) In NaCl liquid culture medium, Halomonas The inoculum size of sp. DYZ-4 is 5% ( v / v 50 mL of moldy corn hydrolysate was used for open fermentation to synthesize PHB at 30℃ and pH 7.0. Samples were continuously taken over 5 days to measure substrate consumption and product accumulation in the fermentation broth. The reducing sugar content was determined using the DNS method and UV spectrophotometer; the total nitrogen residue was determined using the alkaline potassium persulfate method and UV spectrophotometer; the PHB yield was determined by gas chromatography; and the cell dry weight (CDW) was determined by drying. The results are as follows: Figure 4 As shown in Table 2.

[0023] Table 2 Monitoring results during open fermentation synthesis of PHB

[0024] from Figure 4As shown in Table 2, the reducing sugar content in the fermentation broth of the open fermentation system increased from the initial 35.91 g·L⁻¹. -1 It rapidly decreased to 5.36 g·L⁻¹ at 48 h. -1 It was consumed completely after 72 hours; the total nitrogen content decreased from the initial 178.59 mg·L⁻¹. -1 Decreased to 20.24 mg·L⁻¹ at 72 h. -1 The fermentation rate then decreased and slowed down. Meanwhile, the optimal PHB accumulation and percentage were detected at 72 h of fermentation, at 10.97 g·L⁻¹. -1 And 55%. Cell dry weight (CDW) reached a maximum of 19.89 g·L⁻¹ at 72 h. -1 This indicates that strain DYZ-4 can fully utilize the reducing sugars in the enzymatic hydrolysate of moldy corn as a nutrient substrate for fermentation and growth. Therefore, it can be concluded that... Halomona The s sp. DYZ-4 strain can efficiently convert moldy corn hydrolysate to accumulate PHB.

[0025] Comparative Example 1 This comparative example provides a method for synthesizing PHB by treating moldy corn with a compound enzyme preparation. The difference from Example 1 is that in step (2), the compound enzyme preparation is composed of α-amylase and β-amylase in a mass ratio of 1:1. The remaining steps are the same as in Example 1.

[0026] Comparative Example 2 This comparative example provides a method for synthesizing PHB by treating moldy corn with a compound enzyme preparation. The difference from Example 1 is that in step (2), the compound enzyme preparation is composed of β-amylase and glucoamylase in a mass ratio of 1:1. The remaining steps are the same as in Example 1.

[0027] Comparative Example 3 This comparative example provides a method for synthesizing PHB by treating moldy corn with a compound enzyme preparation. The difference from Example 1 is that in step (2), the compound enzyme preparation is composed of α-amylase and glucoamylase in a mass ratio of 1:1. The remaining steps are the same as in Example 1.

[0028] Comparative Example 4 This comparative example provides a method for synthesizing PHB by treating moldy corn with a compound enzyme preparation. The difference from Example 1 is that in step (2), the compound enzyme preparation consists only of α-amylase. The remaining steps are the same as in Example 1.

[0029] Comparative Example 5 This comparative example provides a method for synthesizing PHB by treating moldy corn with a compound enzyme preparation. The difference from Example 1 is that in step (2), the compound enzyme preparation consists only of β-amylase. The remaining steps are the same as in Example 1.

[0030] Comparative Example 6 This comparative example provides a method for synthesizing PHB by treating moldy corn with a compound enzyme preparation. The difference from Example 1 is that in step (2), the compound enzyme preparation consists only of glucoamylase. The remaining steps are the same as in Example 1.

[0031] Comparative Example 7 This comparative example provides a method for synthesizing PHB from moldy corn. The difference from Example 1 is that in step (2), no compound enzyme preparation is added, and the moldy corn is directly fermented at 50°C for 8 hours to obtain moldy corn hydrolysate. The remaining steps are the same as in Example 1.

[0032] Comparative Example 8 This comparative example provides a method for synthesizing PHB from corn. The difference from Example 1 is that in step (2), no compound enzyme preparation is added. Normal corn is directly fermented at 50°C for 8 hours to obtain corn hydrolysate, which is used as the carbon source in step (3). The remaining steps are the same as in Example 1.

[0033] Comparative Example 9 This comparative example provides a method for synthesizing PHB from corn. The difference from Example 1 is that in step (2), moldy corn is replaced with normal corn. The remaining steps are the same as in Example 1.

[0034] Results Analysis In the enzymatic pretreatment process of step (2) of Examples 1 and Comparative Examples 1-9, samples were taken every hour to analyze the reducing sugar content in the hydrolysate of moldy corn using the DNS method. The results are as follows: Figures 5-7 As shown in Table 3.

[0035] Table 3 Reducing sugar content under different enzymatic pretreatment conditions

[0036] Figure 5 The results show the determination of reducing sugar content in the enzymatic hydrolysate at different enzymatic pretreatment times in Examples 1, 1 Comparative Examples, and 4. Figure 5 It can be seen that the reducing sugar content in the hydrolysate of moldy corn increases with the increase of pretreatment time. The reducing sugar content is the highest when a compound enzyme preparation composed of α-amylase, β-amylase and glucoamylase is used and the enzymatic hydrolysis pretreatment time is 6 h.

[0037] Figure 6 The results show the determination of reducing sugar content in the enzymatic hydrolysates of Examples 1 and Comparative Examples 1-3. From... Figure 6As shown in Table 3, the reducing sugar content in the hydrolysate of moldy corn obtained in Example 1 was higher than that in Comparative Examples 1-3. That is, the composite enzyme preparation composed of α-amylase, β-amylase and glucoamylase yielded the highest reducing sugar content (35.91 g·L⁻¹) from the enzymatic hydrolysis of moldy corn. -1 The next most significant combinations were the α-amylase + glucosidase combination in Comparative Example 3, the β-amylase + glucosidase combination in Comparative Example 2, and the α-amylase + β-amylase combination in Comparative Example 1, each with a concentration of 31.16 g·L⁻¹. -1 26.20 g·L -1 19.69 g·L -1 ...

[0038] Figure 7 The results show the reducing sugar content determination in the enzyme hydrolysates of comparative examples 4-8. From... Figure 7 As shown in Table 3, among the three amylases (α-amylase, β-amylase, and glucoamylase), the reducing sugar yield of moldy corn treated with α-amylase (Comparative Example 4) was the highest, at 11.17 g·L⁻¹. -1 The second most abundant enzyme was glucoamylase (comparative example 6, 8.31 g·L⁻¹). -1 ) and β-amylase (Comparative Example 5, 4.49 g·L -1 However, adding any one of these enzymes alone is less effective than adding a compound enzyme preparation composed of all three (Example 1). Furthermore, when fermentation is carried out without the compound enzyme preparation, the starch in the moldy corn is enzymatically hydrolyzed by amylases and other substances produced by molds, resulting in a reducing sugar content of 3.31 g·L⁻¹ in the moldy corn hydrolysate (Comparative Example 7). -1 The reducing sugar content was higher than that in normal corn hydrolysate (Comparative Example 8) (2.01 g·L⁻¹). -1 ).

[0039] Figure 8 This is a comparison chart of reducing sugar content in the hydrolysate of moldy corn from Example 1 and the hydrolysate of normal corn from Comparative Example 9. From... Figure 8 It can be seen that the reducing sugar content in the hydrolysate of moldy corn is 35.91 g·L⁻¹. -1 Slightly lower than normal corn hydrolysate (40.17 g·L⁻¹). -1 This is because the starch and other nutrients in normal corn are not destroyed, so the hydrolysate has a high content of reducing sugars after enzymatic hydrolysis by a compound enzyme preparation composed of three amylases. However, since the reducing sugar content in the hydrolysate of moldy corn is only about 10.6% lower than that of normal corn hydrolysate, and its carbon content is still relatively high, it can still be used as a carbon source for open fermentation to synthesize PHA.

[0040] Comparative Example 10 This comparative example provides a method for synthesizing PHB by treating moldy corn with a compound enzyme preparation. The difference from Example 1 is that in step (2), the amount of compound enzyme preparation added is 0.01 wt%, 0.02 wt%, 0.03 wt%, 0.04 wt%, 0.05 wt%, and 0.06 wt%, respectively. The remaining steps are the same as in Example 1.

[0041] Comparative Example 11 This comparative example provides a method for synthesizing PHB by treating moldy corn with a compound enzyme preparation. The difference between this method and Comparative Example 1 is that in step (2), the amount of compound enzyme preparation added is 0.01 wt%, 0.02 wt%, 0.03 wt%, 0.04 wt%, 0.05 wt%, and 0.06 wt%, respectively. The remaining steps are the same as in Example 1.

[0042] Comparative Example 12 This comparative example provides a method for synthesizing PHB by treating moldy corn with a compound enzyme preparation. The difference between this method and Comparative Example 4 is that in step (2), the amount of compound enzyme preparation added is 0.01 wt%, 0.02 wt%, 0.03 wt%, 0.04 wt%, 0.05 wt%, and 0.06 wt%, respectively. The remaining steps are the same as in Example 1.

[0043] Results Analysis The changes in reducing sugar content in the enzymatic hydrolysate under different amounts of compound enzyme preparation added in Comparative Examples 10-12 are as follows: Figure 9 As shown in Table 4.

[0044] Table 4. Reducing sugar content in enzymatic hydrolysate at different amounts of compound enzyme preparations.

[0045] from Figure 9 As shown in Table 4, in Comparative Examples 10-12, when different compound enzyme preparations were used, the reducing sugar content in the enzymatic hydrolysate initially increased and then stabilized with increasing amounts of the compound enzyme preparation. The highest reducing sugar content (35.91 g·L⁻¹) was observed when a compound enzyme preparation consisting of α-amylase, β-amylase, and glucoamylase was used at an addition amount of 0.05 wt%. -1 Based on the addition of 5 g of moldy corn to the enzymatic hydrolysis system, 50 mL of moldy corn hydrolysate was obtained, with a reducing sugar content of 35.91 g·L⁻¹. -1 Calculations show that on average, each gram of moldy corn can produce 0.36 g of reducing sugar.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for synthesizing PHA by treating moldy corn with a compound enzyme preparation, characterized in that, Includes the following steps: (1) Screening of PHA-producing halophilic bacteria: The low-temperature preserved mud sample was inoculated into an inorganic salt liquid culture medium, shaken, and passaged to obtain a culture medium containing PHA-producing halophilic bacteria. The third-generation culture medium containing PHA-producing halophilic bacteria was diluted and spread on an inorganic salt solid culture medium plate containing Nile Red for further cultivation. Colonies that turned red under ultraviolet light were picked, isolated, purified, and screened to obtain PHA-producing halophilic bacteria strains. (2) Enzymatic pretreatment of moldy corn: Moldy corn was pretreated with a compound enzyme preparation to obtain a hydrolysate of moldy corn. (3) Open-ended PHA synthesis: In an inorganic salt liquid culture medium, moldy corn hydrolysate was used as the fermentation carbon source. PHA-producing halophilic bacteria were inoculated and subjected to open fermentation to accumulate PHA intracellularly.

2. The method for synthesizing PHA by treating moldy corn with a compound enzyme preparation according to claim 1, characterized in that, In step (1), the mud sample is the bottom mud of Yuncheng Salt Lake in Shanxi Province. The mud sample is stored at a temperature of 0~4℃ and for a storage time of 2~4 days.

3. The method for synthesizing PHA by treating moldy corn with a compound enzyme preparation according to claim 1, characterized in that, In step (1), the inorganic salt concentration in the inorganic salt liquid culture medium is 25~35 g·L. -1 The NaCl solution was used, the shaking frequency was 150~250 rpm, the subculture temperature was 25~35℃, and the subculture time was 2~4 days.

4. The method for synthesizing PHA by treating moldy corn with a compound enzyme preparation according to claim 1, characterized in that, In step (1), the diluent for the third-generation culture medium containing PHA-producing halophilic bacteria is sterile water, and the dilution method is to dilute it sequentially at 10... -2 10 -3 10 -4 The inorganic salt was serially diluted by multiples, and the inorganic salt in the inorganic salt solid medium was NaCl. The culture temperature of the inorganic salt solid medium was 25~35℃ and the time was 12~36 h.

5. The method for synthesizing PHA by treating moldy corn with a compound enzyme preparation according to claim 1, characterized in that, In step (1), the screening method for PHA-producing halophilic bacteria strains is as follows: the selected colonies are fermented and cultured with glucose, fructose or glycerol as carbon sources respectively, and the PHA-producing halophilic bacteria strains with the highest PHA production are screened out as inoculum strains for open PHA synthesis.

6. The method for synthesizing PHA by treating moldy corn with a compound enzyme preparation according to claim 1, characterized in that, In step (2), the compound enzyme preparation is one or more of α-amylase, β-amylase, and glucoamylase. The amount of compound enzyme preparation added is 0.01 wt% to 0.06 wt% of the moldy corn. The temperature of the enzymatic hydrolysis pretreatment is 40 to 60℃ and the time is 1 to 8 h.

7. The method for synthesizing PHA by treating moldy corn with a compound enzyme preparation according to claim 1, characterized in that, In step (3), the inorganic salt in the inorganic salt liquid culture medium is NaCl, and the open fermentation temperature is 20~40℃, the time is 4~7d, and the pH is 6.0~8.

0.

8. The application of the method for synthesizing PHA by treating moldy corn with the compound enzyme preparation as described in any one of claims 1 to 7.

9. The application according to claim 8, characterized in that, A PHB-producing halophilic bacterium strain was screened according to the method of claim 5. This strain belongs to the genus *Alkalophilus* and is named... Halomonas sp. DYZ-4, whose 16S rRNA has the NCBI sequence number PV554778, has been deposited at the China Center for Type Culture Collection with the accession number CCTCC M2026502.

10. The application according to claim 9, characterized in that, In an inorganic salt liquid culture medium, moldy corn hydrolysate was used as the fermentation carbon source. Halomonas sp. DYZ-4 is a fermentation strain that synthesizes PHB using open fermentation.

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