Preparation method and application of bran hydrolysate rich in glucose

Abstract

The invention discloses a glucose-rich bran hydrolysate preparation method and application, the method comprises the following steps: bran is used as a matrix to prepare a fungus fermentation medium, the fungus fermentation medium comprises the following components: 60 g / L of ultrafine powder bran, 1 g / L of (NH4) 2SO4, 0.5 g / L of MgSO4, 0.5 g / L of KH2PO4, 1.0 g / L of K2HPO4, 1 mM of Al < 3 + >, 2 mM of Mn < 2 + > and 2 mM of Ba < 2 + >, and the initial pH value is 6.5; inoculating flammulina velutipes strains, and standing and fermenting to obtain the biological bran hydrolysate (BWBH). The sugar composition in the obtained BWBH mainly comprises glucose, the concentration of a microbial growth inhibitor is extremely low, the BWBH serves as a carbon source for bacillus fermentation to produce acetoin, and compared with traditional carbon sources of glucose / xylose and CWBH, the bacterial growth speed, the carbon source utilization rate and the product yield are obviously improved.

Description

Technical Field

[0001] The invention relates to the field of microbial fermentation, and in particular to a preparation method and application of a glucose-rich bran hydrolyzate. Background Art

[0002] Lignocellulosic biomass (such as corn straw, cottonseed hulls, cotton stalks, sawdust, and wheat bran) is one of the most abundant, low-cost, and renewable raw materials. It can be converted into high-value-added bio-based chemicals through hydrolysis and microbial fermentation. As one of the world's three major staple crops, countries such as China, the United States, and Brazil produce large quantities of wheat annually, with wheat bran being a major by-product during flour processing. Wheat bran, due to its high carbohydrate content and loose lignocellulosic structure, is an ideal feedstock for the biorefining industry.

[0003] To improve fermentation efficiency, wheat bran is often pretreated to obtain reducing sugars. Studies have shown that acid hydrolysis, alkaline hydrolysis, enzymatic hydrolysis, and microbial treatment are common wheat bran pretreatment methods. Dilute acid / alkaline pretreatment can yield higher concentrations of fermentable sugars, but requires high temperature and pressure, and the sugar decomposition products and inhibitors produced during the process can reduce subsequent fermentation efficiency. In contrast, microbial pretreatment offers advantages due to its mild conditions and low inhibitor production. While commercial enzyme preparations such as lignin-degrading enzymes, cellulases, and hemicellulases have been used for lignocellulose degradation, their low conversion efficiency and high cost limit their application. Fungi such as white-rot and brown-rot fungi, which secrete hydrolytic enzymes to convert biomass into reducing sugars, have garnered increasing attention due to their economic and environmental advantages.

[0004] Regardless of whether chemical or biological hydrolysis is used, the main components of biomass raw material hydrolysates are usually hexose (such as glucose) and pentose (such as xylose). However, most industrial strains have significantly better utilization efficiency of hexose than pentose, which makes it a challenge to achieve co-fermentation of the two types of sugars. Although metabolic engineering strategies have achieved the co-utilization of hexose and pentose in a few model strains, their lack of universality limits the widespread development of biomass hydrolysates. How to make biomass hydrolysates with glucose as the main sugar has become a key scientific issue. Summary of the Invention

[0005] To address the shortcomings of existing chemical methods for hydrolyzing wheat bran, such as an irrational reducing sugar composition (excessively high xylose content) and high levels of fermentation inhibitors, the present invention provides a method for preparing a glucose-rich wheat bran hydrolysate (BWBH) via fungal fermentation, and its application in microbial fermentation. This method significantly improves the carbon source suitability of the hydrolysate by optimizing fermentation process parameters, resulting in ease of operation, low cost, and suitability for industrialization.

[0006] The present invention solves the above-mentioned technical problem with the following technical solution: a method for preparing a glucose-rich bran hydrolyzate is provided, which comprises the following steps:

[0007] (1) Preparation of fungal fermentation medium: Use ultrafine bran as the matrix material, and the liquid culture medium contains 60 g ultrafine bran, 1 g (NH4)2SO4, 0.5 g MgSO4, 0.5 g KH2PO4, 1.0 g K2HPO4, and 1.0 g Al per liter. 3+ 1mM, Mn 2+ 2mM, Ba 2+ 2mM, and sterilized at 121°C for 20min to obtain the fungal fermentation medium;

[0008] (2) Bran biodegradation: inoculating the fungal fermentation medium obtained in step (1) with the fungus F. velutipes, and fermenting the culture medium at a constant temperature of 22-30° C. (preferably 25° C.) for 6 days. The culture solution obtained is collected, and the supernatant is collected by centrifugation to obtain a biological bran hydrolyzate (BWBH), which is the glucose-rich bran hydrolyzate.

[0009] The volume of the fungal fermentation medium in step (1) is 10-30 mL / 100 mL conical flask, and the initial pH value is 5.5-8.5;

[0010] Preferably, the volume of the fungal fermentation medium in step (1) is 20 mL / 100 mL conical flask (i.e., divided into 100 mL conical flasks, with a liquid volume of 20 mL), and the initial pH value is 6.5.

[0011] The bran particle size specifications include coarse powder, fine powder and ultrafine powder, and the preferred particle size specification is ultrafine bran. The particle size of the ultrafine bran is ≤6.5 μm.

[0012] The fungal fermentation medium has an ultrafine bran substrate concentration of 40-100 g / L, preferably 60 g / L.

[0013] The enoki mushroom strains described in step (2) need to be activated in the PDA culture medium in the early stage; the enoki mushroom activation method is: inoculating the enoki mushroom strains into the PDA culture medium, and culturing at a constant temperature of 25° C. for 5 days until the mycelium completely covers the surface of the culture medium, thereby obtaining an activated strain plate;

[0014] The PDA culture medium contains 200g of potato, 20g of glucose, 3g of peptone and 20g of agar per liter;

[0015] Then, three fungus blocks were taken from the plate covered with mycelium using a 6 mm diameter puncher and inoculated into the fungal fermentation medium for cultivation.

[0016] The specific method of centrifuging the collected culture fluid in step (2) is as follows: centrifuging the collected culture fluid at 10,000 rpm for 10 minutes, collecting the supernatant as the biological bran hydrolyzate (BWBH), and storing it at -20°C for later use.

[0017] The liquid culture medium volume, pH value and culture temperature will affect the secretion of carbohydrate active enzymes (CAZymes) by the mycelium to degrade cellulose and hemicellulose in bran. When the pH value of the liquid culture medium is 6.5, the reducing sugar content of the hydrolyzate increases by 24% relative to that at pH 5.5; at the same time, excessively high liquid volume and culture temperature are not conducive to the degradation of bran.

[0018] The above parameters such as bran substrate concentration, bran particle size, liquid volume, culture temperature and liquid culture medium pH value all have a great influence on the reducing sugar concentration in the obtained bio-based bran hydrolysate. Only within the parameter range can a bio-based bran hydrolysate with better performance be obtained, which is conducive to its later application as a carbon source in microbial industrial fermentation.

[0019] The invention provides a glucose-rich bran hydrolyzate prepared by the preparation method.

[0020] The present invention also provides the use of the bran hydrolyzate in the fermentation production of acetoin by Bacillus BS4481. The obtained BWBH is used as a carbon source in the production of platform compounds by industrial microbial fermentation, specifically comprising the following steps:

[0021] The glucose-rich bran hydrolyzate (i.e., biological bran hydrolyzate BWBH) is vacuum freeze-dried and concentrated to a reducing sugar concentration of 200 g / L, and then a bacterial fermentation medium (pH 7.0, liquid volume 50 mL / 250 mL conical flask) is prepared according to the formula: 20 g reducing sugar, 2 g yeast powder, 5 g KH2PO4, 10 g K2HPO4, 1 g (NH4)2SO4, and 0.5 g MgSO4 per liter is prepared. 1-5% (v / v) pre-activated BS4481 seed liquid is inoculated and cultured at 30°C and 200 rpm for 40-48 h to produce acetoin.

[0022] The Bacillus BS4481 was pre-activated with LB medium and then inoculated into a bacterial fermentation medium. The LB medium was prepared to contain 10 g of peptone, 5 g of yeast powder, and 10 g of NaCl per liter, with a volume of 50 mL / 250 mL conical flask. The activation conditions were 30° C. and 180 rpm shaking for 18 hours to obtain a pre-activated BS4481 seed solution.

[0023] The fermentation inoculum amount is preferably 1% (v / v) pre-activated BS4481 seed liquid.

[0024] The Bacillus BS4481 used in the present invention is from Jiangnan University and is modified based on Bacillus subtilis 168. The modification process refers to patent CN 104498394 A.

[0025] Compared with the prior art, the advantages of the present invention are: by adopting the method of the present invention, bran is used as a matrix to prepare a fungal fermentation medium, and after sterilization, the Enoki mushroom strain is inoculated into the fungal fermentation medium for static fermentation, the sugar composition of the obtained BWBH is mainly glucose, and the content and concentration of microbial growth inhibitors such as phenols and acetic acid are extremely low, and BWBH is used as a carbon source for Bacillus fermentation to produce acetoin, and compared with traditional carbon sources such as glucose / xylose and chemical bran hydrolyzate (CWBH), the bacterial growth rate, carbon source utilization rate and product yield are significantly improved.

[0026] Compared with traditional chemical bran hydrolysate (CWBH), BWBH has significant advantages: glucose accounts for 47.5% of the total reducing sugars, while xylose accounts for only 3.9%, and the content of fermentation inhibitors such as phenols and acetic acid is significantly reduced. When it is used as a carbon source for Bacillus BS4481 to ferment and produce acetoin, the bacterial biomass and product yield are significantly better than traditional carbon sources glucose / xylose and CWBH, with a conversion efficiency of 0.603g acetoin / g total reducing sugar, which is 54.7% higher than glucose, and is completely better than CWBH (increased by 3.08 times). The present invention provides a new and efficient carbon source solution for the high-value utilization of biomass resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Comparison of the ability of different edible fungi strains to degrade bran and accumulate reducing sugars.

[0028] Figure 2 Glucose and xylose contents in static fermentation bran hydrolysate.

[0029] Figure 3 Single factor optimization of biological bran hydrolysate process.

[0030] Figure 4 Comparison of sugar composition between BWBH and CWBH.

[0031] Figure 5 Comparison of inhibitor content between BWBH and CWBH.

[0032] Figure 6 Comparison of acetoin production rates by Bacillus BS4481 using bran hydrolysate. DETAILED DESCRIPTION

[0033] The following examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the examples, where specific conditions are not specified, the experiments were performed under conventional conditions or those recommended by the manufacturer. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0034] Example 1:

[0035] A method for preparing a glucose-rich bran hydrolyzate comprises the following steps:

[0036] (1) Strain activation: The low-temperature preserved edible fungus strain was inoculated into PDA culture medium and cultured at a constant temperature of 25°C for 5-9 days until the mycelium completely covered the surface of the culture medium to obtain an activated strain plate.

[0037] Wherein, the PDA culture medium contains 200g potato, 20g glucose, 3g peptone and 20g agar per liter;

[0038] (2) Bran biodegradation: Take the bacterial plate obtained in step (1), punch it with a sterile puncher (diameter = 6 mm), take out 3 bacterial blocks and inoculate them into fungal fermentation medium, and culture them at 25° C. in the dark for 7 days.

[0039] The fungal fermentation medium contains 50 g of bran, 1 g of (NH4)2SO4, 0.5 g of MgSO4, 0.5 g of KH2PO4, and 1.0 g of K2HPO4 per liter, with an initial pH of 7.0, a liquid volume of 20 mL / 100 mL conical flask, and a medium pH of 7.0;

[0040] (3) Preparation of hydrolyzate: 8 mL of the culture medium from step (2) was taken, centrifuged at 10,000 rpm for 10 min, and the supernatant was collected to obtain a bran hydrolyzate. The reducing sugar concentration in the bran hydrolyzate was determined by the DNS method;

[0041] (4) DNS determination method: After the hydrolyzate is appropriately diluted, 500 μL of the dilution is mixed with an equal volume of DNS reagent, and the mixture is colorized in a boiling water bath for 10 min. After cooling with running water, 4 mL of ultrapure water is added and mixed. The blank control (ultrapure water replaces the sample) is used to adjust the mixture to zero, the OD540 value is measured, and the reducing sugar concentration is calculated using the glucose standard curve.

[0042] The edible fungi strains described in step (1) and the culture method of step (2) were screened and tested, and 8 edible fungi strains, including King Oyster Mushroom, Lentinus edodes, Agrocybe odoratum, Agrocybe tumefaciens, Agaricus oleifera, Volvariella volvacea, Pleurotus ostreatus and Flammulina velutipes, were selected. Parallel experiments were carried out under static culture and shaking culture (180 rpm) conditions, and the reducing sugar concentration in the hydrolyzate was used as an evaluation index to determine the optimal strain and culture mode.

[0043] Example 2:

[0044] A method for preparing a glucose-rich bran hydrolyzate comprises the following steps:

[0045] (1) Activation of Enoki mushrooms: Enoki mushrooms were inoculated into PDA culture medium and cultured at a constant temperature of 25°C for 5 days until the mycelium completely covered the surface of the culture medium to obtain an activated culture plate.

[0046] Wherein, the PDA culture medium contains 200g potato, 20g glucose, 3g peptone and 20g agar per liter;

[0047] (2) Bran biodegradation: Take the bacterial plate obtained in step (1), punch it with a sterile puncher (diameter = 6 mm), take out 3 bacterial blocks and inoculate them into fungal fermentation medium, and culture them at 25°C in the dark for 6 days.

[0048] The fungal fermentation medium contains 50 g of bran, 1 g of (NH4)2SO4, 0.5 g of MgSO4, 0.5 g of KH2PO4, and 1.0 g of K2HPO4 per liter, with an initial pH of 7.0 and a liquid volume of 20 mL / 100 mL conical flask; the bran is ground and passed through a 2000 mesh sieve to form an ultrafine powder (particle size ≤ 6.5 μm);

[0049] (3) Preparation of hydrolyzate: 8 mL of the culture medium from step (2) was taken, centrifuged at 10,000 rpm for 10 min, and the supernatant was collected to obtain a bran hydrolyzate. The reducing sugar concentration in the bran hydrolyzate was determined by the DNS method;

[0050] (4) DNS determination method: After the hydrolyzate is appropriately diluted, 500 μL of the dilution is mixed with an equal volume of DNS reagent, and the mixture is colorized in a boiling water bath for 10 min. After cooling with running water, 4 mL of ultrapure water is added and mixed. The blank control (ultrapure water replaces the sample) is used to adjust the mixture to zero, the OD540 value is measured, and the reducing sugar concentration is calculated using the glucose standard curve.

[0051] The inventors conducted a multi-factor system optimization on the bran biodegradation process described in step (2), and used a single variable gradient test method to examine the effects of parameters such as culture temperature, liquid volume, initial pH, bran particle size, bran concentration, whether metal ions were added, and the types of metal ions added on the accumulation of reducing sugars. The parameters presented in step (2), such as culture temperature of 25°C, liquid volume of 20mL / 100mL conical flask, initial pH 7.0, bran particle size ≤6.5μm, and bran concentration of 50g / L, were adjusted to other corresponding parameters set for a single variable gradient test to obtain the corresponding bran hydrolyzate. The reducing sugar concentration in each bran hydrolyzate was then determined by the DNS method to optimize the best parameters. The above parameters examined by the inventor using the single variable gradient test method are specifically:

[0052] Culture temperature: The culture temperature can be set in 5 gradients (22°C, 25°C, 30°C, 37°C, 45°C), with a temperature control accuracy of ±0.5°C;

[0053] Liquid volume: 6 gradients are set for liquid volume (10mL, 20mL, 30mL, 40mL, 50mL, 60mL / 100mL conical flask);

[0054] Initial pH: The initial pH was set to 7 gradients (5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5) and precisely adjusted using 1 M HCl / NaOH solution;

[0055] Bran particle size: Bran particle size is divided into three levels, namely coarse powder, fine powder passing through a 20-mesh sieve (particle size ≤ 0.85 mm), and ultrafine powder passing through a 2000-mesh sieve (particle size ≤ 6.5 μm);

[0056] Bran concentration: 7 gradients of bran concentration are set (40g / L, 50g / L, 60g / L, 70g / L, 80g / L, 90g / L, 100g / L);

[0057] Metal ions: 1 mM metal ions (Al 3+ 、Mn 2+ 、Cu 2+ 、Fe 2+ 、Zn + 、Ba 2+ 、Fe 3+ , Ca 2 + );

[0058] To investigate the effects of the above factors on reducing sugar accumulation, no additional metal ions were added to the control group. Three biological replicates were set for all treatment groups.

[0059] Example 3:

[0060] A method for preparing a glucose-rich bran hydrolyzate comprises the following steps:

[0061] (1) Activation of Enoki mushrooms: Enoki mushrooms were inoculated into PDA culture medium and cultured at a constant temperature of 25°C for 5 days until the mycelium completely covered the surface of the culture medium to obtain an activated culture plate.

[0062] Wherein, the PDA culture medium contains 200g potato, 20g glucose, 3g peptone and 20g agar per liter;

[0063] (2) Bran biodegradation: Take the bacterial plate obtained in step (1), punch it with a sterile puncher (diameter = 6 mm), take out 3 bacterial blocks and inoculate them into fungal fermentation medium, and culture them at 25°C in the dark for 6 days.

[0064] The fungal fermentation medium contains 50 g of bran, 1 g of (NH4)2SO4, 0.5 g of MgSO4, 0.5 g of KH2PO4, and 1.0 g of K2HPO4 per liter, with an initial pH of 7.0 and a liquid volume of 20 mL / 100 mL conical flask;

[0065] (3) Preparation of bran hydrolyzate: 8 mL of the culture medium from step (2) was taken, centrifuged at 10,000 rpm for 10 min, and the supernatant was collected to obtain a bran hydrolyzate. The reducing sugar concentration in the bran hydrolyzate was determined by the DNS method;

[0066] (4) DNS determination method: After the hydrolyzate is appropriately diluted, 500 μL of the dilution is mixed with an equal volume of DNS reagent, and the mixture is colorized in a boiling water bath for 10 min. After cooling with running water, 4 mL of ultrapure water is added and mixed. The blank control (ultrapure water replaces the sample) is used to adjust the mixture to zero, the OD540 value is measured, and the reducing sugar concentration is calculated using the glucose standard curve.

[0067] According to the test results of Example 2, the six key factors for the biodegradation process of bran in step (2) include bran substrate concentration, culture medium pH, liquid volume, Al 3+ 、Mn 2+ , and Ba 2+ Design L27(3^6) orthogonal test table.

[0068] Example 4:

[0069] A method for preparing a glucose-rich bran hydrolyzate comprises the following steps:

[0070] (1) Activation of Enoki mushrooms: Enoki mushrooms were inoculated into PDA culture medium and cultured at a constant temperature of 25°C for 5 days until the mycelium completely covered the surface of the culture medium to obtain an activated culture plate.

[0071] Wherein, the PDA culture medium contains 200g potato, 20g glucose, 3g peptone and 20g agar per liter;

[0072] (2) Bran biodegradation: Take the bacterial plate obtained in step (1), punch it with a sterile puncher (diameter = 6 mm), take out 3 bacterial blocks and inoculate them into fungal fermentation medium, and culture them at 25°C in the dark for 6 days.

[0073] Wherein, the fungal fermentation medium contains superfine bran 60g / L, (NH4)2SO41 g / L, MgSO40.5g / L, KH2PO40.5 g / L, K2HPO41.0 g / L, Al 3+ 1mM, Mn 2+ 2mM, Ba 2+2 mM, medium volume 20 mL / 100 mL conical flask, initial pH 6.5;

[0074] (3) Preparation of biological bran hydrolysate: The culture medium of step (2) was collected and centrifuged at 10,000 rpm for 10 min. The supernatant was collected as the biological bran hydrolysate (BWBH) and stored at -20°C for later use.

[0075] (4) The concentrations of glucose and xylose in the BWBH from step (3) were quantitatively determined by HPLC. Before the determination, the hydrolyzate was derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) before column derivatization.

[0076] Pre-column derivatization of PMP was performed according to the following specific steps: After the hydrolyzate was diluted a certain number of times, 250 μL was accurately pipetted, 250 μL of NaOH solution (0.3 M) was added, and 250 μL of PMP-methanol solution (0.5 M) was added. The mixture was allowed to react at 70°C for 4 hours. After the reaction, 0.3 M hydrochloric acid solution was added dropwise to neutralize the sample. 500 μL of chloroform was then added for layered extraction and impurity removal. The mixture was vortexed for 1 minute and centrifuged at 10,000 rpm for 2 minutes. The upper layer was extracted repeatedly 3-4 times until the sample was clear and transparent. After filtration through a 0.22 μm filter membrane, the glucose and xylose contents of the sample were determined by HPLC.

[0077] The HPLC quantitative determination was carried out according to the following method: the mobile phase consisted of 85% phosphate buffer (0.1 M, pH 6.7) and 15% methanol, the flow rate was 0.8 mL / min, the column oven temperature was 35°C, the detector wavelength was 250 nm, and the chromatographic column model was C. 18 SB-Aq (2.1×100 mm, 3.5 μm), run time: 40 min. The contents of glucose and xylose in BWBH were calculated based on their standard curves.

[0078] (5) Quantitatively determine the concentrations of inhibitory phenolic compounds, furfural, acetic acid, and formic acid in the BWBH from step (3).

[0079] The phenolic compounds were determined by the Folin phenol colorimetric method, in which 0.5 mL of sample and 2.5 mL of 10% Folin phenol reagent were accurately drawn, mixed and allowed to stand for 5 min, 2.0 mL of 7.5% Na2CO3 solution was added, and the mixture was allowed to stand at room temperature for 60 min. The absorbance was measured at a wavelength of 765 nm, and the concentration of phenolic compounds in the hydrolyzate was calculated based on the gallic acid standard curve. The furfural was determined by the aniline colorimetric method, in which the reaction system included 4.5 mL of glacial acetic acid in an ice bath and 0.5 mL of aniline in an ice bath. After mixing, 5 mL of sample was added, and the mixture was allowed to stand for 1 h. The absorbance at a wavelength of 520 nm was measured with a blank reagent to adjust the concentration to zero, and the furfural concentration in the hydrolyzate was calculated based on the furfural standard curve. The concentrations of formic acid and acetic acid were determined by HPLC, and the specific measurement conditions were as follows: phosphate buffer (50 mM, pH 2.5) was used as the mobile phase for isocratic elution, the flow rate was 0.8 ml / min, the column temperature was 25°C, the detector wavelength was 210 nm, and the chromatographic column model was C 18 SB-Aq, run time: 15 min, convert formic acid and acetic acid concentrations in BWBH based on the formic acid and acetic acid standards.

[0080] Example 5:

[0081] A preparation method and application of a glucose-rich bran hydrolyzate, comprising the following steps:

[0082] (1) Activation of Enoki mushrooms: Enoki mushrooms were inoculated into PDA culture medium and cultured at a constant temperature of 25°C for 5 days until the mycelium completely covered the surface of the culture medium to obtain an activated culture plate.

[0083] Wherein, the PDA culture medium contains 200g potato, 20g glucose, 3g peptone and 20g agar per liter;

[0084] (2) Bran biodegradation: Take the bacterial plate obtained in step (1), punch it with a sterile puncher (diameter = 6 mm), take out 3 bacterial blocks and inoculate them into fungal fermentation medium, and culture them at 25°C in the dark for 6 days.

[0085] Wherein, the fungal fermentation medium contains superfine bran 60g / L, (NH4)2SO41 g / L, MgSO40.5g / L, KH2PO40.5 g / L, K2HPO41.0 g / L, Al 3+ 1mM, Mn 2+ 2mM, Ba 2+ 2 mM, medium volume 20 mL / 100 mL conical flask, initial pH 6.5;

[0086] (3) Preparation of BWBH: The culture medium from step (2) was collected and centrifuged at 10,000 rpm for 10 min. The supernatant was collected as BWBH and stored at -20°C for later use.

[0087] (4) BWBH concentration: The BWBH obtained in step (3) was taken, and the reducing sugar concentration in the hydrolyzate was determined by the DNS method. The BWBH was concentrated by vacuum freeze drying technology to a reducing sugar concentration of 200 g / L in the hydrolyzate.

[0088] (5) Preparation of bacterial fermentation medium: The concentrated BWBH from step (4) was used as a carbon source to prepare a bacterial fermentation medium. The bacterial fermentation medium contained 20 g of reducing sugar, 2 g of yeast powder, 5 g of KH2PO4, 10 g of K2HPO4, 1 g of (NH4)2SO4, and 0.5 g of MgSO4 per liter. The medium was filled in a 250 mL conical flask at an initial pH of 7.0.

[0089] (6) Seed solution preparation: BS4481 stored in a −80°C refrigerator was inoculated into LB medium to prepare pre-activated BS4481 seed solution, wherein the LB medium contained 10 g of peptone, 5 g of yeast powder, and 10 g of NaCl per liter, and the medium was filled in a volume of 50 mL / 250 mL. The culture was shaken at 30°C and 180 rpm for 18 h to obtain the pre-activated BS4481 seed solution.

[0090] (7) Acetoin fermentation: The pre-activated BS4481 seed solution was transferred to the bacterial fermentation medium at a 1% (v / v) inoculation rate and fermented at 30°C and 200 rpm for 48 h. During this period, samples were taken every 6 h to detect the bacterial density (OD600), residual sugar concentration, and acetoin production.

[0091] Comparative Example 1:

[0092] A method for preparing a chemical wheat bran hydrolysate (CWBH) comprises the following steps:

[0093] (1) Accurately weigh 4.5 g of bran and place it in a 250 mL conical flask. Add 75 mL of 1% dilute sulfuric acid and place in an autoclave at 121°C for 60 min.

[0094] (2) After the reaction, 5 M NaOH solution was added dropwise to adjust the pH of the hydrolyzate to 7.0 ± 0.2. The hydrolyzate was collected by filtration and centrifuged at 10,000 rpm for 10 min. The supernatant was obtained as CWBH.

[0095] (3) The CWBH obtained in step (2) was taken, and the reducing sugar concentration in the hydrolyzate was determined by the DNS method. The CWBH was concentrated by vacuum freeze drying technology to concentrate the reducing sugar concentration in the hydrolyzate to 200 g / L.

[0096] (4) The contents of glucose and xylose in CWBH were quantitatively determined according to the method of step (4) of Example 4.

[0097] (5) The concentrations of inhibitors including furfural, phenolic compounds, formic acid, and acetic acid in CWBH were quantified according to the method of step (5) of Example 4.

[0098] Comparative Example 2:

[0099] A preparation method and application of a chemical bran hydrolyzate (CWBH) comprises the following steps:

[0100] (1) Accurately weigh 4.5 g of bran and place it in a 250 mL conical flask. Add 75 mL of 1% dilute sulfuric acid and place in an autoclave at 121°C for 60 min.

[0101] (2) After the reaction, 5 M NaOH solution was added dropwise to adjust the pH of the hydrolyzate to 7.0 ± 0.2. The hydrolyzate was collected by filtration and centrifuged at 10,000 rpm for 10 min. The supernatant was obtained as CWBH.

[0102] (3) The CWBH obtained in step (2) was taken, and the reducing sugar concentration in the hydrolyzate was determined by the DNS method. The CWBH was concentrated by vacuum freeze drying technology to concentrate the reducing sugar concentration in the hydrolyzate to 200 g / L.

[0103] (4) Preparation of bacterial fermentation medium: The concentrated CWBH, glucose, and xylose from step (3) were used as carbon sources to prepare bacterial fermentation medium. The bacterial fermentation medium contained 20 g of concentrated reducing sugar, 2 g of yeast powder, 5 g of KH2PO4, 10 g of K2HPO4, 1 g of (NH4)2SO4, and 0.5 g of MgSO4 per liter. The medium was filled in a 250 mL conical flask at an initial pH of 7.0.

[0104] (5) Seed solution preparation: BS4481 stored in a -80°C refrigerator was inoculated into LB medium to prepare pre-activated BS4481 seed solution, wherein the LB medium contained 10 g of peptone, 5 g of yeast powder, and 10 g of NaCl per liter, and the medium was filled in a volume of 50 mL / 250 mL. The culture was shaken at 30°C and 180 rpm for 18 h to obtain the pre-activated BS4481 seed solution.

[0105] (6) Acetoin fermentation: The pre-activated BS4481 seed solution was transferred to the bacterial fermentation medium at a 1% (v / v) inoculation rate and fermented at 30°C and 200 rpm for 48 h. During this period, samples were taken every 6 h to detect the bacterial density (OD600), residual sugar concentration, and acetoin production.

[0106] Test results:

[0107] Take 1 mL of the bran hydrolyzate obtained in Example 1 in a centrifuge tube and dilute it appropriately to determine the reducing sugar concentration using the DNS method. Figure 1As shown in the results, different edible fungi showed significant differences in the effects of wheat bran biodegradation on reducing sugar production: static fermentation was more conducive to reducing sugar accumulation than dynamic fermentation, and the reducing sugar concentration of Flammulina velutipes hydrolysate was the highest (5.59 g / L), significantly higher than that of Agaricus glutamate (4.47 g / L) and Volvariella volvacea (3.47 g / L). Further quantitative analysis by HPLC revealed that ( Figure 2 ). The glucose content in the hydrolyzates of Flammulina velutipes, Agrocybe tumefaciens, Agrocybe spp., and Pleurotus ostreatus all exceeded that of xylose. The glucose concentration in the hydrolyzate of Flammulina velutipes reached 3.05 g / L (compared to 0.90 g / L for xylose). Xylose predominated in the hydrolyzates of Agrocybe tumefaciens and Volvariella volvacea, at 2.39 g / L and 1.56 g / L, respectively. Based on a comprehensive analysis of reducing sugar concentrations and sugar composition, the static culture system of Flammulina velutipes was ultimately selected for further research.

[0108] Based on the screening results of Example 2, single factor optimization was performed on the key parameters of Enoki mushroom bran degradation (culture temperature, liquid volume, initial pH, bran concentration and particle size), and the effect of metal ions on reducing sugar accumulation was investigated. Figure 3-1 3-6) showed that the best reducing sugar accumulation effect was achieved when the liquid volume was 20 mL, 25°C, initial pH 6.5, bran concentration 60 g / L, and ultrafine powder (particle size ≤ 6.5 μm). The metal ion experiment showed that the addition of 1 mM Al 3+ 、Mn 2+ and Ba 2+ can increase the reducing sugar concentration by 6.60%, 5.06% and 2.64% respectively, while Cu 2+ 、Zn + and Ca 2+ It exhibits an inhibitory effect.

[0109] According to the single factor optimization results, an orthogonal test was designed (Example 3). As shown in Table 1, the 10th group (reducing sugar yield 20.64%, concentration 12.38 g / L) had the best effect. The Minitab 7.0 variance analysis showed that the influence intensity of each factor was ranked as follows: Al 3+ >pH>Mn 2+ > Bran concentration > Liquid volume > Ba 2+ The best combination (222332) corresponds to the following parameters: bran 60g / L, pH 6.5, Al 3+ 1mM, Mn 2+ 2mM, Ba 2+ 2mM, liquid volume 20mL. The final optimized culture medium formula was determined as follows: superfine bran 60g / L, (NH4)2SO41 g / L, MgSO40.5 g / L, KH2PO40.5 g / L, K2HPO41.0 g / L, Al 3+ 1mM, Mn 2+ 2mM, Ba 2+2mM (liquid volume 20mL, pH 6.5). After inoculation with Flammulina velutipes, the mixture was cultured in the dark at 25℃ for 6 days. The reducing sugar concentration in the obtained BWBH was 13.81g / L, and the reducing sugar yield was 23.33%.

[0110] Table 1 Orthogonal test table

[0111]

[0112] Based on the experimental methods of Example 4 and Comparative Example 1, the sugar composition and fermentation inhibitor concentration differences of BWBH and CWBH were compared and analyzed. Figure 4 As shown in the figure, there are significant differences in the sugar composition of the two: BWBH is mainly composed of glucose (accounting for 47.5%), and xylose accounts for only 3.9%; while in CWBH, xylose accounts for as high as 69.1% and glucose accounts for 14.3%. Further quantitative detection found that ( Figure 5 ). The concentrations of fermentation inhibitors (furfural, organic acids, etc.) caused by chemical treatment were significantly higher in CWBH than in BWBH. CWBH contained 0.24 g / L of furfural, 2.67 g / L of acetic acid, and 0.44 g / L of phenolic compounds, while only 0.42 g / L of phenolic compounds was detected in BWBH, with the remaining inhibitor levels below the detection limit. These results indicate that the reducing sugar composition and low inhibitor content of BWBH are more conducive to microbial fermentation conversion.

[0113] Furthermore, the experiment of acetoin production by Bacillus BS4481 was carried out using BWBH and CWBH as carbon sources and glucose and xylose as controls. Figure 6 As shown, BS4481's sugar utilization rate on BWBH was significantly higher than on other carbon sources, with a residual sugar concentration of only 1.39 g / L after 42 hours of fermentation. The bacterial biomass reached 7.70 g / L, representing 2.08 and 2.55 times that of the glucose / xylose and CWBH groups, respectively. Regarding acetoin synthesis, the product concentrations in all four groups showed an initial upward and then downward trend, with peak values of 11.23 g / L for BWBH, 7.26 g / L for glucose, 3.65 g / L for CWBH, and 2.18 g / L for xylose. Of particular note, the acetoin conversion efficiency of BWBH reached 0.603 g / g, a 54.7% increase over the glucose group and significantly superior to that of CWBH (a 3.08-fold increase).

[0114] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing a glucose-rich bran hydrolyzate, characterized in that: It includes the following steps: (1) Preparation of fungal fermentation medium: Use ultrafine bran as the matrix material, and the liquid culture medium contains 60 g ultrafine bran, 1 g (NH4)2SO4, 0.5 g MgSO4, 0.5 g KH2PO4, 1.0 g K2HPO4, and 1.0 g Al per liter. 3+ 1mM, Mn 2+ 2mM, Ba 2+ 2mM to prepare the fungal fermentation medium; (2) Bran biodegradation: inoculating the fungal fermentation medium obtained in step (1) with the fungus F. velutipes, and fermenting the culture medium at a constant temperature of 25° C. for 6 days. The culture solution obtained is collected, and the supernatant is collected by centrifugation to obtain a biological bran hydrolyzate, which is the glucose-rich bran hydrolyzate.

2. The preparation method according to claim 1, wherein: The volume of the fungal fermentation medium in step (1) is 20 mL / 100 mL conical flask, and the initial pH value is 6.

5.

3. The preparation method according to claim 1, wherein: The particle size of the ultrafine bran is ≤6.5 μm.

4. The preparation method according to claim 1, wherein: The enoki mushroom strains described in step (2) need to be activated in the PDA culture medium in the early stage; the enoki mushroom activation method is: inoculating the enoki mushroom strains into the PDA culture medium, and culturing at a constant temperature of 25° C. for 5 days until the mycelium completely covers the surface of the culture medium, thereby obtaining an activated strain plate; The PDA culture medium contains 200g of potato, 20g of glucose, 3g of peptone and 20g of agar per liter; Then, three fungus blocks were taken from the plate covered with mycelium using a 6 mm diameter puncher and inoculated into the fungal fermentation medium for cultivation.

5. The preparation method according to claim 1, wherein: The specific method of centrifuging the collected culture fluid in step (2) is: The collected culture medium was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected as the biological bran hydrolyzate, which was stored at -20°C for later use.

6. A glucose-rich bran hydrolyzate obtained by the preparation method according to any one of claims 1 to 5.

7. Application of the bran hydrolyzate as claimed in claim 6 in the fermentation production of acetoin by Bacillus BS4481.

8. The use according to claim 7, characterized in that: The glucose-rich bran hydrolyzate is vacuum freeze-dried and concentrated to a reducing sugar concentration of 200 g / L, and then used as a carbon source to prepare a bacterial fermentation medium, wherein each liter of the bacterial fermentation medium contains 20 g of concentrated BWBH reducing sugar, 2 g of yeast powder, 5 g of KH2PO4, 10 g of K2HPO4, 1 g of (NH4)2SO4, and 0.5 g of MgSO4; Bacillus BS4481 is inoculated into the bacterial fermentation medium to ferment and produce acetoin.

9. The use according to claim 8, characterized in that: The pH of the bacterial fermentation culture medium is 7.0, and the liquid volume is 50 mL / 250 mL conical flask.

10. The use according to claim 8, characterized in that: The Bacillus BS4481 was pre-activated in LB medium and then inoculated into a bacterial fermentation medium; the LB medium was prepared to contain 10 g of peptone, 5 g of yeast powder, and 10 g of NaCl per liter, with a volume of 50 mL per 250 mL conical flask, and the activation conditions were 30° C., 180 rpm, and shaking culture for 18 hours to obtain a pre-activated BS4481 seed solution; The fermentation inoculum amount is 1% (v / v) pre-activated BS4481 seed solution; The fermentation production conditions for acetoin are 30°C, 200 rpm shaking culture for 40-48 hours.

Images