A method for preparing a carbon source of a microbial culture medium based on enzymatic corn cob powder

Abstract

The present application belongs to the technical field of high-value utilization of biomass resources, and particularly relates to a method for preparing a carbon source for a microbial culture medium based on enzymatic hydrolysis of corncob powder, and more particularly to a method for preparing a low-cost and high-efficiency carbon source suitable for the culture of probiotic bacteria by using a self-produced endoglucanase and xylanase complex enzyme system to enzymatically hydrolyze corncob powder, and applying the method to the large-scale culture of bifidobacteria, and the application of the method in the culture of bifidobacteria and the efficient synthesis of postbiotics.

Description

Technical Field

[0001] This invention belongs to the field of high-value utilization technology of biomass resources, specifically relating to a method for preparing a carbon source for microbial culture medium based on enzymatic hydrolysis of corn cob powder. Background Technology

[0002] In recent years, the probiotic industry has experienced rapid development and widespread application in food, feed, pharmaceuticals, health products, and daily chemicals. Bifidobacterium (Lactobacillus rhamnosus), due to its multiple important physiological functions such as regulating intestinal flora structure, inhibiting harmful bacteria colonization, enhancing immune function, promoting nutrient absorption and utilization, and improving metabolic levels, has become one of the most widely used and in-demand probiotic strains globally. Large-scale, efficient industrial cultivation is the core foundation for the development of the probiotic industry, while the cost of culture medium raw materials is a key factor restricting the low-cost, large-scale production of probiotics.

[0003] In traditional industrial microbial culture systems, carbon sources constitute the largest portion of the culture medium cost, with refined sugars such as glucose, sucrose, and lactose typically accounting for 30% to 50% of the total cost. Excessively high carbon source costs significantly increase the overall investment in fermentation production, reduce product market competitiveness, and severely restrict the large-scale promotion and sustainable development of the probiotic industry. Against this backdrop, developing novel biomass-based carbon sources that are widely available, inexpensive, renewable, non-grain-based, and safe and non-toxic has become an important research direction and urgent industrial need in the fields of microbial culture, fermentation engineering, and green biomanufacturing.

[0004] Corn is my country's largest grain crop, with a vast planting area and enormous annual yield. Corn cobs, as a major agricultural and forestry byproduct after corn harvest, are abundant, widely available, inexpensive, and can be processed on a large scale. The main chemical components of corn cobs are cellulose, hemicellulose, and lignin, with hemicellulose being the most abundant. Primarily structured around xylan, it also contains small amounts of branched groups such as arabinose and glucuronic acid, making it one of the highest-quality natural raw materials for preparing xylooligosaccharides and functional oligosaccharides. Currently, most corn cobs are burned on-site, discarded indiscriminately, or used only as simple fuel, resulting in extremely low resource utilization. This not only causes serious waste of biomass resources but also generates air pollutants such as smoke and particulate matter, posing a dual problem of environmental pollution and resource waste. Existing conventional processes for enzymatic or chemical hydrolysis of corn cobs often employ a combination of common xylanase and acid-base pretreatment, which easily leads to excessive degradation of hemicellulose, generating large amounts of xylooligosaccharides. Meanwhile, most probiotics, such as Bifidobacteria, can specifically metabolize xylooligosaccharides. Traditional hydrolysis methods result in low xylooligosaccharide yields and high xylose content as a byproduct, leading to poor carbon source utilization, low prebiotic quality, and poor fermentation compatibility. Furthermore, conventional commercial enzymes lack substrate specificity, easily further decomposing xylooligosaccharides into xylose during hydrolysis, hindering the targeted enrichment of high-purity xylooligosaccharides and limiting the industrial application of corn cobs in prebiotic preparation, probiotic-specific carbon sources, and high-value biomass utilization. Therefore, developing a dedicated enzymatic hydrolysis technology for corn cobs that yields high-quality xylooligosaccharides with low or no xylose production, enabling the high-value resource utilization of corn cob waste and adapting it for the fermentation and cultivation of probiotics such as Bifidobacterium, has significant economic, environmental, and industrial application prospects.

[0005] Currently, lignocellulose pretreatment and enzymatic hydrolysis technologies still have many insurmountable shortcomings in practical applications:

[0006] 1. Physical pretreatment methods: These include mechanical ball milling, ultrasonic treatment, and microwave treatment. Although they can reduce the crystallinity of cellulose and increase the specific surface area of ​​the substrate to a certain extent, they generally suffer from problems such as high energy consumption, huge equipment investment, small processing capacity, and high industrial operation costs, making it difficult to achieve large-scale industrial application.

[0007] 2. Chemical pretreatment methods: Dilute acid pretreatment is prone to generating microbial growth inhibitors such as furfural and 5-hydroxymethylfurfural under high temperature conditions. It also has problems such as severe equipment corrosion, difficulty in wastewater treatment, and high environmental load. Although traditional concentrated alkali pretreatment can effectively remove lignin, it is difficult to recover alkali, has a high salt load in wastewater, and high subsequent treatment costs, which does not meet the requirements of green chemical development.

[0008] 3. Biological pretreatment method: Lignin degradation is achieved by using lignin-degrading enzyme systems secreted by microorganisms such as white-rot fungi and brown-rot fungi. However, this method has problems such as a long treatment cycle of several days to several weeks, low degradation efficiency, easy contamination by bacteria, and serious sugar loss, which cannot meet the needs of continuous and high-efficiency industrial production.

[0009] 4. Bottlenecks in enzymatic hydrolysis technology: Commercial cellulase preparations are expensive, accounting for 40% to 60% of the total cost of saccharification; at the same time, general-purpose commercial enzyme systems are not very targeted to specific lignocellulose substrates such as corn cobs, resulting in low hydrolysis efficiency and unstable reducing sugar yield, making it difficult to achieve efficient and low-cost saccharification.

[0010] Studies have shown that xylose is difficult for most probiotics to utilize directly, easily leading to carbon source waste, slow strain growth, and low viable bacterial count and metabolite yield. In contrast, xylooligosaccharides (XOS, 2–10 xylose residues), as a high-quality prebiotic, can be specifically recognized and efficiently utilized by Bifidobacteria, significantly promoting their proliferation, increasing the synthesis of metabolites such as lactic acid, and enhancing intestinal probiotic function.

[0011] Bifidobacteria are the most essential and representative beneficial bacteria in the human gut, playing a crucial role in regulating gut microbiota balance, inhibiting pathogenic bacteria, enhancing immunity, reducing inflammation, and improving metabolism. In recent years, the academic community has proposed the concept of postbiotics, which are functional metabolites and bacterial components produced by probiotics after metabolism and inactivation. Postbiotics have advantages such as safety and stability, easy storage, no risk of live bacteria, and well-defined functions, and have become a research hotspot in the food, health product, and feed industries.

[0012] Efficient cultivation of Bifidobacteria is a prerequisite for the preparation of metabiotics. Traditional culture media use refined sugars such as glucose and lactose as carbon sources, which is costly and not conducive to the specific proliferation of Bifidobacteria and the accumulation of functional metabolites.

[0013] Corn cobs are rich in cellulose and hemicellulose (arabinoxylan), which can be directionally hydrolyzed by xylanase to produce a large amount of xylooligosaccharides (XOS). Xylooligosaccharides are recognized as highly effective biostimulants, possessing the following characteristics: they are not digested and absorbed by the human body, are not utilized by harmful bacteria such as E. coli and Salmonella, and can be specifically and efficiently utilized by Bifidobacteria.

[0014] The existing technology has the following shortcomings:

[0015] Enzymatic hydrolysis of corn cobs primarily aims to produce monosaccharides (glucose, xylose), but yields low levels of oligosaccharides and low-sugar content, failing to maximize the compatibility with Bifidobacteria. Inappropriate enzyme formulations hinder the targeted enrichment of xylooligosaccharides. Furthermore, a low-cost integrated process for corn cob hydrolysate → Bifidobacteria culture → postbiotics is lacking.

[0016] Based on this, the present invention provides a method for preparing a carbon source from corn cob enzymatic hydrolysis for the targeted production of xylooligosaccharides, and uses it for Bifidobacterium culture and postbiotic preparation, realizing the high-value utilization of the entire chain of "biomass → prebiotic carbon source → probiotics → postbiotic".

[0017] In summary, existing technologies lack an integrated carbon source preparation process that is tailored to the characteristics of corn cob substrates, operates under mild conditions, is environmentally friendly, utilizes self-produced enzymes, is low-cost, and suitable for probiotic culture. Therefore, this invention provides a method for preparing a carbon source for probiotic culture medium based on the enzymatic hydrolysis of corn cob powder using self-produced enzymes. This method achieves the resource utilization and high-value utilization of corn cob processing waste, while simultaneously solving the industry bottleneck of high carbon source costs in the industrial cultivation of Bifidobacteria. It has significant theoretical value and broad prospects for industrial application. Summary of the Invention

[0018] To address the above problems, this invention provides a method for preparing a carbon source for microbial culture medium based on enzymatic hydrolysis of corn cob powder, comprising the following steps:

[0019] (1) Preparation of corn cob powder raw material and mild alkali pretreatment: After washing and drying the corn cobs to constant weight, they were mechanically crushed to collect corn cob powder with uniform particle size; the corn cob powder was placed in a NaOH solution with a mass concentration of 1% to 4% and treated at 80℃ to 120℃ for 10 min to 90 min; after treatment, it was cooled to room temperature and the pH of the system was adjusted to 4.8 to 5.2 with dilute hydrochloric acid to obtain a pretreated corn cob powder suspension;

[0020] (2) Preparation of a self-produced endoglucanase Cel7B and xylanase XynA complex enzyme system: endoglucanase Cel7B and xylanase XynA; the encoding genes of the two enzymes were constructed into a prokaryotic expression vector, transformed into Escherichia coli BL21, expressed by IPTG, the bacterial cells were sonicated and the supernatant was collected by centrifugation to obtain the self-produced crude enzyme solution, which was then used for enzyme activity determination;

[0021] (3) Synergistic enzymatic hydrolysis with compound enzymes: The pretreated corn cob powder was prepared into a substrate suspension of 4% to 6% w / v using citrate-sodium citrate buffer; a self-produced compound enzyme system was added to the suspension, wherein the mass ratio of endoglucanase Cel7B to xylanase XynA was 1:1.5 to 1:3, and the total enzyme loading was 10 FPU / g to 30 FPU / g dry weight of corn cob powder; the enzymatic hydrolysis reaction was carried out at 45℃ to 55℃, pH 4.8 to 5.2, and 150 rpm for 36 h to 48 h to obtain corn cob hydrolysate;

[0022] (4) Solid-liquid separation: The enzymatic hydrolysate was centrifuged at 4000 rpm for 10 min to remove undegraded solid residues and collect the supernatant liquid phase components to obtain corn cob enzymatic hydrolysate rich in reducing sugars;

[0023] (5) Probiotic-specific trace element nutritional fortification: Add probiotic-specific trace element composition to the enzymatic hydrolysate, based on per liter of enzymatic hydrolysate: sodium thiosulfate 0.02 g, sodium silicate 0.01 g, ammonium molybdate 0.001 g, cobalt chloride 0.0002 g, EDTA-iron 0.005 g;

[0024] (6) Sterilization treatment: The fortified corn cob-based carbon source liquid is sterilized at 115℃~125℃ for 10 min~20 min, and then cooled to room temperature to obtain a probiotic-specific carbon source that can be used directly.

[0025] (7) Preparation of probiotic culture medium: Add the above corn cob-based carbon source to the glucose-removed MRS basal medium at a ratio of 15% to 25% v / v, adjust the pH to 5.5 to 6.0, dispense and sterilize, and use for Bifidobacterium culture.

[0026] Furthermore, the ratio of corn cob powder to NaOH solution in step (1) is 1:20 g:mL.

[0027] Furthermore, the endoglucanase Cel7B described in step (2) is derived from Trichoderma reesei.

[0028] Furthermore, the xylanase XynA mentioned in step (2) is derived from Aspergillus niger.

[0029] Furthermore, the conditions for the synergistic enzymatic hydrolysis of the compound enzyme in step (3) are: a mass ratio of Cel7B to XynA of 1:2.2, a total enzyme loading of 18 FPU / g, a substrate concentration of 5% (w / v), a hydrolysis temperature of 50℃, a pH of 5.0, and a hydrolysis time of 36 h.

[0030] Furthermore, the sterilization conditions described in step (6) are 121°C for 15 min.

[0031] Furthermore, the carbon source addition ratio in step (7) is 20%.

[0032] The present invention has the following beneficial effects:

[0033] 1. Carbon source costs have been significantly reduced.

[0034] Using corn cob processing waste as raw material to replace traditional refined carbon sources such as glucose and sucrose, the carbon source cost is reduced by more than 42.5%, and the cultivation effect is not significantly different from the pure glucose control group (p>0.05), truly achieving cost reduction without reducing efficiency.

[0035] 2. Self-produced enzymes significantly reduce enzyme costs.

[0036] The laboratory-produced Cel7B+XynA complex enzyme system is used to replace expensive commercial cellulase. The enzyme system is highly targeted, has a significant synergistic effect, and has high hydrolysis efficiency, which greatly reduces the cost of the saccharification process.

[0037] 3. The process is gentle and environmentally friendly.

[0038] Low-concentration mild alkali pretreatment is adopted to avoid equipment corrosion, inhibitor formation and high-salt wastewater discharge caused by strong acids and alkalis; the enzymatic hydrolysis conditions are mild and highly specific, with no harmful byproducts generated, and the whole process meets the requirements of green chemical industry and safe production.

[0039] 4. Suitable for high-efficiency probiotic culture

[0040] Through targeted enhancement with trace elements, key metabolic pathways of Bifidobacteria are effectively activated, significantly improving bacterial biomass, viable count, and lactic acid synthesis capacity; the enzymatic hydrolysate is safe, non-toxic, and free of inhibitors, fully meeting the requirements for food-grade probiotic culture.

[0041] 5. Easy to scale up for industrial production

[0042] The process is simple, easy to operate, has strong equipment versatility, and clear process parameters. It can be directly applied to industrial continuous production and has good economic benefits and industrialization prospects. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0044] Figure 1. Flowchart of the process for preparing carbon source for probiotic culture medium by enzymatic hydrolysis of corn cob powder

[0045] Figure 2 shows the effect of different particle sizes on the concentration of reducing sugars after enzymatic hydrolysis. As the particle size of corn cob powder decreases, the concentration of reducing sugars gradually increases, reaching its highest value at 300–400 mesh. Further decreasing the particle size to 800 mesh does not significantly increase the reducing sugar concentration. Considering both the enzymatic hydrolysis effect and the grinding cost, 300–400 mesh was determined to be the optimal particle size.

[0046] Figure 3. Effects of different pretreatment conditions on the concentration of reducing sugar after enzymatic hydrolysis; the results showed that the pretreatment with 1% NaOH, 90℃, and 60 min had the best effect, and the concentration of reducing sugar was significantly higher than that of other groups.

[0047] Figure 4. Effect of different enzyme ratios on the hydrolysis of corn cob powder; experimental results are shown below. Figure 4 As shown, the combination of endoglucanase Cel7B from Trichoderma reesei and xylanase XynA from Aspergillus niger exhibits the best hydrolysis effect on corn cob powder, significantly higher than other combinations.

[0048] Figure 5. Response surface optimization diagram of enzyme ratio and total enzyme load to reducing sugar concentration; the results show that Cel7B:XynA=1:2.2 and total enzyme load of 18 FPU / g are the optimal enzymatic hydrolysis conditions, and the reducing sugar concentration reaches the maximum value.

[0049] Figure 6. Effects of different carbon sources on the growth curves of Bifidobacterium; the results showed that when corn cob-based carbon source was used as the only carbon source, the growth trend of Bifidobacterium was not significantly different from that of the glucose control group, but was significantly better than that of the blank MRS group, indicating that corn cob-based carbon source can completely replace glucose for the culture of Bifidobacterium.

[0050] Figure 7. Effects of different carbon sources on the viable count of Bifidobacteria; as shown in Figure 7, the viable count of the corn cob-based carbon source group can reach 3.80 × 10⁻⁶. 9 CFU / mL, compared with the glucose control group (4.10×10⁻⁶). 9 The CFU / mL ratio showed no significant difference, but was significantly higher than the blank MRS group. This indicates that the enzymatic hydrolysis carbon source from corn cobs can meet the high-efficiency proliferation requirements of Bifidobacteria and has the potential to replace glucose as a carbon source for industrial culture media. Detailed Implementation

[0051] Various exemplary embodiments of the present invention are now described in detail. Unless otherwise specified, the methods used in the embodiments are conventional methods, and the reagents used are commercially available reagents or reagents prepared using conventional methods. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and embodiments of the present invention.

[0052] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0053] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0054] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0055] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0056] Example 1: Effect of corn cob powder particle size on sugar yield from enzymatic hydrolysis

[0057] Corn cob powder with particle sizes of 100 mesh, 200 mesh, 300 mesh, 400 mesh and 800 mesh was taken and treated under the optimal pretreatment conditions (1% NaOH, 90℃, 15min). Then, enzymatic hydrolysis was carried out under the same conditions, and the reducing sugar concentration of the hydrolysate was determined by the DNS method.

[0058] Experimental results show that the highest concentration of reducing sugars in corn cob powder with a mesh size of 300-400 mesh is achieved through enzymatic hydrolysis, reaching 3.60 mg / mL. Overly coarse particle size is not conducive to the binding of enzymes with substrates, while overly fine particle size increases the energy consumption and cost of grinding.

[0059] Example 2: Optimization of Mild Alkali Pretreatment Conditions

[0060] Single-factor experiments were conducted with different NaOH concentrations, treatment temperatures, and treatment times to compare the reducing sugar concentrations after enzymatic hydrolysis in each group.

[0061] Experimental results show that 1% NaOH, 90℃, and 60 min are the optimal pretreatment conditions, and the reducing sugar concentration can reach 6.41 mg / mL, which is significantly higher than that of other treatment groups.

[0062] Example 3: Screening and Ratio Optimization of Self-Produced Compound Enzyme Combinations

[0063] According to the sequences published in GenBank, the Cel7B (XP_006970146.1) and XynA (XP_001390672.1) genes were synthesized, and Nde I and Xho I restriction sites were introduced at both ends of the genes.

[0064] The synthesized gene fragment was digested with enzymes and then cloned into the prokaryotic expression vector pET-28a(+) to construct a recombinant expression plasmid.

[0065] The recombinant expression plasmid was transformed into E. coli BL21(DE3) competent cells using the heat shock method, plated on LB solid medium containing kanamycin, and cultured at 37°C for 12 h to screen for positive clones.

[0066] Positive clones were inoculated into LB liquid medium and cultured at 37°C and 220 rpm until the OD600 reached 0.6. Then, IPTG was added to a final concentration of 0.2 mM, and expression was induced at 16°C and 180 rpm for 16 h.

[0067] After induction, collect the bacterial cells by centrifugation at 8000 rpm for 10 minutes, and resuspend the cells in PBS buffer. Disrupt the cells using an ultrasonic cell disruptor (200W, 2 seconds on, 3 seconds off, total 15 minutes). After disruption, centrifuge at 12000 rpm for 15 minutes, and collect the supernatant as the crude enzyme solution. Store at 4°C for later use.

[0068] By comparing the hydrolysis effects of four commercial cellulases and three hemicellulases on pretreated corn cob powder, the single enzyme combination with the best hydrolysis efficiency was screened.

[0069] Among them, cellulase 1 is a cellulase (carrier is starch, derived from Aspergillus niger, >10U / mg); cellulase 2 is a cellulase (carrier is starch, derived from Trichoderma viride, >20U / mg); cellulase 3 is a cellulase (50U / mg biotechnology grade); and cellulase 4 is a cellulase (50U / mg).

[0070] Hemiglycanase 1 is xylanase (derived from Pichia pastoris, 9025-57-4, industrial grade, >100 U / mg); hemiglycanase 2 is xylanase XynA (derived from Aspergillus niger); hemiglycanase 3 is xylanase (derived from Trichoderma viride).

[0071] The mass ratio of Cel7B to XynA was set to 1:1, 1:1.5, 1:2, 1:2.2, 1:3, and 1:4, with a total enzyme loading of 18 FPU / g, and the enzymatic hydrolysis effect was measured.

[0072] Experimental results show that when Cel7B:XynA = 1:2.2, the synergistic effect of enzymatic hydrolysis is the strongest, and the yield of reducing sugar is the highest, reaching 42.5%.

[0073] Example 4: Optimization of Enzymatic Hydrolysis Process Parameters

[0074] The substrate concentration, hydrolysis temperature, pH, and hydrolysis time were systematically optimized, and the optimal hydrolysis conditions were determined to be: substrate concentration 5% (w / v), temperature 50℃, pH 5.0, rotation speed 150 rpm, and hydrolysis time 36 h. Under these conditions, the reducing sugar concentration was the highest and tended to be stable.

[0075] Example 5: Validation of the effect of corn cob-based carbon source on the culture of Bifidobacteria

[0076] Experimental group: 20% (v / v) corn cob-based carbon source + decarbonized MRS medium

[0077] Control group: isocarbon glucose + intact MRS medium

[0078] Culture conditions: Anaerobic culture at 37℃ for 48 h.

[0079] The test results are as follows:

[0080] Experimental group OD600: 3.28

[0081] Control group OD600: 3.35

[0082] viable bacteria count in the experimental group: 3.8 × 10^9 CFU / mL

[0083] viable bacterial count in the control group: 4.1 × 10^9 CFU / mL

[0084] The results showed that there was no significant difference between the experimental group and the control group (p>0.05), and that corn cob-based carbon source could completely replace glucose for efficient culture of Bifidobacteria.

[0085] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a carbon source for microbial culture medium based on enzymatic hydrolysis of corn cob powder, characterized in that, Includes the following steps: (1) Preparation of corn cob powder raw material and mild alkali pretreatment: After washing and drying the corn cobs to constant weight, they were mechanically crushed to collect corn cob powder with uniform particle size; the corn cob powder was placed in a NaOH solution with a mass concentration of 1% to 4% and treated at 80℃ to 120℃ for 10 min to 90 min; after treatment, it was cooled to room temperature and the pH of the system was adjusted to 4.8 to 5.2 with dilute hydrochloric acid to obtain a pretreated corn cob powder suspension; (2) Preparation of a self-produced endoglucanase Cel7B and xylanase XynA complex enzyme system: endoglucanase Cel7B and xylanase XynA; the encoding genes of the two enzymes were constructed into a prokaryotic expression vector, transformed into Escherichia coli BL21, expressed by IPTG, the bacterial cells were sonicated and the supernatant was collected by centrifugation to obtain the self-produced crude enzyme solution, which was then used for enzyme activity determination; (3) Synergistic enzymatic hydrolysis with compound enzymes: The pretreated corn cob powder was prepared into a substrate suspension of 4% to 6% w / v using citrate-sodium citrate buffer; a self-produced compound enzyme system was added to the suspension, wherein the mass ratio of endoglucanase Cel7B to xylanase XynA was 1:1.5 to 1:3, and the total enzyme loading was 10 FPU / g to 30 FPU / g dry weight of corn cob powder; the enzymatic hydrolysis reaction was carried out at 45℃ to 55℃, pH 4.8 to 5.2, and 150 rpm for 36 h to 48 h to obtain corn cob hydrolysate; (4) Solid-liquid separation: The enzymatic hydrolysate was centrifuged at 4000 rpm for 10 min to remove undegraded solid residues and collect the supernatant liquid phase components to obtain corn cob enzymatic hydrolysate rich in reducing sugars; (5) Probiotic-specific trace element nutritional fortification: Add probiotic-specific trace element composition to the enzymatic hydrolysate, based on per liter of enzymatic hydrolysate: sodium thiosulfate 0.02 g, sodium silicate 0.01 g, ammonium molybdate 0.001 g, cobalt chloride 0.0002 g, EDTA-iron 0.005 g; (6) Sterilization treatment: The fortified corn cob-based carbon source liquid is sterilized at 115℃~125℃ for 10min~20min, and then cooled to room temperature to obtain a probiotic-specific carbon source that can be used directly. (7) Preparation of probiotic culture medium: Add the above corn cob-based carbon source to the glucose-removed MRS basal medium at a ratio of 15% to 25% v / v, adjust the pH to 5.5 to 6.0, dispense and sterilize, and use for Bifidobacterium culture.

2. The method according to claim 1, characterized in that, The ratio of corn cob powder to NaOH solution in step (1) is 1:20 g:mL.

3. The method according to claim 1, characterized in that, The endoglucanase Cel7B mentioned in step (2) is derived from Trichoderma reesei.

4. The method according to claim 1, characterized in that, The xylanase XynA mentioned in step (2) is derived from Aspergillus niger.

5. The method according to claim 1, characterized in that, The conditions for the synergistic enzymatic hydrolysis of the compound enzyme in step (3) are: a mass ratio of Cel7B to XynA of 1:2.2, a total enzyme loading of 18 FPU / g, a substrate concentration of 5% (w / v), a hydrolysis temperature of 50℃, a pH of 5.0, and a hydrolysis time of 36 h.

6. The method according to claim 1, characterized in that, The sterilization conditions described in step (6) are 121°C for 15 min.

7. The method according to claim 1, characterized in that, The carbon source addition ratio in step (7) is 20%.

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