Method for preparing biocompatible raw material by depolymerizing tobacco stems

Abstract

The invention relates to the technical field of resource utilization of tobacco by-products, in particular to a method for preparing a biocompatible raw material by depolymerizing tobacco stems, which comprises the following steps: step (1), mixing the tobacco stems with alkali liquor, and then carrying out alkali thermal reaction to depolymerize lignin to obtain alkali treated slurry; and (2) adjusting the pH value of the alkali-treated slurry to 6 + / -1, and directly adding an enzyme preparation for enzymolysis to obtain enzymatic hydrolysate containing both sugar and aromatic compounds. According to the method disclosed by the invention, the sufficient depolymerization of the lignin is realized under a mild condition by accurately controlling the parameters of the alkali thermal reaction, and the slurry subjected to alkali treatment is innovatively and directly subjected to enzymolysis, so that necessary complicated steps such as solid-liquid separation and residue washing in the traditional process are omitted, the process flow is remarkably simplified, and the cost is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of resource utilization of tobacco by-products, and particularly relates to a method for preparing biocompatible raw materials by depolymerizing tobacco stems. BACKGROUND

[0002] Tobacco stems are the main by-products in the production process of tobacco industry, with a huge annual output. At present, the main utilization way of tobacco stems is to use them as raw materials for reconstituted tobacco leaves. However, due to the fact that the content of aroma components in tobacco stems is significantly lower than that in high-quality tobacco leaves, the addition proportion of tobacco stems in reconstituted tobacco leaves is limited, and excessive use will directly affect the sensory quality and commercial value of reconstituted tobacco leaves. This situation leads to the fact that the traditional application channel cannot completely absorb the large amount of tobacco stems produced every year, and the intrinsic value of tobacco stems cannot be fully tapped, resulting in resource waste.

[0003] In order to realize the high-value utilization of tobacco stems, it is considered as a promising new way to convert them into high-value-added chemicals through biotransformation technology. The core of this technology is to effectively depolymerize the main biomass components (cellulose, hemicellulose and lignin) in tobacco stems into small molecular sugar and aromatic compounds that can be utilized by microorganisms.

[0004] It is well known in the art that lignin and fragments and derivatives produced in the process of depolymerization are strong inhibitors of cellulase, which will seriously hinder the efficiency of the subsequent enzymatic hydrolysis process. In order to solve this contradiction, the prior art uses a sufficient amount or even an excessive amount of alkali under relatively severe reaction conditions to try to depolymerize and dissolve lignin as completely as possible, so as to maximize the elimination of its inhibition to enzymatic hydrolysis from the source. However, this treatment method which pursues complete depolymerization inevitably leads to the fact that the strong alkaline environment remaining in the system will directly cause the inactivation of cellulase while solving the original problem.

[0005] Based on the above technical path, the prior art forms a step-by-step separation process route that first realizes the depolymerization of lignin through strong alkali treatment, and then must be separated to obtain an alkali liquor rich in aromatic compounds; then the solid residue obtained by separation is washed to remove the residual alkali liquor; finally, the cellulose substrate after treatment is subjected to enzymatic hydrolysis to prepare sugars. This process mode can realize the separate utilization of each component, but it has multiple defects: the process flow is complex and long, and the multiple phase separation operations significantly increase the equipment investment and operating cost, in addition, this separation mode disperses the value of each component of tobacco stems, and cannot realize their synergistic utilization, which restricts the improvement of the overall resource utilization value.

[0006] In order to solve the above problems, the present application is proposed. SUMMARY

[0007] In view of the problems of complex process flow, high cost and limited resource value of tobacco stem treatment in the prior art, the purpose of the present application is to provide a method for preparing biocompatible raw materials by depolymerizing tobacco stems. The method realizes the complete depolymerization of lignin under mild conditions by precisely controlling the parameters of alkaline thermal reaction, and innovatively directly performs enzymatic hydrolysis on the slurry after alkaline treatment, thereby eliminating the cumbersome steps such as solid-liquid separation and residue washing which are necessary in the traditional process, significantly simplifying the process flow and reducing the cost.

[0008] The process breaks through the technical limitation of dispersed utilization of tobacco stem components caused by the existing step-by-step treatment mode, integrates the steps of alkaline treatment and enzymatic hydrolysis, and enables lignin, cellulose and hemicellulose in tobacco stems to be efficiently depolymerized in the corresponding steps in a coherent process system, and enables the products to be retained in the final enzymatic hydrolysis liquid, thereby obtaining a complex enzymatic hydrolysis liquid which is rich in fermentable sugars and biocompatible aromatic compounds. The enzymatic hydrolysis liquid can be directly used as a high-quality carbon source for microbial fermentation, and provides a key raw material for efficiently converting tobacco stems into gimalene and other high-value chemicals by biotransformation technology, thereby comprehensively improving the resource value and utilization level of tobacco stems.

[0009] The technical solutions adopted by the present application are as follows:

[0010] The present application provides a method for preparing biocompatible raw materials by depolymerizing tobacco stems, comprising the following steps:

[0011] Step (1), mixing tobacco stems with alkaline solution and performing alkaline thermal reaction to depolymerize lignin, and obtaining alkaline treatment slurry;

[0012] Step (2), adjusting the pH of the alkaline treatment slurry to 6±1, and directly adding enzyme preparation for enzymatic hydrolysis to obtain enzymatic hydrolysis liquid containing sugars and aromatic compounds.

[0013] Preferably, the alkaline solution is sodium hydroxide solution; the mass ratio of tobacco stems to sodium hydroxide is 8-12:0.8-1.2.

[0014] Preferably, the temperature of the alkaline thermal reaction is 110-130℃, and the reaction time is 0.8-1.2h.

[0015] Preferably, before the mixing step, a step of crushing the tobacco stems is further included, and the crushed tobacco particles need to pass through an 18-mesh sieve.

[0016] Preferably, the enzyme preparation is one or both of cellulase and hemicellulase.

[0017] In the enzymatic hydrolysis process, the addition amount of cellulase and hemicellulase is the conventional amount in the art, for example, the addition amount of complex enzyme preparation cellulose Ctec3 is added in an amount of 100 BHU. Cellulase and hemicellulase with equal activity can also be added separately.

[0018] Preferably, the alkali treatment slurry is directly subjected to enzymatic hydrolysis after pH adjustment; wherein no dewatering washing is performed before pH adjustment, and no buffer is added when adjusting pH.

[0019] The second aspect of the present application provides an enzymatic hydrolysate prepared by the method of the first aspect.

[0020] The third aspect of the present application provides a use of the enzymatic hydrolysate of the second aspect as a carbon source for microbial fermentation.

[0021] Preferably, the microorganism is bacteria or yeast.

[0022] The fourth aspect of the present application provides a use of the enzymatic hydrolysate of the second aspect as a carbon source for fermentation of an engineered bacterium for producing germacrene.

[0023] The present application has the following beneficial effects:

[0024] 1. The present application directly connects the alkali treatment and the enzymatic hydrolysis process in the same reaction system, completely eliminating the solid-liquid separation, residue washing and subsequent secondary alkali hydrolysis and other complex steps indispensable in the traditional process. This not only greatly shortens the process cycle and reduces equipment investment, but also avoids a large amount of wastewater generated in the washing process, thereby greatly reducing the overall operating cost and environmental governance pressure.

[0025] 2. In the present application, the main components in tobacco stems are respectively converted into high-value products in a coherent process. This process is based on the essential differences in chemical structure and the significant differences in reaction activity of lignin and cellulose under mild alkaline heat conditions: the ester bonds and β-O-4 ether bonds and other connecting bonds rich in the amorphous three-dimensional network structure of lignin have high reaction activity in an alkaline environment and can preferentially undergo hydrolysis and cleavage; while the crystalline region of cellulose is composed of a large number of intramolecular and intermolecular hydrogen bonds to form a dense structure, which has significant kinetic stability under the mild conditions of the present application and is completely retained. This selective depolymerization mechanism enables lignin to be efficiently converted into biologically active aromatic compounds such as p-coumaric acid, while the glycosidic bond structure of cellulose and hemicellulose is not destroyed, so that it can be efficiently converted into fermentable sugars such as glucose and xylose in the subsequent enzymatic hydrolysis process. The final enzymatic hydrolysate is a composite carbon source rich in fermentable sugars and biocompatible aromatic compounds, realizing efficient resource utilization and value enhancement of the whole components of tobacco stems.

[0026] 3、The reason why the prior art must adopt the "step-by-step separation" mode is that the severe alkaline treatment condition easily causes the recondensation reaction of lignin fragments. According to the known mechanism of lignin chemistry, under the combined action of high temperature and strong alkali, the degradation path of lignin tends to be complicated, not only the rupture of β-O-4 ether bond occurs, but also the high-activity phenoxy radical and aldehyde intermediates are generated through the reverse aldol condensation, benzaldehyde condensation and free radical homolysis and other side reaction paths. When the concentration of these intermediates reaches a certain level, complex polymers can be formed through C-O / C-C coupling or electrophilic substitution reactions. Such polymers are confirmed to be strong inhibitors of cellulase due to their significant hydrophobicity and non-specific adsorption to proteins, thereby causing the subsequent enzymatic hydrolysis process to be unable to effectively proceed.

[0027] The core breakthrough of the present application is to guide the reaction path to the ideal hydrolysis direction by precisely controlling the alkali-thermal reaction parameters. Under specific conditions, the system not only ensures the effective rupture of the β-O-4 ether bond in lignin, but also inhibits the side reactions at a relatively low level. At this time, the ionic hydrolysis path exhibits a significant kinetic advantage compared to the free radical condensation and other side reactions. The moderate alkali concentration not only provides the necessary nucleophilic environment to promote hydrolysis, but also avoids excessive promotion of side reactions. This precise regulation of the reaction path enables lignin to be effectively depolymerized into low-molecular-weight aromatic monomers, while the generation of inhibitory polymers is controlled below the critical threshold, thereby successfully integrating the alkali treatment and enzymatic hydrolysis steps in a single system. The innovation of this technical path lies in that it solves the long-standing technical bottleneck restricting the utilization of whole components of lignocellulose by regulating the competition relationship of the intermediate path in the reaction process.

[0028] 4、The prepared complex enzymatic hydrolysate does not require any subsequent separation and purification and can be directly used as a high-quality carbon source for culturing industrial microorganisms such as bacteria and yeast, especially for producing engineering bacteria of high-value chemicals such as gimalene, thereby opening up a new and economically feasible technical path for the high-value conversion of tobacco waste. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following will briefly introduce the drawings needed to be used in the description of the embodiments. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative labor.

[0030] Figure 1 The liquid chromatogram of the enzymatic hydrolysate obtained according to the embodiment 1 of the present application. DETAILED DESCRIPTION

[0031] The application is further illustrated by the following examples, which are not intended to limit the application. The experimental methods in the examples, unless otherwise specified, are generally performed according to conventional conditions and conditions described in manuals, or using general equipment, materials, reagents, etc. recommended by manufacturers, which are commercially available.

[0032] Example 1

[0033] This example provides a method for preparing a biocompatible raw material from depolymerized tobacco stems, comprising the following steps:

[0034] (1) Raw material pretreatment: dry tobacco stems were crushed by a crusher and passed through an 18-mesh sieve to obtain tobacco stem powder for use;

[0035] (2) Alkaline thermal reaction: 10 g of the above tobacco stem powder was placed in a 500 mL reaction kettle, and an alkali solution prepared by dissolving 1 g of sodium hydroxide in 200 mL of deionized water was added and stirred uniformly. The reaction kettle was sealed and reacted at 120°C for 1 hour, with continuous stirring during the reaction;

[0036] (3) pH adjustment: after the reaction was completed, the obtained alkali-treated slurry was cooled to room temperature, and the pH was adjusted to 6.0 with dilute hydrochloric acid;

[0037] (4) Enzymatic hydrolysis: a complex enzyme preparation was added to the slurry after pH adjustment for enzymatic hydrolysis, and the enzymatic hydrolysis conditions were 50°C, 200 rpm, and 48 hours. The complex enzyme preparation cellulose Ctec3 was added at 100 BHU per gram of dry tobacco stem;

[0038] (5) Product analysis: after the enzymatic hydrolysis was completed, the composition of the enzymatic hydrolysate was analyzed. High-performance liquid chromatography detection showed that the glucose concentration in the enzymatic hydrolysate was 15.5 mg / mL, the xylose concentration was 6.80 mg / mL, and the p-coumaric acid yield was 11.4 mg / g of tobacco stems.

[0039] Example 2

[0040] This example provides a method for fermenting an engineered E. coli strain using an enzymatic hydrolysate to produce germacrene, comprising the following steps:

[0041] (1) Construction of engineered bacteria: an engineered E. coli strain producing germacrene was constructed according to the method of patent CN103409400A. The gene expression vectors pACYC-mvaE-mvaS, pTrc-ERG12-ERG8-ERG19, and pET28a-HaGS-IspA-IDI were introduced into E. coli BL21(DE3) by heat shock transformation to obtain an engineered E. coli strain producing germacrene;

[0042] (2) Seed liquid preparation: single colony was picked from the plate and inoculated into LB medium containing corresponding antibiotics, and incubated at 37°C, 200 rpm for 12 hours;

[0043] (3) Fermentation medium preparation: enzyme hydrolysate prepared in Example 1 was used as the main carbon source to prepare the fermentation medium, wherein the enzyme hydrolysate was added in an amount of 60% of the total volume of the medium;

[0044] (4) Fermentation culture: the seed liquid was inoculated into the fermentation medium at an inoculation amount of 1%, and incubated at 37°C, 200 rpm until the OD600 value was about 0.8. 6 μL of 20% inducer IPTG and 10 mL of extractant n-dodecane were added, and the culture was continued at 30°C, 200 rpm for 48 hours;

[0045] (5) Product detection: a sample of the fermentation broth was taken, and the gimmerene yield was detected by gas chromatography. The results showed that the gimmerene yield reached 1.1 g / L.

[0046] Example 3

[0047] This example provides a method for fermenting Saccharomyces cerevisiae engineering bacteria for producing gimmerene using enzyme hydrolysate, which comprises the following steps:

[0048] (1) Engineering bacteria construction: Saccharomyces cerevisiae engineering bacteria for producing gimmerene were constructed according to the method of patent CN103409400A. The gene expression vector pESC-His-HaGS was introduced into Saccharomyces cerevisiae CEN.PK2-1C-F6 by electroporation to obtain Saccharomyces cerevisiae engineering bacteria for producing gimmerene;

[0049] (2) Fermentation medium preparation: enzyme hydrolysate prepared in Example 1 was used as the main carbon source to prepare the fermentation medium, wherein the enzyme hydrolysate was added in an amount of 50% of the total volume of the medium;

[0050] (3) Fermentation culture: the seed liquid of Saccharomyces cerevisiae engineering bacteria for producing gimmerene was inoculated into the fermentation medium, and the initial OD600 value was 0.5. After 12 hours of fermentation culture at 30°C, 200 rpm, 20% (v / v) dodecane was added for in-situ extraction fermentation, and the culture was continued for 48 hours;

[0051] (4) Product detection: a sample of the fermentation broth was taken, and the gimmerene yield was detected by gas chromatography. The results showed that the gimmerene yield reached 25 mg / L.

[0052] Comparative Example 1

[0053] This comparative example shows the effect of low-temperature alkali treatment on depolymerization:

[0054] The conditions are the same as Example 1 except that the alkali reaction temperature is changed to 110°C. Specifically, 10 g of tobacco stem powder is placed in a 500 mL reaction kettle, and an alkali solution prepared by dissolving 1 g of sodium hydroxide in 200 mL of deionized water is added, and the reaction is carried out at 110°C for 1 hour. The glucose and xylose concentrations in the enzymatic hydrolysate are 10.2 mg / mL and 5.2 mg / mL, respectively, and the p-coumaric acid yield is 9 mg / g of tobacco stem.

[0055] The enzymatic hydrolysate prepared in this comparative example is used for fermentation by the E. coli engineering strain according to the method of Example 2. Gas chromatography detection shows that the yield of gimmerene is 0.7 g / L, which is significantly lower than 1.1 g / L in Example 2.

[0056] Comparative Example 2

[0057] This comparative example shows the effect of high-temperature alkali treatment on depolymerization:

[0058] The conditions are the same as Example 1 except that the alkali reaction temperature is changed to 130°C. Specifically, 10 g of tobacco stem powder is placed in a 500 mL reaction kettle, and an alkali solution prepared by dissolving 1 g of sodium hydroxide in 200 mL of deionized water is added, and the reaction is carried out at 130°C for 1 hour. The glucose and xylose concentrations in the enzymatic hydrolysate are 11.2 mg / mL and 6.2 mg / mL, respectively, and the p-coumaric acid yield is about 11 mg / g of tobacco stem.

[0059] The enzymatic hydrolysate prepared in this comparative example is used for fermentation by the E. coli engineering strain according to the method of Example 2. Gas chromatography detection shows that the yield of gimmerene is 0.8 g / L, which is lower than 1.1 g / L in Example 2.

[0060] Comparative Example 3

[0061] This comparative example shows the effect of high alkali dosage on depolymerization:

[0062] The tobacco stem dosage is 8 g, the NaOH dosage is 1.2 g, the water is 200 mL, the alkali reaction temperature is 120°C, and the reaction time is 1 hour, and the other conditions are the same as in Example 1. Specifically, 8 g of tobacco stem powder is placed in a 500 mL reaction kettle, and an alkali solution prepared by dissolving 1.2 g of sodium hydroxide in 200 mL of deionized water is added, and the reaction is carried out at 120°C for 1 hour. The glucose and xylose concentrations in the enzymatic hydrolysate are 12.2 mg / mL and 6.7 mg / mL, respectively, and the p-coumaric acid yield is 9 mg / g of tobacco stem.

[0063] The enzymatic hydrolysate prepared in this comparative example is used for fermentation by the E. coli engineering strain according to the method of Example 2. Gas chromatography detection shows that the yield of gimmerene is 0.6 g / L, which is significantly lower than 1.1 g / L in Example 2.

[0064] Comparative Example 4

[0065] The present comparative example shows the influence of low alkali dosage on depolymerization effect:

[0066] The tobacco stem dosage was 10 g, the NaOH dosage was 0.8 g, the water was 200 mL, the alkali reaction temperature was 120℃, the reaction time was 1 hour, and the rest of the conditions were the same as in Example 1. Specifically, 10 g of tobacco stem powder was weighed into a 500 mL reaction kettle, and an alkali solution prepared by dissolving 0.8 g of sodium hydroxide in 200 mL of deionized water was added, and the reaction was carried out at 120℃ for 1 hour. The glucose and xylose concentrations in the enzymatic hydrolysate were 13 mg / mL and 6.2 mg / mL, respectively, and the coumaric acid yield was 11.0 mg / g of tobacco stem.

[0067] The enzymatic hydrolysate prepared in the present comparative example was used for fermentation by E. coli engineering bacteria according to the method of Example 2. Gas chromatography detection showed that the gimmerene yield was 0.9 g / L, which was lower than 1.1 g / L in Example 2.

[0068] The results of the above examples and comparative examples show that the specific process parameter range provided by the present application can achieve efficient depolymerization of the whole components of tobacco stem, and the obtained enzymatic hydrolysate can be successfully used for microbial fermentation to produce gimmerene. The results of the comparative examples further demonstrate that deviating from the preferred parameter range of the present application will result in a decrease in product yield, highlighting the advantages of the technical solution of the present application.

[0069] The above has exemplarily described the present application, and it should be noted that any simple modification, modification or equivalent replacement that does not deviate from the core of the present application and can be easily replaced by those skilled in the art without creative labor falls within the protection scope of the present application.

Claims

1. A method for preparing biocompatible raw materials by depolymerizing tobacco stems, characterized in that, The method comprises the following steps: Step (1), mixing tobacco stems with alkali solution and performing alkali thermal reaction to depolymerize lignin, to obtain alkali treatment slurry; Step (2), adjusting the pH of the alkali treatment slurry to 6±1, and directly adding enzyme preparation to perform enzymolysis, to obtain enzymolysis liquor containing both sugar and aromatic compounds.

2. The method of claim 1, wherein, The alkali solution is sodium hydroxide solution; the mass ratio of tobacco stems to sodium hydroxide is 8-12:0.8-1.

2.

3. The method of claim 1, wherein, The temperature of the alkali thermal reaction is 110-130℃, and the reaction time is 0.8-1.2h.

4. The method of claim 1, wherein, Before the mixing step, a step of crushing the tobacco stems is further included, and the tobacco particles after crushing need to be able to pass through an 18-mesh sieve.

5. The method of claim 1, wherein, The enzyme preparation is one or both of cellulase and hemicellulase.

6. The method of claim 1, wherein, The alkali treatment slurry is directly subjected to enzymolysis after pH adjustment; wherein, no water washing is performed before pH adjustment, and no buffer solution is added when adjusting the pH.

7. Enzymolysis liquor prepared by the method in any one of claims 1 to 6.

8. Use of the enzymatic hydrolysate according to claim 7, characterized in that, for use as a carbon source for microbial fermentation.

9. Use according to claim 8, characterized in that, The microorganism is bacteria or yeast.

10. Use of the enzymolysis liquor in claim 7 as a fermentation carbon source for engineering bacteria producing germacrene.

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