CN118580990B - Clostridium tyrobutyricum TGL-A236 and method for producing fatty acids by fermenting stem juice - Google Patents
Clostridium tyrobutyricum TGL-A236 and method for producing fatty acids by fermenting stem juice Download PDFInfo
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12P7/00—Preparation of oxygen-containing organic compounds
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Abstract
本发明涉及发酵工程技术领域，具体公开了酪丁酸梭菌TGL‑A236及利用茎秆汁液发酵生产脂肪酸的方法。本发明公开了一株酪丁酸梭菌(Clostridium tyrobutyricum)TGL‑A236，其保藏编号为：CCTCC NO：M 2024002。本发明还公开了应用该菌株和甜高粱茎秆汁液进行发酵生产脂肪酸的方案。本发明的酪丁酸梭菌TGL‑A236在发酵生产丁酸时产量高，此外，本发明可成功将甜高粱茎秆汁中的糖组分转化为丁酸和乙酸，实现生物质资源向化工产品的绿色转化，且方法简便、环保高效、适合工业化生产，为甜高粱综合利用开拓了新的应用前景。
The present invention relates to the technical field of fermentation engineering, and specifically discloses Clostridium tyrobutyricum TGL‑A236 and a method for producing fatty acids by fermenting stalk juice. The present invention discloses a strain of Clostridium tyrobutyricum TGL‑A236, and its preservation number is: CCTCC NO: M 2024002. The present invention also discloses a scheme for fermenting and producing fatty acids using the strain and sweet sorghum stalk juice. The Clostridium tyrobutyricum TGL‑A236 of the present invention has a high yield when fermenting and producing butyric acid. In addition, the present invention can successfully convert sugar components in sweet sorghum stalk juice into butyric acid and acetic acid, realizing the green conversion of biomass resources into chemical products, and the method is simple, environmentally friendly and efficient, suitable for industrial production, and opens up new application prospects for the comprehensive utilization of sweet sorghum.
Description
Technical Field
The invention relates to the technical field of fermentation engineering, in particular to clostridium tyrobutyrate TGL-A236 and a method for producing fatty acid by utilizing stalk juice fermentation.
Background
Sweet Sorghum (Sorgum bicolor (L.) Moench) is succulent and rich in sugar, and has the advantages of drought resistance, waterlogging resistance, saline-alkali resistance, short growth period, rapid accumulation of sugar, high biological yield and the like. The planting popularization rate of sweet sorghum is not high, the seeds of the sweet sorghum are more prone to be used as main food or white spirit brewing, juice extracted from stems of the sweet sorghum is often used as a raw material for preparing syrup in the food industry, and high-value deep processing application is lacking. The juice yield of the sweet sorghum stalks is generally not more than 55%, and a large amount of non-structural soluble sugar such as sucrose (53-85%), glucose (9-33%) and fructose (6-21%), starch, amino acid, inorganic salt and secondary products including aconitic acid, organic acid, phenolic substances and the like are contained in the juice, so that the added value of the sweet sorghum products can be improved to a greater extent, and the deep-processed industrial chain structure of the sweet sorghum products can be prolonged and perfected if the sweet sorghum products are developed and utilized as carbon sources for microbial fermentation.
Studies have shown that sweet sorghum stalk juice can be used as a substrate for microbial growth and metabolism. The Chinese patent No. CN103740568A discloses a white spirit brewing method which uses aroma-producing yeast as a fermentation strain and sweet sorghum stalk juice as a unique carbon source, and high-quality sweet sorghum white spirit with the purity of 50-68 degrees is obtained, and the Chinese patent No. CN101230371A prepares a yeast glucan product with the purity of more than 80 percent by fermenting the sweet sorghum juice. The sweet sorghum stalk juice can also be mixed with other substrates to participate in the growth metabolism of microorganisms and the synthesis of products, for example, chinese patent CN104694334A and Chinese patent CN104673590A respectively disclose brewing processes for brewing fruit juice wine by taking sweet sorghum stalk juice and wild hawthorn pulp as substrates and preparing fruit wine by taking sweet sorghum stalk juice and dogwood as raw materials, chinese patent CN102321679A discloses a method for fermenting acetone, butanol and ethanol by mixing sweet sorghum juice with straw enzymatic hydrolysate thereof, and Chinese patent CN108374024A discloses an intensive production process for coproducing ethanol, glucose syrup and fructose products by sweet sorghum stalk juice and corn flour.
Clostridium tyrobutyrate (Clostridium tyrobutyricum) is strictly anaerobic, gram positive, has very high butyric acid tolerance and butyric acid selectivity, and is the best cell factory for producing butyric acid. As biomass substrates for clostridium tyrobutyrate fermentation of butyric acid, many studies and researches have been made by the former, such as hydrolysis treatment of raw materials of corn fiber, cane molasses, beet molasses or wheat straw. However, the agricultural raw materials for producing sucrose at the present stage mainly depend on corns, sugarcanes, sugarbeets and the like, and a large amount of the raw materials are used for industrial fermentation, so that not only can the supply of raw materials of bulk agricultural products and animal feeds be reduced, but also the market price of the sucrose can be influenced, and therefore, the non-food sugar material carbon source, namely sweet sorghum stalk juice, has huge potential as a fermentable sugar source for synthesizing the biological butyric acid.
Disclosure of Invention
It is an object of the present invention to provide clostridium tyrobutyrate which can efficiently produce butyric acid, and a method for producing fatty acids from stem juice.
In order to achieve the object, the technical scheme of the invention is as follows:
In a first aspect, the invention provides clostridium tyrobutyricum (Clostridium tyrobutyricum) TGL-A236 with a preservation number of CCTCC NO: M2024002.
According to the invention, clostridium tyrobutyrate is subjected to heavy ion irradiation treatment, a new clostridium tyrobutyrate TGL-A236 is obtained by unexpected screening, butyric acid can be produced by fermentation, the yield is increased by more than one time compared with that of a starting strain, and unexpected effects are obtained.
In a second aspect, the present invention provides a microbial agent comprising clostridium tyrobutyrate (Clostridium tyrobutyricum) TGL-a236 as described above.
The clostridium tyrobutyricum TGL-A236 microbial inoculum of the invention can be in a liquid state or a solid state.
Preferably, clostridium tyrobutyrate TGL-a236 may be prepared as a bacterial suspension by culture, or further prepared as a solid microbial inoculum for subsequent fermentation production of butyric acid.
In a third aspect, the invention provides the use of clostridium tyrobutyricum (Clostridium tyrobutyricum) TGL-a236 or a microbial inoculum as described above in the fermentative production of fatty acids.
Preferably, the fatty acid is butyric acid and/or acetic acid.
In the application of the invention, the carbon source used in fermentation is sweet sorghum stalk juice, preferably, the culture medium used in fermentation comprises a nitrogen source and sweet sorghum stalk juice, the nitrogen source is yeast extract and/or peptone, more preferably, the concentration of the nitrogen source is 2-8 g/L (further preferably, 2.8-3.2 g/L).
Yeast extract and peptone (Peptone) are all commercial conventional products in the present invention. The yeast extract is a brown yellow soluble product prepared by degrading proteins, nucleic acids and the like in yeast cells and refining. Peptone is a pale yellow powder with the appearance of meat, casein or gelatin hydrolyzed with acid or protease and dried.
As a specific embodiment, the yeast extract used in the present invention is available under the product designation OXOID LP0021B. The product brand of peptone is Beijing aobosin AOBOX,01-001.
In the application of the invention, the fermentation condition is that the inoculum size is 8-12% v/v, and the fermentation is carried out for 11-13 hours at 37+ -1 ℃ and then is carried out for 72-120 hours in shaking at 140-160rpm at 37+ -1 ℃.
Preferably, the culture medium in the fermentation process of the invention also comprises water, the mixed solution of water and sweet sorghum stalk juice is used as a solvent of the culture medium, and the proportion of the water to the sweet sorghum stalk juice can be adjusted by a person skilled in the art according to the fermentation requirement so as to ensure that the fermentation is normally carried out and the carbon source is sufficient, and more preferably, the proportion of the water to the sweet sorghum stalk juice is 1:1v/v.
In the application of the invention, the preparation method of the sweet sorghum stalk juice comprises the following steps:
(1) Preprocessing, namely squeezing sweet sorghum stalks, centrifuging, collecting supernatant, adjusting the pH value to 4.8-5.2, and sterilizing to obtain a solution;
(2) Non-reducing sugar hydrolysis, namely, carrying out enzymolysis on the solution by sucrose invertase to obtain hydrolysate;
(3) Degumming, namely removing colloid particles in the hydrolysate by diatomite to obtain a solution to be decontaminated;
(4) And (3) removing impurities, namely standing the solution to be removed, centrifuging, adjusting the pH value to 6.2-6.5, and filtering.
According to the invention, the juice obtained after juice extraction of the sweet sorghum stalks is directly used as a culture medium component of clostridium tyrobutyrate, clostridium tyrobutyrate cannot grow, and clostridium tyrobutyrate cannot directly utilize high-proportion sucrose contained in the sweet sorghum stalk juice to perform growth metabolism, so that a large amount of sucrose in fermentation liquor remains.
In the prior art, the sweet sorghum stalk juice is generally treated by a chemical method, such as an acid hydrolysis method (such as adding a strong acid reagent such as sulfuric acid) to hydrolyze sucrose, and adding chemical reagents such as lime milk, calcium hydroxide, a polyanionic flocculant and the like to remove colloid and impurities. A conventional sweet sorghum juice treatment method comprises a hot lime clarification method based on cane molasses, specifically, the sweet sorghum juice is heated firstly, colloid particles and soluble proteins are coagulated and form natural floccules, then lime Milk (MOL) is added to the sweet sorghum juice, pH is adjusted to 6.5 to neutralize acid, and a polyanionic flocculant is added to enable the floccules to settle, so that clarified sweet sorghum stalk juice is obtained. The turbidity removal rate of sweet sorghum juice treated by the conventional method can reach more than 90%, but the lime milk and the flocculant are often industrial chemical reagents, the safety and the environmental protection of the lime milk and the flocculant are still to be evaluated, and the lime milk and the flocculant are not green and clean treatment processes.
According to the invention, sucrose is converted by an enzyme hydrolysis method, the hydrolysis liquid is degummed by the diatomite serving as a food additive, the removal rate of colloid is as high as more than 99%, the efficiency is higher, the sucrose conversion efficiency of the obtained hydrolysis liquid is high, the loss of converted sugar (fructose and glucose) is low, and the environment-friendly butyric acid which can be produced by taking the substrate as a carbon source can be further applied to more high-value fields such as foods, biological medicines and the like, and the added value of the butyric acid is improved.
In the invention, the sucrose invertase is a commercially available conventional product, and a person skilled in the art can select the enzymolysis condition according to the selected sucrose invertase, preferably, the sucrose invertase is purchased from Hongrunbaoshun (China) so as to be beneficial to improving the sucrose hydrolysis rate. In the application of the invention, the centrifugation condition in the step (1) is 3800-4200rpm,8-12min;
In the step (1), 1M phosphoric acid solution can be adopted to adjust the pH, and the sterilization condition is that the sterilization is carried out at the high temperature of 110-120 ℃ for 15-25min.
And/or in the step (2), the adding amount of the sucrose invertase is 0.008-0.012%w/v, the enzyme activity of the sucrose invertase is more than or equal to 200units/mg, and the enzymolysis condition is that the sucrose invertase is treated for 18-24 hours under the oscillation of 41-43 ℃ and 140-160 rpm;
and/or, in the step (3), the adding amount of the diatomite is 4.8-5.2% w/v, and the degumming condition is that the diatomite is treated for 1.5-3 hours under the vibration of 40-45 ℃ and 140-160rpm (preferably, the diatomite is treated for 1.8-2.2 hours under the vibration of 41-43 ℃ and 140-160 rpm);
in the step (3), 1M NaOH can be used for adjusting the pH.
And/or, the standing time in the step (4) is 25-35min, the centrifugation condition is 3800-4200rpm,8-12min, and the pore size at the time of filtration is 0.45-1 μm (preferably, 0.7 μm).
In the application of the invention, after removing the colloid particles in the hydrolysate, the method further comprises a step of decoloring;
Preferably, the activated carbon is used for decolorization, the addition amount of the activated carbon is 0.8-1.2% w/v, and the decolorization treatment condition is that the activated carbon is treated for 1.5-3 hours under the conditions of 40-45 ℃ and 140-160rpm (preferably, the activated carbon is treated for 1.8-2.2 hours under the conditions of 41-43 ℃ and 140-160 rpm).
In a fourth aspect, the present invention provides a method for fermentative production of fatty acids using sweet sorghum stalk juice as carbon source, the method for preparing sweet sorghum stalk juice being as described above.
The method of the invention adopts a specific preparation method of sweet sorghum stalk juice, so that the sweet sorghum stalk juice can be successfully used as a carbon source for producing fatty acid, is used for fermenting fatty acid by clostridium tyrobutyricum, and expands the utilization value of sweet sorghum stalk.
In the method of the present invention, the fermentation bacteria used for fermentation are Clostridium casei Cirm BIA 2237 or Clostridium casei (Clostridium tyrobutyricum) TGL-A236, clostridium casei (Clostridium tyrobutyricum) TGL-A236 as described above, and/or the medium and conditions of fermentation are as described above.
The invention has the advantages that:
The invention provides a novel clostridium tyrobutyrate TGL-A236 which can efficiently produce butyric acid and is beneficial to industrial fermentation production of butyric acid.
The invention also obtains a method for successfully converting sugar components in sweet sorghum stalk juice into butyric acid and acetic acid through specific treatment of the sweet sorghum stalk. The method realizes the conversion of the biological butyric acid by taking sweet sorghum stalk juice as the only carbon source. The sweet sorghum stalk treatment method disclosed by the invention has the advantages of high sucrose hydrolysis conversion rate, low sucrose loss rate and excellent colloid removal effect, can improve the centrifugal efficiency and mass transfer rate so as to improve the butyric acid fermentation efficiency, realizes the green conversion of biomass resources to chemical products, is simple, environment-friendly and efficient, is suitable for industrial production, and opens up a new application prospect for the comprehensive utilization of sweet sorghum.
Preferably, the sweet sorghum stalk juice prepared by the specific method is used for producing butyric acid by the strict anaerobic fermentation of clostridium tyrobutyrate TGL-A236, so that the yield of butyric acid is further improved, and the sweet sorghum stalk juice has wide application prospect.
Drawings
FIG. 1 is a schematic process flow diagram of the treatment method of the present invention.
Fig. 2 shows the change of the appearance of the sweet sorghum stalk juice during the green treatment, and the sweet sorghum juice is sequentially subjected to coarse liquid separation, degumming and decoloration, sedimentation and impurity removal from left to right.
FIG. 3 is a graph showing statistics of sugar component content at various time points of sucrose invertase treatment of sweet sorghum juice. In the figure, the lower case letters on the data histogram are different, representing significant differences.
FIG. 4 is a kinetic model of non-reducing enzymatic hydrolysis in sweet sorghum juice.
Fig. 5 shows particle size distribution of sweet sorghum stalk juice in green treatment, and the detection results of sweet sorghum juice after coarse separation, degumming and decoloring and sedimentation and film passing are shown in sequence from top to bottom.
Fig. 6 shows the solid/liquid culture growth of clostridium tyrobutyicum Cirm BIA and 2237 in the pre-treatment/post-treatment sweet sorghum stalk juice, the top panel shows the solid/liquid culture of the pre-treatment sweet sorghum stalk juice, and the bottom panel shows the solid/liquid culture of the post-treatment sweet sorghum stalk juice.
FIG. 7 shows the solid/liquid culture growth of Clostridium tyrobutyrate TGL-A236 before/after treatment, the upper graph shows the solid/liquid culture of sweet sorghum stalk juice before treatment, and the lower graph shows the solid/liquid culture of sweet sorghum stalk juice after treatment.
FIG. 8 is a graph showing the 24h growth curve of Clostridium tyrobutyrate Cirm BIA 2237 in sweet sorghum stalk juice medium liquid culture before/after treatment.
FIG. 9 is a graph showing the growth curve of Clostridium tyrobutyrate TGL-A236 in sweet sorghum stalk juice medium liquid culture for 24h before/after treatment.
FIG. 10 is a graph showing the synthesis of butyric acid and acetic acid produced by Clostridium tyrobutyrate Cirm BIA 2237 in sweet sorghum stalk juice after treatment.
FIG. 11 is a graph showing the synthesis of butyric acid and acetic acid as the products of Clostridium tyrobutyrate TGL-A236 in sweet sorghum stalk juice after treatment.
FIG. 12 shows the production of butyric acid by shake flask fermentation of Clostridium tyrobutyrate Cirm BIA 2237 and TGL-A236 in RCM medium at different carbon source concentrations. In the figure, ns represents that the difference is not significant, and x represents that the difference is significant.
FIG. 13 shows the butyrate production of Clostridium tyrobutyrate Cirm BIA 2237 and TGL-A236 by shake flask fermentation in treated sweet sorghum broth peptone medium containing varying concentrations of nitrogen source. In the figure, the difference is significant.
Detailed Description
Preferred embodiments of the present invention will be described in detail below with reference to examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention may be made by those skilled in the art without departing from the spirit and scope of this invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents and the like used in the examples below, unless otherwise indicated, are all those available commercially or may be prepared by methods conventional in the art.
The strain of the invention is as follows:
Clostridium casei (Clostridium tyrobutyricum) Cirm BIA 2237 is provided by recent physical research institute of China academy of sciences, stored in biophysical room of biomedical center, disclosed in ：Whole-Genome Sequencing and Annotation of Clostridium tyrobutyricum Strain Cirm BIA 2237,Isolated from Silage.DOI:https://doi.org/10.1128/mra.00492-19.
Clostridium tyrobutyrate (Clostridium tyrobutyricum) TGL-A236 is a mutant strain which is bred by combining solid-liquid adaptive evolution culture with IV generation directional screening (optimal acid production) strategy in a sweet sorghum juice culture medium after 300Gy dose 12C6+ ion beam irradiation mutagenesis of clostridium tyrobutyrate (Clostridium tyrobutyricum) Cirm BIA 2237.
Clostridium tyrobutyrate (Clostridium tyrobutyricum) TGL-A236 is preserved in China center for type culture collection (CCTCC, address: china, university of Wuhan, and mail code: 430072) at 1 month and 2 days 2024, and classified and named clostridium tyrobutyrate TGL-A236Clostridium tyrobutyricum TGL-A236 with a preservation number of CCTCC NO: M2024002.
In the following examples, clostridium tyrobutyrate (Clostridium tyrobutyricum) TGL-A236 is abbreviated as clostridium tyrobutyrate TGL-A236, clostridium tyrobutyrate (Clostridium tyrobutyricum) Cirm BIA 2237 is abbreviated as clostridium tyrobutyrate Cirm BIA 2237, and the same meaning is given.
Example 1 Green treatment Process and treatment Effect of sweet sorghum stalks
In the embodiment, the sweet sorghum stalks are treated according to the following green process flow:
1. Squeezing juice and coarse separation of raw materials
Squeezing the harvested sweet sorghum stalks by a three-hub squeezer, centrifuging the juice at 4000rpm for 10min, collecting supernatant, adjusting the pH to 5.0,115 ℃ by using a 1M phosphoric acid solution, sterilizing at high temperature for 20min, and freezing in a-20 ℃ refrigerator for later use.
2. Catalytic non-reducing sugar hydrolysis
Sucrose invertase (CAS NO:9001-57-4, > 200units/mg, hongrungshun, china) was added to the sweet sorghum juice stock solution in an amount of 0.01% (w/v; equivalent to 20 units/mL), and hydrolyzed in a constant temperature shaker at 42℃and 150rpm for 24 hours.
3. Degumming and decoloring treatment
5% (W/v) of diatomaceous earth (CAS NO:61870-53-2, metallocene, china) was added to the hydrolysate, after 2h in a 42℃and 150rpm thermostatic shaker, 1% (w/v) of food grade powdered activated carbon was added, and the mixture was also placed in a 42℃and 150rpm thermostatic shaker for 2h (the step of treating with activated carbon may be omitted if there was NO requirement for the chromaticity of the solution).
4. Sedimentation, filtration and impurity removal
Standing for 30min by sedimentation, centrifuging at 4000rpm for 10min, regulating pH of supernatant with 1M NaOH solution to 6.3, filtering with water filter (glass fiber, 0.7 μm×25mm, new Star, china) with pore diameter of 0.7 μm, collecting filtrate, packaging, and storing in-20deg.C refrigerator.
The schematic diagram of the treatment process of fresh sweet sorghum stalks is shown in fig. 1, the appearance changes of the sweet sorghum juice after coarse liquid separation, degumming and decoloration and the sweet sorghum juice after sedimentation and impurity removal are shown in fig. 2, the color value, turbidity and the like of the sweet sorghum juice after treatment by the method are obviously reduced, and the sweet sorghum juice is converted from brown suspension with suspended particles into clear and transparent light yellow solution and can be used as a preferred carbon source of clostridium microorganisms.
Example 2 analysis of sucrose conversion Effect of sweet sorghum stalk juice
1. Experimental method
The sucrose invertase described in example 1 was added to sweet sorghum stalk juice (prepared by the method of step 1 of example 1) in an amount of 0.01% (w/v) for enzymolysis, and the enzymolysis was performed in a constant temperature shaker at 42 ℃ and 150rpm, samples were taken at 0, 6, 12, 18, and 24 hours, and the main sugar component in the hydrolysate was measured by High Performance Liquid Chromatography (HPLC) and the degree of sucrose hydrolysis and inversion was analyzed.
The detection method comprises the following steps:
(1) Zafex Carbohydrate ES chromatography columns (250X 4.6mm,5 μm).
(2) Chromatographic conditions, mobile phase A-25% ultrapure water, mobile phase B-75% acetonitrile, flow rate 1.0ml/min, chromatographic column temperature 40 ℃, RI differential detector temperature 30 ℃, sample injection amount 15.00 mu L, and running time 25min.
2. Experimental results
As can be seen from the fact that the hydrolysis and conversion conditions of sucrose in the sweet sorghum juice before and after treatment are shown in FIG. 3, after the sucrase is added into the sweet sorghum juice stock solution for reaction for 24 hours, the concentration of sucrose is converted from 59.59g/L to the remaining 1.02g/L through hydrolysis, and the total amount of converted sugar reaches 133.69g/L. The sucrose hydrolysis rate of the sweet sorghum juice under the action of sucrose invertase reaches 98.29 percent, the sucrose invertase rate is 99.13 percent, and the sucrose loss rate is only 0.87 percent. Meanwhile, trace sucrose still remains unhydrolyzed when the reaction is carried out for 24 hours, so that no sucrose invertase remains after the enzymolysis reaction, and most sucrose is hydrolyzed and converted when the reaction is carried out for 18 hours, and the total amount of invert sugar (fructose and glucose) is not significantly different from the value when the reaction is carried out for 24 hours, so that the sucrose hydrolysis treatment time of the process can be optimized to 18 hours.
This example also performed a first/second order enzymatic hydrolysis reaction kinetic model fit based on sweet sorghum juice sucrose inversion experimental data, wherein the reaction rate is expressed as invert sugar production per unit time after sucrose hydrolysis (fig. 4). The conversion sugar production amount and the enzymatic reaction time at different time points are subjected to nonlinear fitting, so that the fact that the fitting effect of the sucrose hydrolysis reaction of the sucrase in the sweet sorghum juice substrate in a primary dynamic model (R 2 > 0.999) and a secondary dynamic model (R 2 > 0.999) is good is verified, the experimental value of the final conversion sugar production amount of the sweet sorghum juice stock solution is slightly lower than the predicted value of the primary/secondary dynamic model, and the reasonable technological enzymolysis reaction condition setting is proved, and no sucrose invertase residue exists.
Example 3 analysis of degumming Effect of sweet sorghum straw juice
In this example, the particle size distribution of the juice of sweet sorghum stalks, which was not treated (obtained by the method of example 1, step 1) and treated by the method of each step described in example 1, was measured by a wet full-automatic laser particle size analyzer. The test parameters are that the refractive index of the sample is 1.61-0.1i, the refractive index of the medium is 1.33, the dispersion medium is water, the test particle size range is 0.1-800 mu m, the ultrasonic power is 50W, and the circulation flow is 1L/min. After the background collection procedure is completed, the untreated and treated sweet sorghum stalk juice is prepared and is detected on a 50mL machine. Coarse separation of sweet sorghum juice, degumming and decoloring, settling and impurity removing, and grain size distribution of sweet sorghum stalk juice. The results are shown in FIG. 5.
The diameter range of the colloid particles is generally 0.001-1 mu m (namely 1-1000 nm) (Gregory, 2005), and the particle size is differentiated according to the cumulative distribution curve of the particle size of the suspended particles, so as to obtain the volume distribution ratio of the particles of the sweet sorghum stalk juice before/after treatment in different particle size ranges.
The ratio of the colloidal particles was found to be 18.57% in the crude sweet sorghum juice chaotropic (particle size <1 μm) as compared with the ratio of the colloidal particles (particle size <1 μm) in the crude sweet sorghum juice chaotropic (upper graph in FIG. 5), and the ratio of the colloidal particles in the degummed and decolorized sweet sorghum juice (middle graph in FIG. 5) was directly reduced to 0.07%, and the removal rate of the colloidal particles was found to be 99.62%. Finally, the grain size of the sweet sorghum juice (the lower graph in fig. 5) subjected to sedimentation and impurity removal is almost free of grains with the grain size of more than 0.1 mu m, which shows that the flocculation and impurity removal effect of the process is good.
EXAMPLE 4 solid/liquid culture of Clostridium tyrobutyrate Cirm BIA 2237 and Clostridium tyrobutyrate TGL-A236 in sweet sorghum stalk juice after untreated/treated
Clostridium intensified culture medium (RCM) 10.0g/L peptone, 10.0g/L beef powder, 3.0g/L yeast extract, 5.0g/L glucose, 1.0g/L soluble starch, 5.0g/L sodium chloride, 3.0g/L, L-cysteine hydrochloride, 0.5g/L sodium acetate.
The treated sweet sorghum juice culture medium comprises 3g/L of yeast extract, the treated sweet sorghum stalk juice (prepared by the method of the example 1) is added as a solvent, and agar 20g/L is added when the solid culture medium is prepared.
Untreated (pre-treatment) sweet sorghum juice culture medium: 3g/L of yeast extract, untreated sweet sorghum stalk juice (substantially the same preparation method as the method step 1 of example 1 except that the pH was adjusted to 6.3) was added as a solvent, and agar 20g/L was added at the time of preparation of the solid culture medium.
Two types of clostridium tyrobutyrate are cultivated and activated in a clostridium intensified culture medium until the bacterial liquid OD 600 is 0.8, then clostridium tyrobutyrate bacterial liquid is inoculated into untreated and treated sweet sorghum stalk juice solid and liquid culture mediums according to the inoculum size of 5% (v/v), anaerobic culture is carried out under the conditions of 37 ℃ and 150rpm, and the growth condition of the strain before and after treatment of the sweet sorghum stalk juice culture mediums is observed. The results are shown in FIGS. 6 to 9.
FIGS. 6 and 7 show the growth of Clostridium tyrobutyrate Cirm BIA 2237 and Clostridium tyrobutyrate TGL-A236 in untreated/treated sweet sorghum stalk juice solid and liquid media, the strains showed no signs of growth after 3d of the pre-treated sweet sorghum stalk juice plate and liquid media, while round colonies with milky white, neat edges, smooth and moist surfaces and diameters of 1-3 mm were formed after dilution of 1000X coated for 3d in the treated sweet sorghum stalk juice plate, the strains grew well after 1d in the treated sweet sorghum stalk juice medium, the cell concentration increased significantly, the maximum OD 600 reached 1.4030 (Cirm BIA 2237) and 1.4737 (TGL-A236), the bacterial liquid surface had significantly white fine foam, and the average gas yield of 10mL bacterial liquid reached above 60 mL.
As can be seen from fig. 8 and 9, the cell density (OD 600) of clostridium tyrobutyricum Cirm BIA 2237 in the untreated sweet sorghum stalk juice decreases from the initial 0.3473 to 8 hours to 0.3153, the maximum OD 600 appearing in clostridium tyrobutyricum TGL-a236 at 4 hours is 0.4223 until 24 hours, and then the cell density fluctuates in the interval of 0.35-0.37 all the time, which indicates that the cells of the two strains can not grow and reproduce normally in the growing environment of the untreated sweet sorghum stalk juice.
When the strain is inoculated into the treated sweet sorghum stalk juice culture medium, the complete growth cycle of clostridium tyrobutyrate Cirm BIA 2237 can be observed from the growth curve, after a delay period of 4 hours, the strain is in a logarithmic phase of high-speed growth until 12 hours, then the growth rate gradually slows down to 20 hours, OD 600 reaches a maximum value 1.4030, finally the decay period OD 600 is slowly reduced, and the maximum OD 600 (1.4737) of clostridium tyrobutyrate TGL-A236 appears in 24 hours and still does not reach a plateau period, and the growth performance of the strain is stronger than that of clostridium tyrobutyrate Cirm BIA 2237. In conclusion, two strains of clostridium tyrobutyrate can normally grow and reproduce in sweet sorghum stalk juice treated by the green process, and the feasibility of the process is proved.
EXAMPLE 5 production of butyric acid in juice of sweet sorghum stalks by Clostridium tyrobutyrate Cirm BIA 2237 and Clostridium tyrobutyrate TGL-A236
Fermentation medium, 3g/L of yeast extract, treated sweet sorghum stalk juice (obtained by the method of example 1) is diluted 1:1 with pure water, and the diluted solution is added as a solvent to prepare.
Clostridium tyrobutyrate Cirm BIA and Clostridium tyrobutyrate TGL-A236 are inoculated into clostridium intensified culture medium (see example 4), standing and culturing for 20-24 h in a 37 ℃ incubator, aligning activated bacterial liquid OD 600 to 0.8, transferring into anaerobic fermentation bottle containing 200mL fermentation medium with 10% (v/v) inoculation amount, introducing sterile nitrogen gas, and sealing the bottle mouth. After the fermentation flask was allowed to stand in a 37℃incubator for 12 hours, it was transferred to a 37℃incubator with a constant temperature shaker at 150rpm for 120 hours. Samples were taken at fermentation times 0, 24, 48, 72, 96, 120h and the concentrations of the product butyric acid and acetic acid in the fermentation broth were measured at different time points.
The experimental results are shown in fig. 10 and 11, and the concentration of butyric acid and acetic acid increases exponentially from 12h to 72h after a very short delay period. The maximum butyric acid concentration of clostridium tyrobutyrate Cirm BIA 2237 during the whole batch fermentation process was 12.0448g/L, the butyric acid selectivity was 88.78%, the maximum butyric acid concentration of the mutant clostridium tyrobutyrate TGL-a236 was 24.1364g/L, and the butyric acid selectivity was 86.36%.
The concentration of acetic acid by-products of clostridium tyrobutyrate Cirm BIA, 2237 and clostridium tyrobutyrate TGL-a236 reached the highest levels 1.4413g/L and 3.8798g/L respectively at 96 hours, followed by gradual decrease in concentration. From the synthetic curve of the product butyric acid/acetic acid, the sweet sorghum juice treated by the process of the invention can be used for fermentation production of the main product butyric acid.
In summary, the invention provides clostridium tyrobutyrate (Clostridium tyrobutyricum) TGL-A236 with ideal butyric acid production performance. The invention also provides a treatment process of the sweet sorghum stalk juice, which has high sucrose hydrolysis conversion rate, wherein the sucrose hydrolysis rate of the sweet sorghum stalk juice reaches more than 98 percent under the action of sucrose invertase, the sucrose conversion rate is 99.13 percent, the sucrose loss rate is only 0.87 percent, and the colloid removal effect is excellent, the colloid particle removal rate reaches 99.62 percent, and the treatment process plays an important role in improving the centrifugal efficiency and the mass transfer rate so as to improve the butyric acid fermentation efficiency.
The solution obtained by the sweet sorghum stalk juice treatment process is suitable for culturing clostridium butyricum, and the green treatment process of sweet sorghum stalk juice which can be used for culturing clostridium bacteria and fermenting butyric acid is provided, and under the conditions of 37 ℃ of culture temperature, 24-72 h of culture time and strict anaerobism, the normal growth and propagation of clostridium tyrobutyricum strains in sweet sorghum stalk juice/solid culture medium can be observed.
The treatment process of sweet sorghum stalk juice is suitable for producing butyric acid, the activated clostridium tyrobutyrate Cirm BIA A2237 and TGL-A236 bacterial solutions are inoculated into a sweet sorghum stalk juice culture medium for 5d shake flask fermentation experiments, the highest concentration of butyric acid is detected to be 12.0448g/L and 24.1364g/L respectively, the highest concentration of acetic acid is 1.4413g/L and 3.8798g/L respectively, sugar components in sweet sorghum stalk juice are converted into butyric acid and acetic acid, green conversion of biomass resources to chemical products is realized, and the method is simple, environment-friendly and efficient, is suitable for industrial production, and opens up a new application prospect for comprehensive utilization of sweet sorghum.
EXAMPLE 6 shake flask fermentation experiments of Clostridium tyrobutyrate Cirm BIA 2237 and Clostridium tyrobutyrate TGL-A236 in Clostridium enhanced Medium (RCM)
1. Experimental method
The experimental strain is clostridium tyrobutyrate Cirm BIA 2237 and TGL-A236.
The clostridium intensified culture medium (RCM) comprises peptone 10.0g/L, beef powder 10.0g/L, yeast extract 3.0g/L, soluble starch 1.0g/L, sodium chloride 5.0g/L, sodium acetate 3.0g/L, L-cysteine hydrochloride 0.5g/L. The carbon source is glucose, and three gradients of 5.0g/L,20.0g/L and 60.0g/L are set.
Culturing and activating two clostridium tyrobutyrate bacteria in RCM culture medium containing 5g/L glucose until the bacterial liquid OD 600 is 0.8, inoculating clostridium tyrobutyrate bacterial liquid into RCM liquid culture medium with glucose concentration of 5.0g/L,20.0g/L and 60.0g/L respectively according to 5% (v/v) inoculum size, performing anaerobic shake flask fermentation at 37 ℃ and 150rpm for 72h, and detecting butyric acid production condition of each strain in RCM culture medium under three carbon source gradients by adopting gas chromatography.
2. Experimental results
The fermentation results are shown in FIG. 12. As can be seen from the above, when the glucose concentration was 5g/L, clostridium casei Cirm BIA 2237 (control group) and mutant TGL-A236 produced 2.1397g/L and 2.3124g/L butyric acid, respectively, and the butyric acid production of the two strains was at the same level due to the lower carbon source concentration, without significant difference. When the glucose concentration is raised to 20g/L, the final butyric acid concentration of clostridium casei Cirm BIA 2237 is 6.2410g/L, and clostridium casei TGL-A236 can synthesize 9.7580g/L butyric acid in 72h, which is significantly higher than that of the control group. When the carbon source concentration is increased to 60g/L, the final concentration of butyric acid of clostridium tyrobutyrate TGL-A236 shake flask fermentation reaches 22.3568g/L, which is 1.33 times of the final concentration (16.7726 g/L) of clostridium tyrobutyrate Cirm BIA 2237 butyric acid. The experimental results demonstrate that the mutant strain TGL-a236 has a significant advantage in the production of butyric acid under culture conditions with sufficient carbon source.
Example 7 shake flask fermentation experiments of two strains in sweet sorghum peptone Medium
1. Experimental method
The experimental strain is clostridium tyrobutyrate Cirm BIA 2237 and TGL-A236.
Sweet sorghum juice peptone liquid medium comprising peptone (set up two gradients of 2.0 and 8.0 g/L) and treated sweet sorghum juice (prepared by the method of example 1) after 1:1 dilution with pure water.
Culturing and activating two strains of clostridium tyrobutyricum in RCM culture medium until the bacterial liquid OD 600 is 0.8, inoculating clostridium tyrobutyricum bacterial liquid into sweet sorghum peptone liquid culture medium with peptone concentration of 2.0g/L and 8.0g/L according to the inoculum size of 5% (v/v), performing anaerobic shake flask fermentation at 37 ℃ and 150rpm for 72h, and detecting the butyric acid production condition of the strains under the gradient of two nitrogen sources by adopting a gas chromatography method.
2. Experimental results
Referring to FIG. 13, it is understood that Clostridium tyrobutyrate Cirm BIA 2237 (control group) and mutant TGL-A236 produced 5.6247g/L and 9.6618g/L of butyric acid, respectively, at a peptone concentration of 2g/L, and that mutant TGL-A236 produced significantly higher amounts of butyric acid than Clostridium tyrobutyrate Cirm BIA 2237. When the peptone concentration is 8g/L, the butyric acid final concentration of clostridium tyrobutyrate Cirm BIA 2237 is 12.3412g/L, and clostridium tyrobutyrate TGL-A236 can synthesize 24.4276g/L butyric acid at 72h of fermentation, which is also significantly higher than that of the control group. The experimental result proves that the peptone can be used as a nitrogen source in a sweet sorghum juice culture medium for clostridium tyrobutyricum to grow and metabolize. In addition, under the culture condition of sufficient carbon source, the peptone addition amount is in the range of 2-8 g/L, the mutant strain TGL-A236 has a remarkable advantage in terms of producing butyric acid, and the butyric acid yield is improved with the increase of the peptone addition amount.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
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