Clostridium tyrobutyricum TGL-A236 and method for producing fatty acids by fermenting stem juice

Abstract

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

[0001] The invention relates to the technical field of fermentation engineering, in particular to Clostridium tyrobutyricum TGL-A236 and a method for producing fatty acids by fermenting stem juice. Background Art

[0002] Sweet sorghum (Sorghum bicolor (L.) Moench) has juicy stalks, rich in sugar, and has the advantages of drought and waterlogging resistance, salt and alkali resistance, short growth cycle, fast sugar accumulation, and high biological yield. However, the planting rate of sweet sorghum is not high, and people tend to use its grains as a staple food or brew liquor. The juice extracted from its stalks is often used as a raw material for syrup in the food industry, lacking high-value deep processing applications. The juice extraction rate of sweet sorghum stalks generally does not exceed 55%. The juice contains a large amount of non-structural soluble sugars, such as sucrose (53-85%), glucose (9-33%) and fructose (6-21%), starch, amino acids, inorganic salts, and secondary products including aconitic acid, organic acids, phenolic substances, etc. If it is developed and utilized as a carbon source for microbial fermentation, it will greatly increase the added value of sweet sorghum products and extend and improve its deep processing industry chain structure.

[0003] Studies have shown that sweet sorghum stalk juice can be used as a substrate for microbial growth and metabolism. Chinese invention patent CN103740568A discloses a liquor brewing method using aroma yeast as a fermentation strain and sweet sorghum stalk juice as the sole carbon source, obtaining high-quality sweet sorghum liquor with a temperature of 50-68°; Chinese invention patent CN101230371A uses yeast extract obtained by fermenting sweet sorghum juice to prepare a yeast glucan product with a purity of >80%. Sweet sorghum stalk juice can also be mixed with other substrates to participate in the growth metabolism and product synthesis of microorganisms: for example, Chinese patent CN104694334A and Chinese patent CN104673590A disclose brewing processes for brewing fruit wine using sweet sorghum stalk juice and wild hawthorn pulp as substrates, and preparing fruit wine using sweet sorghum stalk juice and Cornus officinalis as raw materials; Chinese patent CN102321679A discloses a method for mixing sweet sorghum juice with its straw hydrolyzate for acetone, butanol and ethanol fermentation; Chinese patent CN108374024A discloses an intensive production process for co-producing ethanol, fructose syrup and fructose products using sweet sorghum stalk juice, corn flour and glucose syrup.

[0004] Clostridium tyrobutyricum is a strict anaerobic bacterium, Gram-positive, with high butyrate tolerance and butyrate selectivity, and is the best cell factory for producing butyrate. Many studies and explorations have been done on the biomass substrates for butyrate fermentation by Clostridium tyrobutyricum, such as hydrolysis of corn fiber, sugarcane molasses, beet molasses or wheat straw. However, the agricultural raw materials for sucrose production at this stage mainly rely on corn, sugarcane and beets. The large-scale use of these raw materials for industrial fermentation will not only reduce the supply of bulk agricultural products and animal feed raw materials, but may also affect the stability of sucrose market prices. Therefore, the non-food sugar carbon source, sweet sorghum stalk juice, has great potential as a fermentable sugar source for bio-butyrate synthesis. Summary of the invention

[0005] One of the purposes of the present invention is to provide a Clostridium tyrobutyricum that can efficiently produce butyric acid, and a method for preparing fatty acids using stem juice.

[0006] In order to achieve this object, the technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a strain of Clostridium tyrobutyricum TGL-A236, whose deposit number is: CCTCC NO: M 2024002.

[0008] The present invention subjects Clostridium tyrobutyricum to heavy ion irradiation treatment, and unexpectedly screens out a new Clostridium tyrobutyricum TGL-A236, which can ferment and produce butyric acid, and the output is more than doubled compared with the starting strain, achieving an unexpected effect.

[0009] In a second aspect, the present invention provides a bacterial agent comprising the above-mentioned Clostridium tyrobutyricum TGL-A236.

[0010] The Clostridium tyrobutyricum TGL-A236 bacterial agent of the present invention may be in liquid or solid form.

[0011] Preferably, Clostridium tyrobutyricum TGL-A236 can be cultured to prepare a bacterial suspension, or the bacterial suspension can be further prepared into a solid bacterial agent for subsequent fermentation production of butyric acid.

[0012] In a third aspect, the present invention provides the use of the above-mentioned Clostridium tyrobutyricum TGL-A236 or bacterial agent in the fermentation production of fatty acids.

[0013] Preferably, the fatty acid is butyric acid and / or acetic acid.

[0014] In the application of the present invention, the carbon source used during fermentation is sweet sorghum stalk juice. Preferably, the culture medium during fermentation includes a nitrogen source and sweet sorghum stalk juice, and the nitrogen source is yeast extract and / or peptone. More preferably, the concentration of the nitrogen source is 2 to 8 g / L (further preferably 2.8-3.2 g / L).

[0015] In the present invention, yeast extract and peptone are both commercially available conventional products. Yeast extract is a brown-yellow soluble product obtained by degrading and refining proteins and nucleic acids in yeast cells. Peptone is a light yellow powder obtained by hydrolyzing meat, casein or gelatin with acid or protease and then drying.

[0016] As a specific embodiment, the product brand of the yeast extract used in the present invention is OXOID LP0021B. The product brand of the peptone is Beijing Aobox AOBOX, 01-001.

[0017] In the application of the present invention, the fermentation conditions are: inoculation amount 8-12% v / v, first static culture at 37±1°C for 11-13h, and then culture at 37±1°C and 140-160rpm with shaking for 72-120h.

[0018] Preferably, the culture medium during fermentation of the present invention also includes water, and a mixture of water and sweet sorghum stalk juice is used as the solvent of the culture medium. Those skilled in the art can adjust the ratio of water to sweet sorghum stalk juice according to fermentation requirements to ensure normal fermentation and sufficient carbon source. More preferably, the ratio of the two is 1:1 v / v.

[0019] In the application of the present invention, the method for preparing the sweet sorghum stalk juice comprises:

[0020] (1) Pretreatment: The sweet sorghum stalks were squeezed and centrifuged, and the supernatant was collected and the pH value was adjusted to 4.8-5.2, and the solution was obtained by sterilization;

[0021] (2) non-reducing sugar hydrolysis: enzymatically hydrolyzing the solution with sucrose invertase to obtain a hydrolyzate;

[0022] (3) degumming: removing the colloidal particles in the hydrolyzate with diatomaceous earth to obtain a solution to be degummed;

[0023] (4) Impurity removal: The solution to be impurity-removed is allowed to stand, centrifuged, adjusted to a pH value of 6.2-6.5, and then filtered.

[0024] The present invention has found during research that directly using juice squeezed from sweet sorghum stalks as a culture medium component for Clostridium tyrobutyricum cannot enable the growth of Clostridium tyrobutyricum, and Clostridium tyrobutyricum cannot directly utilize the high proportion of sucrose contained in the sweet sorghum stalk juice for growth and metabolism, resulting in a large amount of sucrose remaining in the fermentation liquid. However, after the sweet sorghum stalk juice is treated by the specific method of the present invention, the growth of Clostridium tyrobutyricum is good, the sweet sorghum stalk juice can be successfully utilized for butyric acid production, and the use of the sweet sorghum stalk juice is expanded.

[0025] The prior art generally uses chemical methods to treat sweet sorghum stalk juice, such as acid hydrolysis (such as adding strong acid reagents such as sulfuric acid) to hydrolyze sucrose, and adding chemical reagents such as lime milk, calcium hydroxide, and polyanionic flocculants to remove colloids and impurities. For example, the conventional sweet sorghum juice treatment method includes a hot lime clarification method based on sugarcane molasses, which is specifically: the sweet sorghum juice is first heated to coagulate the colloidal particles and soluble proteins to form natural floccules, and then lime milk (MOL) is added to the sweet sorghum juice, the pH is adjusted to 6.5 to neutralize the acid, and a polyanionic flocculant is added to settle the floccules, thereby obtaining clarified sweet sorghum stalk juice. The turbidity removal rate of sweet sorghum juice treated by conventional methods can generally reach more than 90%, but the lime milk and flocculants involved are often industrial chemical reagents, and their safety and environmental protection still need to be evaluated, and it is not a green and clean treatment process.

[0026] The present invention converts sucrose by enzymatic hydrolysis and degummes the hydrolyzate by using diatomaceous earth as a food additive, so that the removal rate of colloids is as high as over 99%, which is more efficient. The obtained hydrolyzate has high sucrose conversion efficiency and low conversion sugar (fructose+glucose) loss. The environmentally friendly butyric acid produced by using the substrate as a carbon source can be applied to more high-value fields such as food and biomedicine, thereby increasing the added value of butyric acid.

[0027] In the present invention, the sucrose invertase is a conventional commercial product, and those skilled in the art can select the enzymatic hydrolysis conditions according to the selected sucrose invertase; preferably, the sucrose invertase of the present invention is purchased from Hongrun Baoshun (China) to improve the sucrose hydrolysis rate. In the application of the present invention, the centrifugation conditions in step (1) are: 3800-4200rpm, 8-12min;

[0028] In step (1), 1 M phosphoric acid solution can be used to adjust the pH, and the sterilization conditions are: high temperature sterilization at 110-120° C. for 15-25 min.

[0029] And / or, in step (2), the amount of sucrose invertase added is 0.008-0.012% w / v, and the enzyme activity of sucrose invertase is ≥200 units / mg; the conditions for enzymatic hydrolysis are: 41-43° C., 140-160 rpm shaking for 18-24 h;

[0030] And / or, in step (3), the amount of diatomaceous earth added is 4.8-5.2% w / v, and the degumming conditions are: 40-45° C., 140-160 rpm shaking for 1.5-3 h (preferably, 41-43° C., 140-160 rpm shaking for 1.8-2.2 h);

[0031] In step (3), 1 M NaOH can be used to adjust the pH.

[0032] And / or, the standing time in step (4) is 25-35 min; the centrifugation conditions are 3800-4200 rpm, 8-12 min; and the pore size during filtration is 0.45-1 μm (preferably, 0.7 μm).

[0033] In the application of the present invention, after removing the colloidal particles in the hydrolyzate, a decolorization step is also included;

[0034] Preferably, activated carbon is used for decolorization, the amount of activated carbon added is 0.8-1.2% w / v, and the decolorization conditions are: 40-45°C, 140-160rpm shaking for 1.5-3h (preferably, 41-43°C, 140-160rpm shaking for 1.8-2.2h).

[0035] In a fourth aspect, the present invention provides a method for producing fatty acids by fermentation, wherein sweet sorghum stalk juice is used as a carbon source for the fermentation production of fatty acids, and the method for preparing the sweet sorghum stalk juice is as described above.

[0036] The method of the invention adopts a specific sweet sorghum stalk juice preparation method, so that the sweet sorghum stalk juice can be successfully used as a carbon source for the production of fatty acids and used for the application of Clostridium tyrobutyricum in fermenting fatty acids, thereby expanding the utilization value of the sweet sorghum stalks.

[0037] In the method of the present invention, the fermentation bacteria used for fermentation is Clostridium tyrobutyricum Cirm BIA 2237 or Clostridium tyrobutyricum TGL-A236, and the Clostridium tyrobutyricum TGL-A236 is as described above; and / or, the culture medium and fermentation conditions during fermentation are as described above.

[0038] The beneficial effects of the present invention are at least:

[0039] The invention provides a new Clostridium tyrobutyricum TGL-A236, which can efficiently produce butyric acid and is beneficial to the industrial fermentation production of butyric acid.

[0040] The present invention also obtains a method for successfully converting the sugar components in the sweet sorghum stalk juice into butyric acid and acetic acid by specifically treating the sweet sorghum stalks. The conversion of biological butyric acid using the sweet sorghum stalk juice as the sole carbon source is achieved. The sweet sorghum stalk treatment method of the present invention has a high sucrose hydrolysis conversion rate, a low sucrose loss rate, and an excellent colloid removal effect. It can improve the centrifugal efficiency and mass transfer rate to enhance the butyric acid fermentation efficiency, and realize the green conversion of biomass resources into chemical products. The method is simple, environmentally friendly, efficient, and suitable for industrial production, which opens up new application prospects for the comprehensive utilization of sweet sorghum.

[0041] Preferably, the sweet sorghum stalk juice prepared by the specific method of the present invention is used in the strict anaerobic fermentation of Clostridium tyrobutyricum TGL-A236 to produce butyric acid, and the butyric acid yield is further improved, which has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The figure is a schematic diagram of the process flow of the treatment method of the present invention.

[0043] Figure 2 The changes in the appearance of sweet sorghum stalk juice during the green processing; from left to right: sweet sorghum juice crude separation, sweet sorghum juice after degumming and decolorization, and sweet sorghum juice after sedimentation and impurity removal.

[0044] Figure 3 The statistical results of the content of each sugar component in sweet sorghum juice at different time points when sucrose invertase was used to treat the juice. In the figure, the lowercase letters on the data bar graph are different, indicating significant differences.

[0045] Figure 4 This is the kinetic model for the enzymatic hydrolysis of non-reducing sugars in sweet sorghum juice.

[0046] Figure 5 This is the particle size distribution of sweet sorghum stalk juice during the green processing process; from top to bottom: the test results of sweet sorghum juice crude separation solution, sweet sorghum juice after degumming and decolorization, and sweet sorghum juice after sedimentation and membrane.

[0047] Figure 6 The growth of Clostridium tyrobutyricum Cirm BIA 2237 in solid and liquid cultures of sweet sorghum stalk juice before and after treatment; the upper picture shows the solid-liquid culture of sweet sorghum stalk juice before treatment; the lower picture shows the solid-liquid culture of sweet sorghum stalk juice after treatment.

[0048] Figure 7 The growth of Clostridium tyrobutyricum TGL-A236 in solid and liquid cultures of sweet sorghum stalk juice before and after treatment; the upper figure shows the solid-liquid culture of sweet sorghum stalk juice before treatment; the lower figure shows the solid-liquid culture of sweet sorghum stalk juice after treatment.

[0049] Figure 8This is the 24-h growth curve of Clostridium tyrobutyricum Cirm BIA 2237 in liquid culture in sweet sorghum stalk juice medium before and after treatment.

[0050] Fig. 9 This is the 24-h growth curve of Clostridium tyrobutyricum TGL-A236 in liquid culture in sweet sorghum stalk juice medium before and after treatment.

[0051] Fig.10 This is the synthesis curve of butyric acid and acetic acid products in sweet sorghum stalk juice after treatment by Clostridium tyrobutyricum Cirm BIA 2237.

[0052] Fig.11 This is the synthesis curve of butyric acid and acetic acid products in sweet sorghum stalk juice after treatment by Clostridium tyrobutyricum TGL-A236.

[0053] Fig.12 The butyric acid yields of Clostridium tyrobutyricum Cirm BIA 2237 and TGL-A236 in shake flask fermentation in RCM medium with different carbon source concentrations. In the figure, ns means no significant difference, and * means significant difference.

[0054] Fig.13 Butyric acid production of Clostridium tyrobutyricum Cirm BIA 2237 and TGL-A236 in shake flask fermentation in sweet sorghum juice peptone medium containing different concentrations of nitrogen source. In the figure, * indicates significant difference. DETAILED DESCRIPTION

[0055] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0056] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0057] The strains involved in the present invention are as follows:

[0058] Clostridium tyrobutyricum Cirm BIA 2237 was provided by the Institute of Modern Physics, Chinese Academy of Sciences, and preserved in the Biophysics Laboratory of the Biomedical Center. It is published at: Whole-Genome Sequencing and Annotation of Clostridium tyrobutyricum Strain Cirm BIA 2237, Isolated from Silage. DOI: https: / / doi.org / 10.1128 / mra.00492-19.

[0059] Clostridium tyrobutyricum TGL-A236 is a 300 Gy-dose Clostridium tyrobutyricum Cirm BIA2237. 12 C 6+ After ion beam irradiation mutagenesis, the mutant strain was selected through "solid-liquid" adaptive evolution culture in sweet sorghum juice medium combined with the IV generation directed screening (optimal acid production) strategy.

[0060] Clostridium tyrobutyricum TGL-A236 was deposited in the China Center for Type Culture Collection (CCTCC, address: Wuhan University, Wuhan, China, Postal Code: 430072) on January 2, 2024, and was classified and named Clostridium tyrobutyricum TGL-A236Clostridium tyrobutyricum TGL-A236, and the preservation number is: CCTCC NO: M2024002.

[0061] In the following examples, Clostridium tyrobutyricum TGL-A236 is abbreviated as Clostridium tyrobutyricum TGL-A236, and Clostridium tyrobutyricum Cirm BIA 2237 is abbreviated as Clostridium tyrobutyricum Cirm BIA 2237, which have the same meanings.

[0062] Example 1 Green treatment process and treatment effect of sweet sorghum stalks

[0063] In this embodiment, the sweet sorghum stalks are processed according to the following green process flow:

[0064] 1. Juicing and separation of raw materials

[0065] The harvested sweet sorghum stalks were pressed with a three-hub press, the juice was centrifuged at 4000 rpm for 10 min, the supernatant was collected, the pH was adjusted to 5.0 with 1 M phosphoric acid solution, sterilized at 115°C for 20 min, and then frozen in a -20°C refrigerator for later use.

[0066] 2. Catalyze the hydrolysis of non-reducing sugars

[0067] Sucrose invertase (CAS NO: 9001-57-4, ≥ 200 units / mg, Hongrun Baoshun, China) was added to the sweet sorghum juice at an addition amount of 0.01% (w / v; equivalent to 20 units / mL) and hydrolyzed in a constant temperature oscillator at 42°C and 150 rpm for 24 h.

[0068] 3. Degumming and decolorization

[0069] 5% (w / v) diatomaceous earth (CAS NO: 61870-53-2, Damao, China) was added to the hydrolyzate and placed in a constant temperature oscillator at 42°C and 150 rpm for 2 h. Then, 1% (w / v) food-grade powdered activated carbon was added and placed in a constant temperature oscillator at 42°C and 150 rpm for 2 h (if there is no requirement for the color of the solution, the step of treating with activated carbon can be omitted).

[0070] 4. Sedimentation filtration and impurity removal

[0071] The sedimentation method was used. After standing for 30 min, the supernatant was centrifuged at 4000 rpm for 10 min. The pH of the supernatant was adjusted to 6.3 with 1 M NaOH solution and then filtered through a water filter with a pore size of 0.7 μm (glass fiber, 0.7 μm×25 mm, Xinxing, China). The filtrate was collected and packaged and stored in a -20°C refrigerator for later use.

[0072] Schematic diagram of the processing process of fresh sweet sorghum stalks Figure 1 As shown in the figure, the appearance changes of the sweet sorghum juice after crude separation, degumming and decolorization, and sedimentation and impurity removal are as follows: Figure 2 As shown, the color value and turbidity of the sweet sorghum juice were significantly reduced after being treated by the method, and the brown suspension with suspended particles was transformed into a clear and transparent light yellow solution, which can be used as a preferred carbon source for Clostridium microorganisms.

[0073] Example 2 Analysis of sucrose conversion effect of sweet sorghum stalk juice

[0074] 1. Experimental Methods

[0075] The sucrose invertase described in Example 1 was added to sweet sorghum stalk juice (prepared by step 1 of the method of Example 1) in an amount of 0.01% (w / v) for enzymatic hydrolysis. The enzymatic hydrolysis reaction was carried out in a constant temperature oscillator at 42°C and 150 rpm. Samples were taken at 0, 6, 12, 18, and 24 h. The main sugar components in the hydrolyzate were determined by high performance liquid chromatography (HPLC) and the degree of sucrose hydrolysis and conversion was analyzed.

[0076] The detection method is as follows:

[0077] (1) Chromatographic column: Zafex Carbohydrate ES (250×4.6 mm, 5 μm).

[0078] (2) Chromatographic conditions: mobile phase A-25% ultrapure water, mobile phase B-75% acetonitrile; flow rate: 1.0 ml / min; column temperature: 40°C; RI differential detector temperature: 30°C; injection volume: 15.00 μL; running time: 25 min.

[0079] 2. Experimental results

[0080] The hydrolysis and transformation of sucrose in sweet sorghum juice before and after treatment can be found in Figure 3 It can be seen that after 24 hours of reaction with sucrase in the sweet sorghum juice, the sucrose concentration was hydrolyzed from 59.59 g / L to 1.02 g / L, and the final total amount of inverted sugar reached 133.69 g / L. Under the action of invertase, the sucrose hydrolysis rate of sweet sorghum juice reached 98.29%, the sucrose conversion rate was 99.13%, and the sucrose loss rate was only 0.87%. At the same time, there was still a trace amount of sucrose that was not hydrolyzed after 24 hours of reaction, so there was no residual invertase after the enzymatic reaction; at 18 hours, most of the sucrose had been hydrolyzed and converted, and the total amount of inverted sugar (fructose + glucose) was not significantly different from the value at 24 hours, so the sucrose hydrolysis treatment time of this process can be optimized to 18 hours.

[0081] This example also fitted the first-order / second-order enzymatic hydrolysis reaction kinetic model based on the experimental data of sucrose conversion in sweet sorghum juice, where the reaction rate is expressed as the amount of invert sugar produced after sucrose hydrolysis per unit time ( Figure 4 The nonlinear fitting of the invert sugar production at different time points and the enzymatic reaction time verified that the sucrose hydrolysis reaction of sucrase in sweet sorghum juice substrate was in the first-order kinetic model (R 2 >0.999) and the second-order kinetic model (R 2 >0.999) had good fitting effects. The experimental value of the final invert sugar production of sweet sorghum juice was slightly lower than the predicted value of the first-order / second-order kinetic model, indicating that the enzymatic reaction conditions of the process were reasonably set and there was no sucrose invertase residue.

[0082] Example 3 Analysis of the effect of degumming treatment of sweet sorghum stalk juice

[0083] In this example, a wet-method fully automatic laser particle size analyzer is used to determine the particle size distribution of untreated sweet sorghum stalk juice (prepared by step 1 of method in example 1) and sweet sorghum stalk juice treated according to each step of the method described in example 1. The test parameters are as follows: the sample refractive index is 1.61-0.1i, the medium refractive index is 1.33, the dispersion medium is water, the test particle size range is 0.1-800μm, the ultrasonic power is 50W, and the circulation flow rate is 1L / min. After the background acquisition program is completed, 50mL of untreated and treated sweet sorghum stalk juice are prepared for testing on the machine. The particle size distribution of the crude sweet sorghum juice, the sweet sorghum juice after degumming and decolorization, and the sweet sorghum stalk juice after sedimentation and impurity removal. The results are as follows Figure 5 shown.

[0084] The diameter of colloidal particles is generally in the range of 0.001 to 1 μm (i.e., 1 to 1000 nm) (Gregory, 2005). The particle size is differentiated according to the cumulative distribution curve of suspended particle size to obtain the volume distribution percentage of particles in different particle size ranges of sweet sorghum stalk juice before and after treatment.

[0085] The proportion of colloidal particles can be observed from the left part of the gray dotted line in the figure. Figure 5 In the figure above), the colloidal particles (particle size <1 μm) accounted for 18.57%. Figure 5 The percentage of colloidal particles in the middle figure of the figure was directly reduced to 0.07%, and the colloidal particle removal rate reached 99.62%. Finally, after sedimentation and impurity removal, the sweet sorghum juice ( Figure 5 There are almost no particles with a particle size larger than 0.1μm, indicating that the flocculation and impurity removal effect of this process is good.

[0086] Example 4 Solid / liquid culture of Clostridium tyrobutyricum Cirm BIA 2237 and Clostridium tyrobutyricum TGL-A236 in untreated / treated sweet sorghum stalk juice

[0087] Reinforced Clostridium medium (RCM): peptone 10.0 g / L, beef powder 10.0 g / L, yeast extract 3.0 g / L, glucose 5.0 g / L, soluble starch 1.0 g / L, sodium chloride 5.0 g / L, sodium acetate 3.0 g / L, L-cysteine ​​hydrochloride 0.5 g / L.

[0088] Treated sweet sorghum juice medium: 3 g / L yeast extract, treated sweet sorghum stalk juice (prepared by the method of Example 1) was added as a solvent, and 20 g / L agar was added during the preparation of the solid medium.

[0089] Untreated (before treatment) sweet sorghum juice medium: 3 g / L yeast extract, untreated sweet sorghum stalk juice (prepared in the same manner as in step 1 of method of Example 1, except that the pH value is adjusted to 6.3) is added as a solvent, and 20 g / L agar is added when preparing the solid medium.

[0090] The two Clostridium tyrobutyricum were cultured and activated in Clostridium fortified medium until the bacterial solution OD 600 The concentration of tyrobutyric acid was 0.8, and then the Clostridium tyrobutyricum was inoculated into the untreated and treated sweet sorghum stalk juice solid and liquid culture medium at a rate of 5% (v / v), and anaerobically cultured at 37°C and 150rpm to observe the growth of the strain in the sweet sorghum stalk juice culture medium before and after treatment. Figures 6 to 9 .

[0091] Figure 6 , Figure 7 The growth of Clostridium tyrobutyricum Cirm BIA 2237 and Clostridium tyrobutyricum TGL-A236 in untreated / treated sweet sorghum stalk juice solid and liquid culture media was shown. The strains showed no signs of growth on the sweet sorghum stalk juice plates and liquid culture media before treatment for 3 days, but grew milky white, round colonies with neat edges, smooth and moist surfaces, and a diameter of 1 to 3 mm after being diluted 1000× and coated on the treated sweet sorghum stalk juice plates for 3 days. The strains grew well in the treated sweet sorghum stalk juice liquid culture media after 1 day, and the bacterial concentration increased significantly, with the maximum OD 600 The values ​​reached 1.4030 (Cirm BIA 2237) and 1.4737 (TGL-A236), with obvious white fine foam on the surface of the bacterial liquid, and the average gas production of 10 mL of bacterial liquid was more than 60 mL.

[0092] from Figure 8 , Fig. 9 It can be seen that the cell density (OD 600 ) decreased from the initial 0.3473 to 0.3153 at 8 h, and then remained below 0.32 until 24 h. The maximum OD of Clostridium tyrobutyricum TGL-A236 at 4 h 600 It was 0.4223, and then fluctuated in the range of 0.35-0.37, indicating that the cells of the two strains could hardly grow and reproduce normally in the growth environment of untreated sweet sorghum stalk juice.

[0093] When the strain was inoculated into the treated sweet sorghum stem juice medium, it could be observed from the growth curve that Clostridium tyrobutyricum Cirm BIA 2237 completed a complete growth cycle within 24 h. After a lag period of 4 h, the strain was in a high-speed logarithmic growth phase until 12 h, and then the growth rate gradually slowed down to OD 0. 600 Reaching the maximum value of 1.4030, and finally entering the decay stage OD 600 The maximum OD of Clostridium tyrobutyricum TGL-A236 600 (1.4737) appeared at 24h and had not yet reached the plateau phase, showing stronger growth performance than Clostridium tyrobutyricum Cirm BIA 2237. In summary, both strains of Clostridium tyrobutyricum could grow and reproduce normally in the sweet sorghum stalk juice treated by the green process of the present invention, which confirmed the feasibility of the process method.

[0094] Example 5 Production of Butyric Acid in Treated Sweet Sorghum Stalk Juice by Clostridium tyrobutyricum Cirm BIA 2237 and Clostridium tyrobutyricum TGL-A236

[0095] Fermentation medium: 3 g / L yeast extract, treated sweet sorghum stalk juice (prepared by the method of Example 1) and pure water were diluted 1:1, and the diluted solution was added as a solvent for preparation.

[0096] The Clostridium tyrobutyricum Cirm BIA 2237 and Clostridium tyrobutyricum TGL-A236 were inoculated into the Clostridium fortified culture medium (see Example 4), and cultured in a 37°C incubator for 20 to 24 hours. 600 After aligning to 0.8, transfer to an anaerobic fermentation bottle containing 200 mL of fermentation medium at an inoculum of 10% (v / v), and seal the bottle mouth after passing sterile nitrogen. After the fermentation bottle is statically cultured in a 37°C constant temperature box for 12 hours, it is transferred to a constant temperature oscillator at 37°C and 150 rpm for 120 hours. Samples are taken at 0, 24, 48, 72, 96, and 120 hours of fermentation to measure the concentrations of butyric acid and acetic acid products in the fermentation broth at different time points.

[0097] The experimental results are as follows Fig.10 , Fig.11 As shown in the figure, after a short lag phase, the concentrations of butyric acid and acetic acid increased exponentially from 12h, and entered the steady state at 72h. During the whole batch fermentation process, the maximum butyric acid concentration of Clostridium tyrobutyricum Cirm BIA 2237 was 12.0448g / L, and the butyric acid selectivity was 88.78%; the maximum butyric acid concentration of mutant Clostridium tyrobutyricum TGL-A236 was 24.1364g / L, and the butyric acid selectivity was 86.36%.

[0098] The concentrations of byproduct acetic acid of Clostridium tyrobutyricum Cirm BIA 2237 and Clostridium tyrobutyricum TGL-A236 reached the highest levels of 1.4413 g / L and 3.8798 g / L respectively at 96 h, and then the concentrations gradually decreased. From the product butyric acid / acetic acid synthesis curve, it can be seen that the sweet sorghum juice treated by the process of the present invention can be used for the fermentation production of the main product butyric acid.

[0099] In summary, the present invention first provides a strain of Clostridium tyrobutyricum TGL-A236 with ideal butyric acid production performance. The present invention also provides a process for treating sweet sorghum stalk juice, which has a high sucrose hydrolysis conversion rate: the sucrose hydrolysis rate of the sweet sorghum stalk juice under the action of sucrose invertase reaches more than 98%, the sucrose conversion rate is 99.13%, the sucrose loss rate is only 0.87%, and the colloid removal effect is excellent: the colloid particle removal rate reaches 99.62%, which plays an important role in improving the centrifugal efficiency and mass transfer rate, thereby improving the butyric acid fermentation efficiency.

[0100] The solution obtained by the sweet sorghum stalk juice processing process of the present invention is suitable for culturing butyric acid-producing Clostridium: a green processing process for sweet sorghum stalk juice that can be used to cultivate Clostridium bacteria and ferment butyric acid is provided. Under a culture temperature of 37°C, a culture time of 24 to 72 hours, and strict anaerobic conditions, it can be observed that the Clostridium tyrobutyricum strain can grow and reproduce normally in the liquid / solid culture medium of sweet sorghum stalk juice.

[0101] The sweet sorghum stalk juice processing process of the present invention is suitable for producing butyric acid: two bacterial liquids of activated Clostridium tyrobutyricum Cirm BIA 2237 and TGL-A236 are inoculated into a sweet sorghum stalk juice culture medium for a 5-day shake flask fermentation experiment, and the maximum butyric acid concentrations detected are 12.0448 g / L and 24.1364 g / L, respectively, and the maximum acetic acid concentrations are 1.4413 g / L and 3.8798 g / L, respectively; the present invention converts sugar components in the sweet sorghum stalk juice into butyric acid and acetic acid, realizes 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.

[0102] Example 6 Shake flask fermentation experiment of Clostridium tyrobutyricum Cirm BIA 2237 and Clostridium tyrobutyricum TGL-A236 in Clostridium reinforced medium (RCM)

[0103] 1. Experimental methods

[0104] Experimental strains: Clostridium tyrobutyricum Cirm BIA 2237 and TGL-A236.

[0105] Culture medium: The ingredients of Clostridium reinforced medium (RCM) are: 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, with three gradients of 5.0g / L, 20.0g / L and 60.0g / L.

[0106] Culture conditions: The two Clostridium tyrobutyricum were cultured and activated in RCM medium containing 5 g / L glucose until the bacterial solution OD 600 After the pH value was 0.8, the Clostridium tyrobutyricum liquid was inoculated at an inoculum size of 5% (v / v) into RCM liquid culture medium with glucose concentrations of 5.0 g / L, 20.0 g / L and 60.0 g / L, respectively. Anaerobic shake flask fermentation was carried out at 37°C and 150 rpm for 72 h. Gas chromatography was used to detect butyric acid production of each strain in RCM culture medium under three carbon source gradients.

[0107] 2. Experimental results

[0108] Fermentation results Fig.12 . It can be seen that when the glucose concentration is 5g / L, Clostridium tyrobutyricum Cirm BIA 2237 (control group) and mutant strain TGL-A236 can produce 2.1397g / L and 2.3124g / L of butyric acid, respectively. Due to the low carbon source concentration, the butyric acid production of the two strains is at the same level at this time, and there is no significant difference. When the glucose concentration is increased to 20g / L, the final butyric acid concentration of Clostridium tyrobutyricum Cirm BIA 2237 is 6.2410g / L, while Clostridium tyrobutyricum TGL-A236 can synthesize 9.7580g / L of butyric acid within 72h, which is significantly higher than the butyric acid production of the control group. When the carbon source concentration increased to 60 g / L, the final butyric acid concentration of Clostridium tyrobutyricum TGL-A236 in shake flask fermentation reached 22.3568 g / L, which was 1.33 times the final butyric acid concentration (16.7726 g / L) of Clostridium tyrobutyricum Cirm BIA 2237. The experimental results proved that under the culture conditions with sufficient carbon source, the mutant strain TGL-A236 has a significant advantage in producing butyric acid.

[0109] Example 7 Shake flask fermentation experiment of two strains in sweet sorghum juice peptone medium

[0110] 1. Experimental methods

[0111] Experimental strains: Clostridium tyrobutyricum Cirm BIA 2237 and TGL-A236.

[0112] Sweet sorghum juice peptone liquid culture medium: comprising peptone (two gradients of 2.0 and 8.0 g / L) and treated sweet sorghum juice (prepared by the method of Example 1) diluted with pure water in a ratio of 1:1.

[0113] Culture conditions: Two strains of Clostridium tyrobutyricum were cultured and activated in RCM medium until the bacterial solution OD 600 After the pH value was 0.8, the Clostridium tyrobutyricum was inoculated into sweet sorghum juice peptone liquid culture medium with peptone concentrations of 2.0 g / L and 8.0 g / L, respectively, at a rate of 5% (v / v). Anaerobic shake flask fermentation was carried out at 37°C and 150 rpm for 72 h. The butyric acid production of the strain under two nitrogen source gradients was detected by gas chromatography.

[0114] 2. Experimental results

[0115] See also Fig.13 , it can be seen that when the peptone concentration is 2g / L, Clostridium tyrobutyricum Cirm BIA 2237 (control group) and mutant strain TGL-A236 can produce 5.6247g / L and 9.6618g / L of butyric acid, respectively, and the butyric acid production of mutant strain TGL-A236 is significantly higher than that of Clostridium tyrobutyricum Cirm BIA 2237. When the peptone concentration is 8g / L, the final butyric acid concentration of Clostridium tyrobutyricum Cirm BIA2237 is 12.3412g / L, while Clostridium tyrobutyricum TGL-A236 can synthesize 24.4276g / L of butyric acid at 72h of fermentation, which is also significantly higher than the butyric acid production of the control group. The experimental results prove that peptone can also be used as a nitrogen source in sweet sorghum juice medium for the growth and metabolism of Clostridium tyrobutyricum. In addition, under the culture conditions with sufficient carbon source, when the amount of peptone added was in the range of 2 to 8 g / L, the mutant strain TGL-A236 had a significant advantage in producing butyric acid, and the butyric acid production increased with the increase of the amount of peptone added.

[0116] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A strain of Clostridium tyrobutyricum TGL-A236, characterized in that: The deposit number is: CCTCC NO: M 2024002.

2. A bacterial agent comprising the Clostridium tyrobutyricum TGL-A236 according to claim 1.

3. Use of the Clostridium tyrobutyricum TGL-A236 according to claim 1 or the bacterial agent according to claim 2 in fermentation production of fatty acids; the fatty acids are butyric acid and / or acetic acid; The carbon source used in the fermentation is sweet sorghum stalk juice, and the nitrogen source is yeast extract and / or peptone, and the concentration of the nitrogen source is 2-8 g / L; The method for preparing the sweet sorghum stalk juice comprises: (1) Pretreatment: The sweet sorghum stalks were squeezed and centrifuged, and the supernatant was collected and the pH value was adjusted to 4.8-5.2, and the solution was obtained by sterilization; (2) Non-reducing sugar hydrolysis: the solution is enzymatically hydrolyzed with sucrose invertase to obtain a hydrolyzate; the amount of sucrose invertase added is 0.008-0.012% w / v, and the enzymatic activity of sucrose invertase is ≥200 units / mg; the enzymatic hydrolysis conditions are: 41-43° C., 140-160 rpm shaking for 18-24 hours; (3) Degumming: removing the colloidal particles in the hydrolyzate with diatomaceous earth to obtain a solution to be degummed; the amount of diatomaceous earth added is 4.8-5.2% w / v, and the degumming conditions are: treating at 40-45° C. and 140-160 rpm for 1.5-3 h; (4) Impurity removal: The solution to be impurity-removed is allowed to stand, centrifuged, adjusted to a pH value of 6.2-6.5, and then filtered.

4. The use according to claim 3, characterized in that: The fermentation conditions are as follows: inoculation amount 8-12% v / v, first static culture at 37±1°C for 11-13 hours, and then culture at 37±1°C, 140-160 rpm with shaking for 72-120 hours.

5. The use according to claim 3, characterized in that: The centrifugation conditions in step (1) are: 3800-4200 rpm, 8-12 min; And / or, the standing time in step (4) is 25-35 min; the centrifugation conditions are 3800-4200 rpm, 8-12 min; and the pore size during filtration is 0.45-1 μm.

6. The use according to any one of claims 3 to 5, characterized in that: After removing the colloidal particles in the hydrolyzate, the method further includes a decolorization step.

7. The use according to claim 6, characterized in that: Activated carbon is used for decolorization, the added amount of activated carbon is 0.8-1.2% w / v, and the decolorization treatment conditions are: 40-45° C., 140-160 rpm shaking for 1.5-3 hours.

8. A method for producing fatty acids by fermentation, characterized in that: Sweet sorghum stalk juice is used as a carbon source for fermentation production of fatty acids. The preparation method of the sweet sorghum stalk juice is as described in any one of claims 3 to 7; the nitrogen source used in the fermentation is yeast extract and / or peptone, and the concentration of the nitrogen source is 2 to 8 g / L; the fermentation bacteria used in the fermentation is Clostridium tyrobutyricum TGL-A236, and the Clostridium tyrobutyricum TGL-A236 is as described in claim 1.

9. The method according to claim 8, characterized in that The fermentation conditions during fermentation are as described in claim 4.

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