阅读说明：本技术 菊糖芽孢乳杆菌及发酵制备d-乳酸的方法 (Lactobacillus inulinus and method for preparing D-lactic acid by fermentation ) 是由 胡金龙 杨哲浩 杨若 于 2020-12-31 设计创作，主要内容包括：本发明涉及一株菊糖芽孢乳杆菌(Sporolactobacillus inulinus)DJ01 CCTCC NO:M2020763,于2020年11月19日保藏在中国典型培养物保藏中心,保藏地址湖北省武汉市武昌区八一路299号。该菊糖芽孢乳杆菌DJ01能够利用水稻秸秆粉进行发酵,并且通过控制水稻秸秆粉中硅元素含量,使得其促进菊糖芽孢乳杆菌发酵酶链降解水稻秸秆粉中碳水化合物,促进其有效转化成为D-乳酸,从而提高对秸秆的利用率,实现资源的有效利用,环保又经济。(The invention relates to a strain of lactobacillus inulinus DJ01 CCTCC NO: M2020763, which is preserved in China Center for Type Culture Collection (CCTCC) at 11-19 th of 2020, with the preservation address of No. 299 in the Wuchang district, Wuhan city, Hubei province. The lactobacillus inulin DJ01 can be fermented by rice straw powder, and by controlling the content of silicon element in the rice straw powder, the lactobacillus inulin fermentation enzyme chain is promoted to degrade carbohydrates in the rice straw powder, and the effective transformation of the lactobacillus inulin fermentation enzyme chain into D-lactic acid is promoted, so that the utilization rate of the straw is improved, the effective utilization of resources is realized, and the method is environment-friendly and economical.)

1. A strain of Lactobacillus inulinus DJ01 is characterized in that the strain is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2020763.

2. The method for preparing D-lactic acid by fermentation of Lactobacillus inulinus according to claim 1, comprising the steps of preparing fermentation seeds, fermenting D-lactic acid and extracting D-lactic acid.

3. The method of claim 2, wherein the formulation of the medium used in the preparation of the fermented seed is: 10g/L of glucose, 4 g/L of beef extract, 0.5 g/L of calcium carbonate, 0.5 g/L of magnesium sulfate, 20g/L of agar and 6-7 of pH.

4. The method according to claim 2, wherein the D-lactic acid fermentation process comprises the following steps:

s1, crushing and sieving the rice straws, soaking the rice straws in a sodium hydroxide solution, performing ultrasonic treatment, taking out the rice straws, and drying the rice straws to obtain rice straw powder;

and S2, inoculating the seed liquid prepared by fermenting the seeds into a large fermentation container, preparing a fermentation culture medium from the rice straw powder obtained in the step S1, and placing the fermentation culture medium in an incubator at the temperature of 35-40 ℃ for stirring and culturing for 50-80 hours without aeration, wherein the content of the rice straw powder in the fermentation culture medium is 45-100 g/L.

5. The method according to claim 4, wherein the fermentation medium further comprises 8-15 g/L of yeast extract powder and 45-65 g/L of calcium carbonate.

6. The method according to claim 4, wherein in the step S1, the concentration of the alkali solution is 0.5-2 mol/L, and the treatment time is 1.5-4 h.

7. The method according to claim 4, wherein in the step of S1, the ultrasonic treatment conditions are as follows: 0.8 to 2.2W/cm²，30～40min。

8. The method of claim 4, wherein the rice straw powder obtained in the step S1 contains 5-11 wt% of silicon.

Technical Field

The invention relates to the technical field of D-lactic acid preparation by fermentation, in particular to lactobacillus inulinus and a method for preparing D-lactic acid by fermentation.

Background

Lactic acid is classified into D-lactic acid and L-lactic acid according to its optical activity. L-lactic acid can be degraded by human body, so it is widely used in food, medical treatment and chemical industry. D-lactic acid can not be degraded by human body, is mostly used in chemical industry at present, is used for pesticide intermediates and the like, and has very high requirement on optical purity which is generally more than 99.5%. D-lactic acid is produced by using starch agricultural products such as corn and the like as raw materials, using L-lactobacillus or Lactobacillus delbrueckii as strains and adopting a microbial fermentation method. With the gradual implementation of national plastic constraints, the market has an increasing demand for degradable materials. Polylactic acid synthesized from L-lactic acid or D-lactic acid is a very ideal degradable material and is one of the main degradable raw materials at present. With the increase of the demand of polylactic acid, if corn and the like are used as raw materials to produce D-lactic acid all the time, the national food safety is threatened inevitably.

Disclosure of Invention

In view of the above, there is a need for a method for producing D-lactic acid by fermentation of lactobacillus inulinus, which is environmentally friendly and economical for industrial mass production.

The invention provides a strain of Lactobacillus inulinus DJ01 which is preserved in China center for type culture Collection with the preservation number of CCTCC NO: M2020763.

In a second aspect of the invention, the method for preparing D-lactic acid by fermenting the lactobacillus inulinus comprises the steps of preparing fermentation seeds, fermenting the D-lactic acid and extracting the D-lactic acid.

Specifically, the formula of the culture medium adopted in the preparation process of the fermented seeds comprises 10g/L of glucose, 4 g/L of beef extract, 0.5 g/L of calcium carbonate, 0.5 g/L of magnesium sulfate, 20g/L of agar and the pH value of 6-7.

Specifically, the D-lactic acid fermentation process specifically comprises the following steps:

s1, crushing and sieving the rice straws, soaking the rice straws in a sodium hydroxide solution, performing ultrasonic treatment, taking out the rice straws, and drying the rice straws to obtain rice straw powder;

and S2, inoculating the seed liquid prepared by fermenting the seeds into a large fermentation container, preparing a fermentation culture medium from the rice straw powder obtained in the step S1, and placing the fermentation culture medium in an incubator at the temperature of 35-40 ℃ for stirring and culturing for 50-80 hours without aeration, wherein the content of the rice straw powder in the fermentation culture medium is 45-100 g/L.

Further, the fermentation medium further comprises 8-15 g/L of yeast extract powder and 45-65 g/L of calcium carbonate.

Specifically, in the step S1, the concentration of the alkali liquor is 0.5-2 mol/L, and the treatment time is 1.5-4 h.

Specifically, in the step S1, the ultrasonic treatment conditions are as follows: 0.8 to 2.2W/cm²，30～40min。

Specifically, the rice straw powder obtained in the step S1 contains 5-11 wt% of silicon element.

Has the advantages that:

the bacillus inulin provided by the invention can be fermented by utilizing the rice straw powder, and the content of the silicon element in the rice straw powder is controlled, so that the bacillus inulin can promote the lactobacillus inulin fermentation enzyme chain to degrade carbohydrates in the rice straw powder and promote the carbohydrates to be effectively converted into D-lactic acid, thereby improving the utilization rate of the straw, realizing the effective utilization of resources, and being environment-friendly and economical.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The embodiment of the invention provides a Lactobacillus inulinus DJ01(Sporolactobacillus inulinus DJ01) CCTCC NO: M2020763, belonging to the genus of Bacillus. The 16S rDNA sequence is shown in SEQ ID No. 1. The sequence is subjected to Blast comparison on Genbank, and the identification result shows that the strain is highly homologous with lactobacillus inulin (Sporolactobacillus inulinus), is determined and named as lactobacillus inulin DJ01, and is preserved in China center for type culture Collection, Wuhan city Wuchang Lojia mountain, postal code at 11/19 of 2020: 430072, preservation address eight-way 299 in Wuchang district, Wuhan city, Hubei province.

The Lactobacillus inulin DJ01(Sporolactobacillus inus) has the following properties:

1) morphological characteristics: rod-shaped, single or paired, can form endospores, a small amount of peritrichous flagellum, and motility. Fine needle-shaped colonies were formed on agar plates containing glucose, yeast extract, beef extract, and proteoglobus sp.

2) Physiological and biochemical characteristics: gram positive, slightly oxygen consuming. No indole is formed, no gelatin is liquefied, no nitrate is reduced, and litmus milk is not changed.

3) Can produce acid by utilizing fructose, glucose, inulin, raffinose, maltose, mannose, trehalose, galactose and sucrose, and can not produce acid by utilizing xylose and arabinose.

4) The growth does not occur below 10 ℃ and above 45 ℃, and the optimal growth temperature is 35-40 ℃.

The method for producing the D-lactic acid by fermenting the rice straws by adopting the lactobacillus inulinus DJ01, which is provided by the embodiment of the invention, comprises the steps of preparing fermentation seeds, fermenting the D-lactic acid and extracting the D-lactic acid.

The formula of the culture medium adopted in the preparation process of the fermented seeds comprises 10g/L of glucose, 4 g/L of beef extract, 0.5 g/L of calcium carbonate, 0.5 g/L of magnesium sulfate, 20g/L of agar and the pH value of 6-7.

Wherein the D-lactic acid fermentation process specifically comprises the following steps:

s1, crushing and sieving the rice straws, soaking the rice straws in a sodium hydroxide solution at the temperature of 45-55 ℃ for 1.5-4 h, taking out the rice straws, and drying the rice straws to obtain rice straw powder;

and S2, inoculating the seed liquid prepared by fermenting the seeds into a large fermentation container, preparing a fermentation culture medium from the rice straw powder obtained in the step S1, and placing the fermentation culture medium in an incubator at the temperature of 35-40 ℃ for stirring and culturing for 50-80 hours without aeration, wherein the content of the rice straw powder in the fermentation culture medium is 45-100 g/L.

Further, the fermentation medium also comprises 10g/L of yeast extract powder and 60g/L of calcium carbonate.

Furthermore, the rice straw powder obtained in the step S1 contains 5-11 wt% of silicon element.

For statistics and analysis of examples of media and fermentation conditions during fermentation, the examples are listed in table 1. The seed medium is listed in Table 1, and the formulation Z1 is: 10g/L of glucose, 4 g/L of beef extract, 0.5 g/L of calcium carbonate, 0.5 g/L of magnesium sulfate, 20g/L of agar and 6-7 of pH; the formulation Z2 is conventional and is described in detail in Kosaki.M.production of high optical purity D-lactic acid (P). US5466588,1995-11-14. Watch (A)The step S1 of medium concentration sequentially comprises the treatment temperature, the sodium hydroxide concentration (0.5-2 mol/L), the alkali liquor soaking treatment time (1.5-4 h) and the ultrasonic intensity (0.8-2.2W/cm)²) And ultrasonic treatment time (30-40 min). In the fermentation medium list in table 1, F1 represents 50-100 g/L of rice straw powder, 10g/L of yeast extract powder and 60g/L of calcium carbonate, and the percentage after F1 is the content of silicon element in the rice straw powder; f2 represents 50g/L corn straw powder (adopting the same processing method as the step S1), a fermentation medium formula of 120g/L glucose and 60g/L protein, and the silicon element in the medium represented by F2 is 0.5-1.5%; the fermentation medium column also shows the content of silicon element in the rice straw powder (the content of silicon element can be measured by a potassium fluosilicate volumetric method or other existing methods). The fermentation conditions in Table 1 are listed as fermentation control temperature and fermentation time in this order.

TABLE 1

After the fermentation is finished, the concentration of the D-lactic acid and the L-lactic acid in the fermentation liquor is detected by an HPLC method, and the concentration of the lactic acid, the optical purity of the D-lactic acid and the conversion rate of the sugar D-lactic acid are measured.

The conversion rate of the sugar D-lactic acid is equal to the mass of the D-lactic acid/the total sugar content in the straw powder multiplied by 100 percent when the fermentation is finished;

wherein the total sugar content in the straw powder refers to the total amount of carbohydrates in the rice straw powder or the corn straw powder. The total sugar content is determined by 3, 5-dinitrosalicylic acid (DNS) color method.

TABLE 2

As can be seen from tables 1 and 2:

1. in table 1, examples 2 to 4 changed the treatment conditions of step S1 from example 1; examples 5-6 the conditions of the fermentation medium were changed from those of example 3; examples 7 to 8 the fermentation conditions were changed from example 3; comparative example 1 the seed medium was changed relative to example 1; comparative examples 2 to 5 changed the treatment conditions of the step S1 from example 1 and were not within the range defined by the present invention, resulting in the use of rice straw powders in which silicon was not within the range defined by the present invention; comparative example 6 used F2 as the culture medium relative to example 1; comparative examples 7 to 8 changed the fermentation conditions relative to example 1 and were not within the scope of the present invention.

2. In Table 2, the optical degree of D-lactic acid prepared in the examples and comparative examples is not much different. However, the lactic acid concentration and the sugar D-lactic acid conversion rate of the fermentation liquid obtained in the example 1 are both obviously higher than those of the fermentation liquids obtained in the comparative examples 2-6, which shows that the seed culture medium provided by the invention can obtain higher fermentation lactic acid concentration, and can also effectively utilize carbohydrate in the straw powder to produce D-lactic acid. Specifically, in example 1, compared with comparative examples 2 to 6, because the content of the silicon element in the straw powder used in the comparative examples 2 to 6 is unreasonably processed, when the straw powder participates in the fermentation and utilization of the lactobacillus inulinus, the carbohydrate in the straw powder cannot be effectively absorbed and converted, which may be related to the degradation of the carbohydrate by the silicon element on the enzymes of the lactobacillus inulinus in the fermentation process, so that the carbohydrate such as cellulose in the straw powder can be fully degraded, and the effective conversion of the carbohydrate into D-lactic acid is improved. In addition, although the fermentation conditions of the comparative examples 7 to 8 are unreasonable, the conversion rate of the sugar D-lactic acid is still remarkably higher than that of the comparative examples 2 to 6 by controlling the silicon element in the rice straw powder within the range limited by the invention.

In conclusion, the bacillus inulin provided by the invention can be fermented by rice straw powder, and the content of silicon element in the rice straw powder is controlled, so that the bacillus inulin can promote the lactobacillus inulin fermentation enzyme chain to degrade carbohydrate in the rice straw powder and promote the carbohydrate to be effectively converted into D-lactic acid, thereby improving the utilization rate of the straw, realizing the effective utilization of resources, and being environment-friendly and economical.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Sequence listing

<110> Wuhan City Dajiang Green Innovative materials science and technology Limited liability company

<120> Lactobacillus inulinus and method for preparing D-lactic acid by fermentation

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aagaacgagt gccagaggaa atgctggtgc tgtgacggta tccggccaga aagccacggc 480

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gcggtgaaat gcgtagagat gtggaggaat accagtggcg aaggcggctc tctggtctgt 720

tactgacgct gaggtgcgaa agcatgggga gcaaacagga ttagataccc tggtagtcca 780

tgccgtaaac gatgaatgct aggtgttagg ggggtccaac cccttagtgc tgaagttaac 840

acattaagca ttccgcctgg ggagtacgac cgcaaggttg aaactcaaag gaattgacgg 900

gggcccgcac aagcagtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca 960

ggtcttgaca tcctctgaca agtctagaga taggccgttc cccttcgggg gacagagtga 1020

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ccgaagggga cgaagggtg 1459