CN100554405C - A kind of method and special-purpose lactobacillus rhamnosus thereof that produces L-lactic acid - Google Patents

Abstract

The invention discloses a method for producing L-lactic acid and a special-purpose Lactobacillus rhamnosus. The Lactobacillus rhamnosus provided by the present invention is Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC No. 2183. L-lactic acid can be obtained by culturing Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183. The fermentation medium of the strain contains carbon source, nitrogen source and a neutralizer for controlling the pH of the fermentation broth; the carbon source is glucose 150-200 g/liter (initial concentration of fermentation); the nitrogen source is soybean meal hydrolysis liquid, soybean meal hydrolyzate + corn steep liquor or soybean meal powder; when the nitrogen source is soybean meal powder, it may also contain protease 0.05-0.1 g/L; the neutralizer is calcium carbonate 75-100 g/L, the balance For water, the pH of the fermentation medium is 5.5-7. Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 uses glucose as a substrate to produce L-lactic acid with an optical purity of 97.6%-98.7% at a conversion rate of 94.5%-96.5% at 35°C-45°C , and its concentration can reach up to 235 g/L.

Description

A method for producing L-lactic acid and its special-purpose Lactobacillus rhamnosus

technical field

The present invention relates to a method for producing L-lactic acid and its special bacterial strain, in particular to a method for producing L-lactic acid and its special Lactobacillus rhamnosus.

Background technique

Lactic acid, also known as dihydroxypropionic acid, is one of the three largest organic acids in the world. As a traditional multi-purpose fine chemical, lactic acid can be used as sour agent, fragrance, preservative, plant growth regulator, biodegradable material, drug and pesticide, etc., used in food, pharmaceutical, brewing, tanning, Textile, environmental protection and agriculture. Lactic acid is a chiral molecule and can be divided into L-lactic acid, D-lactic acid and DL-lactic acid. Polylactic acid made of L-lactic acid as the main monomer has good biocompatibility and biodegradability, and can be made into medical sustained-release materials, biodegradable fibers and biodegradable plastics. L-lactic acid is becoming a research hotspot in the lactic acid industry at home and abroad.

Lactic acid production methods mainly include chemical synthesis, enzymatic synthesis and fermentation. Lactic acid directly synthesized by chemical method is DL-lactic acid, and optical resolution is required to obtain D-lactic acid or L-lactic acid. Moreover, since the raw materials used in the synthesis method are acetaldehyde and highly toxic hydrocyanic acid, the application of synthetic lactic acid in food cannot be widely accepted, and its production cost is also relatively high. Enzymatic catalysis can specifically obtain optically pure lactic acid, but its reaction process is very difficult to study and has not been applied to industrial production. The production of lactic acid by microbial fermentation can obtain D-lactic acid, L-lactic acid or a mixture of the two in a certain proportion through the selection of strains and culture conditions, and can be obtained by decomposing starch, cellulose and other renewable resources. Fermentation of raw materials to produce lactic acid, with low production cost and high product safety, is the main method for large-scale production of lactic acid.

The Chinese patent application number 200480036931.1 discloses a method for producing lactic acid using Lactobacillus paracasei, using glucose, yeast extract and peptone as fermentation substrates to produce 181 g/L of lactic acid; using glucose, peptone and corn steeping powder as fermentation substrates , producing 179.1 g/L of lactic acid. The Chinese patent application number 200510119041.3 discloses a method for fermenting and producing L-lactic acid using Lactobacillus caseisubsp.rhamnosus-Lc-F34. Lactic acid was produced by fed-batch fermentation, which produced 177.6 g/L lactic acid; corn steep liquor and glucose were used as fermentation substrates, and lactic acid was produced by fed-batch fermentation in a 30-liter fermenter, which produced 154.6 g/L L-lactic acid.

The production cost of L-lactic acid is one of the key factors restricting the application of L-lactic acid. Increasing the concentration of L-lactic acid in the fermentation broth in the process of producing L-lactic acid by fermentation is beneficial to reduce the cost. In the process of producing L-lactic acid by fermentation, the nitrogen source in the culture medium is another important factor affecting the production cost of L-lactic acid. Nitrogen-containing raw materials with a wide range of sources and low prices are used as yeast powder and peptone, which are relatively expensive. The substitution of nitrogen source is another effective way to control the production cost of L-lactic acid.

Contents of the invention

One object of the present invention is to provide a strain of Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL producing L-lactic acid.

The Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL provided by the present invention is screened from the soil near a milk factory in Jinan, Shandong, and identified as Lactobacillus rhamnosus (Lactobacillus rhamnosus). Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL was preserved on September 24, 2007 in the General Microbiology Center of China Committee for Culture Collection of Microorganisms (CGMCC for short, address: Datun Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences ), the deposit registration number is CGMCC № 2183.

Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 is a Gram-positive bacterium with long rod-shaped cells, 2.5-3.5 μm long and 0.7-0.8 μm wide. The bacteria can utilize trehalose, sorbitol, glucose, lactose, ribose, cellobiose, maltose and fructose, etc.

The second object of the present invention is to provide a method for producing L-lactic acid using Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183.

The method for producing L-lactic acid provided by the present invention is to ferment Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to obtain L-lactic acid.

The fermentation medium of the Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 contains a carbon source, a nitrogen source and a neutralizer for controlling the pH of the fermentation broth, and the carbon source is industrial glucose (Zibo Hongyu Industry Co., Ltd. Sugar Co., Ltd.) 150-200 grams per liter, the nitrogen source is selected from any one of the following 1) to 3):

1) Soybean meal hydrolyzate 100-300 g/L;

2) Soybean meal powder 20-30 g/L;

3) Soybean meal hydrolyzate 100-300 g/L, corn steep liquor 10-30 g/L;

The pH of the fermentation medium is 5.5-7.

The soybean meal powder can be directly used as a nitrogen source, or acid hydrolysis or enzymatic hydrolysis can be carried out first, and the obtained soybean meal hydrolyzate can be used as a nitrogen source.

The soybean meal hydrolyzate can be prepared according to the following method. Weigh the soybean meal powder (Beijing Kangmingwei Medium Technology Co., Ltd.), fully mix with the ratio of 1g soybean meal powder: 5ml of 2% sulfuric acid solution, and put it in a 90°C water bath for 15 Hour.

When the nitrogen source is soybean meal, the fermentation medium may further contain 0.05-0.1 g/L of protease.

The neutralizing agent can specifically be calcium carbonate.

The fermentation conditions of the Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 can specifically be 35-50°C for 48-80 hours, such as 35-45°C for 50-68 hours.

In the method, Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMC C№2183 can also be first inserted into the following seed medium, cultivated at 35-40°C for 12-20 hours, and then inserted into the fermentation medium Each liter of the seed medium contains: 20 grams of glucose, 5 grams of yeast powder, 5 grams of peptone, 10 grams of calcium carbonate, and the balance is water; the pH of the seed medium is 6.5.

The culture mode of described fermentation can specifically be intermittent vibrating culture (such as with Erlenmeyer flask fermentation culture) or intermittent stirring culture (such as adopting fermentor to cultivate), and the interval time is 12 hours; The rotating speed of described stirring is 30-100 rpm. minutes, the radius of rotation is 33 or 42mm, and the stirring time is 20-40 minutes; the stirring speed is preferably 50 rpm, and the stirring time is preferably 30 minutes.

The invention utilizes Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to produce L-lactic acid, uses industrial glucose as a substrate, and produces optical purity by high-efficiency fermentation at a conversion rate of 94.5%-96.5% under the condition of 35°C-45°C. 97.6%-98.7% L-lactic acid, of which the highest concentration of L-lactic acid can reach 235 g/L. The present invention utilizes Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to produce L-lactic acid. When soybean meal powder is used as nitrogen source, protease can be added at the same time, so that protein hydrolysis and lactic acid fermentation can be carried out simultaneously, and the utilization efficiency of soybean meal powder is improved. , simplifies the raw material processing process, and can also control the production cost of L-lactic acid.

Description of drawings

Figure 1 is a high-performance liquid chromatogram of L-lactic acid fermentation broth produced by Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 fermentation, L-lactic acid standard product, and D-lactic acid standard product. Wherein, A is the high-performance liquid chromatogram of the L-lactic acid standard product, and B is the high-performance liquid chromatogram of the D-lactic acid standard product.

Fig. 2 is the curve of glucose and L-lactic acid changing with time during the fermentation of Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to produce L-lactic acid.

Detailed ways

The steps involved in the method for producing L-lactic acid by fermentation of Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 of the present invention are as follows:

(1) Slant culture: inoculate Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 strains on a solid slant medium containing 1.5g/100ml agar, and culture at 35-40°C for 24-48 hours;

(2) Seed culture: inoculate the slant culture of step (1) into 30ml seed culture medium under aseptic conditions, and culture it statically for 10-24 hours at 35-40°C, and add neutralizer to control the fermentation broth pH, the seed culture solution was prepared;

(3) Fermentation culture: with the inoculum amount of 5-15% volume ratio, inoculate the seed culture liquid in the fermentation medium, cultivate under the condition of 35-50 ℃ for 48-80 hours, wherein, the fermentation in the triangular flask adopts manual vibration, Vibrate once every 12 hours, the fermenter adopts intermittent stirring culture, stir once every 12 hours, the stirring speed is 30-100 rpm, the radius of rotation is 33 or 42mm, the stirring time is 20-40 minutes, and the neutralizer is added to control the fermentation The pH of the liquid is obtained to obtain a fermentation broth containing L-lactic acid;

(4) Sample processing: take the fermentation broth and centrifuge it at 6,000 rpm for 5 minutes, and take the supernatant;

(5) Sample detection: take the supernatant and dilute it, and detect the content of glucose, L-lactic acid and D-lactic acid;

Wherein, the cell culture temperature described in step (1) may specifically be 37-40°C.

Wherein, the cell culture temperature described in step (2) may specifically be 37-40°C.

Wherein, the cell culture temperature described in step (3) may specifically be 35-45°C.

Wherein, the cell culture time described in step (1) may specifically be 24-36 hours.

Wherein, the cell culture time described in step (2) may specifically be 12-20 hours.

Wherein, the cell culture time described in step (3) may specifically be 50-68 hours.

Wherein, the neutralizing agent added in the cultivation process described in steps (2) and (3) is calcium carbonate, and the pH is controlled to be 5.5-7.

Wherein, the determination method of glucose is that the fermented liquid is diluted after centrifugation, and then measured by a biosensing analyzer SBA-40C (Institute of Biology, Shandong Academy of Sciences).

Biosensing analyzer SBA-40C is an analysis instrument with immobilized enzyme as the sensor. Glucose, oxygen and water are catalyzed by glucose oxidase to generate gluconic acid and hydrogen peroxide. The hydrogen peroxide released from the reaction is in contact with the platinum-silver electrode to generate a current signal, which is linearly proportional to the glucose concentration, and the glucose concentration can be obtained by measuring the intensity of the current signal.

The content of L-lactic acid and D-lactic acid was determined by Agilent 1100 liquid chromatograph (Agilent Technologies Co., Ltd.), chiral separation column (Mitsubishi Chemical Corporation, MCI GEL-CRS10W (3μ) 4.6ID × 50mm, for optical isomer separation) , the mobile phase is 0.002mol/L copper sulfate, the flow rate is 0.5ml/min, the injection volume is 20μL, the ultraviolet detector, the detection wavelength is 254nm, and the operating temperature is 25°C. L-lactic acid standard product is the product of Sigma-Aldrich Company, the article number is L1750. D-lactic acid standard product is the product of Sigma-Aldrich Company, the product number is L0625.

Under the above chromatographic conditions, the high performance liquid chromatography detection results of the standard substance are shown in accompanying drawing 1. Among them, A is the high-performance liquid chromatogram of the L-lactic acid standard product, B is the high-performance liquid chromatogram of the D-lactic acid standard product, and C is the L-lactic acid fermentation broth produced by the fermentation of Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 High performance liquid chromatography. Under the chromatographic conditions, the retention time of the L-lactic acid standard was 11.566 minutes, and the retention time of the D-lactic acid standard was 9.724 minutes.

L-lactic acid optical purity calculation method: L-lactic acid optical purity (%)=[L-lactic acid concentration (g/L)-D-lactic acid concentration (g/L)]÷[L-lactic acid concentration (g/L)+ D-lactic acid concentration (g/L)]×100%

Conversion rate calculation method: conversion rate (%) = L-lactic acid concentration (g/L) ÷ glucose consumption (g/L) × 100%

The present invention is further illustrated below by way of examples.

Example 1. Isolation, mutagenesis screening and identification of Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183

1. Isolation and mutagenesis screening of strains

1) Separation of strains

Obtain a soil sample near a milk factory in Jinan, Shandong, weigh 5 grams of soil sample into a 250ml Erlenmeyer flask, add 100ml of normal saline, mix well, dilute 10,000 times with sterile water and spread on MRS solid medium, 40℃ Cultivate, after the growth of single colonies, part of the single colonies will form a transparent circle due to acid production and the dissolution of calcium carbonate in the medium around the colonies. Pick a single colony with a large transparent circle, inoculate it in 10ml of fermentation medium, and culture it at 40°C for 72 hours, measure the concentration of L-lactic acid in the fermentation broth, pick a strain with the highest L-lactic acid production, and inoculate it in MRS Refrigerate on medium slant for later use.

2) Ion beam mutagenesis screening

Inoculate the above-obtained bacterial strains into MRS liquid medium, culture statically at 40°C until the mid-logarithmic phase, and suspend with physiological saline after centrifugation to make a bacterial suspension. Draw 100 μL of bacterial suspension and spread it on a sterile blank culture dish with a diameter of 3.5 cm. ×10 ¹⁴ ions/cm ² , 5×10 ¹⁴ ions/cm ² , 10×10 ¹⁴ ions/cm ² , 50×10 ¹⁴ ions/cm ² , 100×10 ¹⁴ ions/cm ² . The plate after ion implantation was eluted with 1 mL of physiological saline. Draw 100 μL of different injection doses, dilute and spread on the MRS solid medium. After a single colony grows, select the colony with a larger transparent circle, inoculate it in 10ml of fermentation medium, and culture it at 40°C for 72 hours. Determination of glucose concentration, L-lactic acid concentration and D-lactic acid concentration in the fermentation broth. After multiple screenings, a strain with significantly improved L-lactic acid production than the strain before ion beam mutagenesis screening, an L-lactic acid optical purity of over 98%, and a transformation rate of over 95% was obtained, that is, Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183, after multiple passages, the yield of L-lactic acid, the optical purity of L-lactic acid and the conversion rate did not change significantly.

The MRS solid medium used in the screening of Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC №2183 is:

Glucose 20 g/L, yeast extract 5 g/L, peptone 10 g/L, beef extract 10 g/L, dipotassium hydrogen phosphate 2 g/L, diammonium citrate 2 g/L, sodium acetate 5 g/L liter, Tween 801 ml/liter, magnesium sulfate heptahydrate 0.58 grams/liter, manganese sulfate tetrahydrate 0.25 grams/liter, calcium carbonate 15 grams/liter, agar powder 15 grams/liter. The pH value of the medium is 6.5. Sterilize at 115° C. for 20 minutes.

The MRS liquid medium used in the screening of Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 is:

Glucose 20 g/L, yeast extract 5 g/L, peptone 10 g/L, beef extract 10 g/L, dipotassium hydrogen phosphate 2 g/L, diammonium citrate 2 g/L, sodium acetate 5 g/L liter, Tween 801 ml/liter, magnesium sulfate heptahydrate 0.58 g/liter, manganese sulfate tetrahydrate 0.25 g/liter. The pH value of the medium is 6.5. Sterilize at 115° C. for 20 minutes.

The fermentation medium used in the screening of Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 is:

Industrial glucose (Zibo Hongyu Industrial Sugar Co., Ltd.) 180 g/liter, soybean meal hydrolyzate (preparation method of soybean meal hydrolyzate: soybean meal powder (Beijing Kangmingwei Medium Technology Co., Ltd.), with 1g soybean meal powder: 2% of 5ml ( Volume percentage content) the ratio of sulfuric acid solution is fully mixed, 90 ℃ of water baths 15 hours, obtain soybean meal hydrolyzate) 100 g/liter, corn steep liquor (Shanghai Xiwang Starch Sugar Co., Ltd.) 10 g/liter, calcium carbonate 90 grams /Lift. The pH value of the medium is 6.5. Sterilize at 115° C. for 20 minutes.

2. Identification of strains

According to the method described in "Berger's Bacteria Identification Handbook" (the eighth edition) and "Common Bacteria System Identification Handbook" (edited by Dong Xiuzhu, Cai Miaoying, etc., Beijing: Science Press, 2001.2), the bacterial strain was observed and analyzed for its morphological characteristics. Identification of physiological and biochemical characteristics;

The identification results showed that Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 was a Gram-positive bacterium with long rod-shaped cells, 2.5-3.5 μm in length and 0.7-0.8 μm in width. The bacteria can use trehalose, sorbitol, glucose, lactose, ribose, cellobiose, maltose, fructose, mannitol, melezitose, rhamnose, galactose, escin, mannose, but can not use nectar Sugar, Laetrile, Arabinose, Inositol, Raffinose. Oxidase negative. Hemolysis test was negative. Capable of growing at 15°C and 45°C.

Based on the above characteristics, the L-lactic acid producing bacterium of this strain was identified as Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL, and was preserved in the General Microorganism Center of China Committee for Culture Collection of Microorganisms (CGMCC for short, address: : Datun Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), the deposit registration number is CGMCC № 2183.

Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 and "Lactic Acid Bacteria Classification Identification and Experimental Methods" (Edited by Ling Daiwen; edited by Ling Daiwen and Dong Xiuzhu, Beijing: China Light Industry Press, 1999.3) The physiological and biochemical characteristics of Lactobacillus rhamnosus are compared in Table 1.

Table 1

Physiological and biochemical characteristics Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 Lactobacillus rhamnosus (Lactobacillus rhamnosus) described in "Lactic acid bacteria classification and identification and experimental methods"

Laetrile - + Arabic candy - d Cellobiose + + Escin (escin) + + fructose + + Galactose + + Glucose + + Lactose + + Maltose + + Mannitol + + Mannose + + Melezitose + + Melibiose - - Raffinose - - D + + Ribose + + trehalose + + Grow at 15°C + +

In Table 1, + indicates that the test is positive or grows, - indicates that the test is negative or does not grow, and d indicates that 11%-89% of the strains are positive.

Embodiment 2, utilize Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to produce L-lactic acid in Erlenmeyer flask fermentation method

The composition of the medium used in this embodiment is as follows:

Slant medium: glucose 20 g/L, yeast extract 5 g/L, peptone 10 g/L, beef extract 10 g/L, dipotassium hydrogen phosphate 2 g/L, diammonium citrate 2 g/L, acetic acid Sodium 5 g/L, Tween 801 mL/L, magnesium sulfate heptahydrate 0.58 g/L, manganese sulfate tetrahydrate 0.25 g/L, calcium carbonate 15 g/L, agar powder 15 g/L. The initial pH of the medium was 6.5. Sterilization was performed at 115°C for 20 minutes.

Seed medium: glucose 20 g/L, yeast powder 5 g/L, peptone 5 g/L, calcium carbonate 10 g/L. The initial pH of the medium was 6.5. Sterilization was carried out at 115° C. for 20 minutes.

Fermentation medium: industrial glucose (Zibo Hongyu Industrial Sugar Co., Ltd.) 150 g/liter, soybean meal hydrolyzate (preparation method is the same as in Example 1) 100 g/liter, calcium carbonate 75 grams/liter. The initial pH of the medium was 6.5. Sterilization was carried out at 115° C. for 20 minutes.

The method for producing L-lactic acid by fermentation method of the present invention comprises the following steps:

(1) Slant culture: Inoculate Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 on a solid slant medium and culture at 37°C for 36 hours;

(2) Seed culture: the bacterial strain cultivated in step (1) is placed in a 100ml Erlenmeyer flask with 30ml seed culture medium under aseptic conditions with an inoculation loop, and cultured at 37°C for 20 hours to obtain seeds culture medium;

(3) Fermentation culture: 10 ml of the seed culture solution of step (2) was inserted into a 300 ml Erlenmeyer flask equipped with 90 ml of fermentation medium, and cultured at 40° C. for 72 hours to finish the fermentation. Wherein, shake and stir once every 12 hours. The experiment was repeated 3 times.

At the end of the fermentation, take the fermentation broth, centrifuge at 6,000 rpm for 5 minutes, take the supernatant, detect the concentration of glucose, L-lactic acid and D-lactic acid in the fermentation broth according to the above method, and calculate the optical purity and conversion rate of L-lactic acid . The results showed that the concentration of glucose was 10 g/L±0.5 g/L, the concentration of L-lactic acid was 132 g/L±3 g/L, the conversion rate was 94.5%±0.3%, and the optical purity of L-lactic acid was 98.5%±0.4%.

Embodiment 3, utilize Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to produce L-lactic acid by fermentation in Erlenmeyer flask

The composition of the medium used in this embodiment is as follows:

The slant medium and seed medium are the same as in Example 2.

Fermentation medium: industrial glucose (Zibo Hongyu Industrial Sugar Co., Ltd.) 150 grams per liter, soybean meal hydrolyzate (preparation method is the same as in Example 1) 300 grams per liter, calcium carbonate 75 grams per liter. The initial pH of the medium was 7. Sterilization was carried out at 115° C. for 20 minutes.

The method for producing L-lactic acid by fermentation method of the present invention comprises the following steps:

(1) inclined plane culture: with embodiment 2;

(2) seed culture: with embodiment 2;

(3) Fermentation culture: 15 ml of the seed culture solution of step (2) was inserted into a 300 ml Erlenmeyer flask equipped with 85 ml of fermentation medium, and cultured at 37° C. for 65 hours to finish the fermentation. Wherein, shake and stir once every 12 hours. The experiment was repeated 3 times.

At the end of the fermentation, take the fermentation broth, centrifuge at 6,000 rpm for 5 minutes, take the supernatant, and detect the concentration of glucose, L-lactic acid and D-lactic acid in the fermentation broth according to the above detection method, and calculate the optical purity and Conversion rate. The results showed that the concentration of glucose was 2 g/L±0.3 g/L, the concentration of L-lactic acid was 140 g/L±4 g/L, the conversion rate was 95.2%±0.5%, and the optical purity of L-lactic acid was 98.0%±0.2%.

Embodiment 4, utilize Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to produce L-lactic acid in Erlenmeyer flask fermentation method

The composition of the medium used in this embodiment is as follows:

The slant medium and seed medium are the same as in Example 2.

Fermentation medium: industrial glucose (Zibo Hongyu Industrial Sugar Co., Ltd.) 150 grams per liter, soybean meal hydrolyzate (preparation method is the same as in Example 1) 220 grams per liter, calcium carbonate 75 grams per liter. The initial pH of the medium was 5.5. Sterilization was carried out at 115° C. for 20 minutes.

The method for producing L-lactic acid by fermentation method of the present invention comprises the following steps:

(1) inclined plane culture: with embodiment 2;

(2) seed culture: with embodiment 2;

(3) Fermentation culture: Insert 5 ml of the seed nutrient solution of step (2) into a 300 ml Erlenmeyer flask equipped with 95 ml of fermentation medium, and culture at 40° C. for 50 hours to finish the fermentation. Wherein, shake and stir once every 12 hours. The experiment was repeated 3 times.

At the end of the fermentation, take the fermentation broth, centrifuge at 6,000 rpm for 5 minutes, take the supernatant, and detect the concentration of glucose, L-lactic acid and D-lactic acid in the fermentation broth according to the above detection method, and calculate the optical purity and Conversion rate. The results showed that the concentration of glucose was 1 g/L±0.3 g/L, the concentration of L-lactic acid was 142 g/L±2 g/L, the conversion rate was 95.3%±0.3%, and the optical purity of L-lactic acid was 98.5%±0.5%.

Embodiment 5, utilize Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to produce L-lactic acid in Erlenmeyer flask fermentation method

The composition of the medium used in this embodiment is as follows:

The slant medium and seed medium are the same as in Example 2.

Fermentation medium: industrial glucose (Zibo Hongyu Industrial Sugar Co., Ltd.) 180 grams per liter, soybean meal hydrolyzate (preparation method is the same as in Example 1) 100 grams per liter, corn steep liquor (Shanghai Xiwang Starch Sugar Co., Ltd.) 30 grams per liter Liter, calcium carbonate 90 g/liter. The initial pH of the medium was 6. Sterilization was carried out at 115° C. for 20 minutes.

The method for producing L-lactic acid by fermentation method of the present invention comprises the following steps:

(1) inclined plane culture: with embodiment 2;

(2) seed culture: with embodiment 2;

(3) Fermentation culture: 10 ml of the seed culture solution of step (2) was inserted into a 300 ml Erlenmeyer flask equipped with 90 ml of fermentation medium, and cultured at 37° C. for 60 hours to finish the fermentation. Wherein, shake and stir once every 12 hours. The experiment was repeated 3 times.

At the end of the fermentation, take the fermentation broth, centrifuge at 6,000 rpm for 5 minutes, take the supernatant, and detect the concentration of glucose, L-lactic acid and D-lactic acid in the fermentation broth according to the above detection method, and calculate the optical purity and Conversion rate. The results showed that the concentration of glucose was 8 g/L±1.0 g/L, the concentration of L-lactic acid was 165 g/L±5 g/L, the conversion rate was 96.0%±0.5%, and the optical purity of L-lactic acid was 98.2%±0.3%.

Embodiment 6, utilize Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to produce L-lactic acid in the Erlenmeyer flask fermentation method

The composition of the medium used in this embodiment is as follows:

The slant medium and seed medium are the same as in Example 2.

Fermentation medium: industrial glucose (Zibo Hongyu Industrial Sugar Co., Ltd.) 200 grams/liter, soybean meal hydrolyzate (preparation method is the same as Example 1) 300 grams/liter, corn steep liquor (Shanghai Xiwang Starch Sugar Co., Ltd.) 10 grams/liter Liter, calcium carbonate 100 g/liter. The initial pH of the medium was 6.5. Sterilization was carried out at 115° C. for 20 minutes.

The method for producing L-lactic acid by fermentation method of the present invention comprises the following steps:

(1) inclined plane culture: with embodiment 2;

(2) seed culture: with embodiment 2;

(3) Fermentation culture: 15 ml of the seed culture solution of step (2) was inserted into a 300 ml Erlenmeyer flask equipped with 85 ml of fermentation medium, and cultured at 35° C. for 48 hours to finish the fermentation. Wherein, shake and stir once every 12 hours. The experiment was repeated 3 times.

At the end of the fermentation, take the fermentation broth, centrifuge at 6,000 rpm for 5 minutes, take the supernatant, and detect the concentration of glucose, L-lactic acid and D-lactic acid in the fermentation broth according to the above detection method, and calculate the optical purity and Conversion rate. The results showed that the concentration of glucose was 1 g/L±0.5 g/L, the concentration of L-lactic acid was 192 g/L±2 g/L, the conversion rate was 96.5%±0.4%, and the optical purity of L-lactic acid was 97.6%±0.2%.

Example 7, Utilizing Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to produce L-lactic acid by fermentation in Erlenmeyer flask

The composition of the medium used in this embodiment is as follows:

The slant medium and seed medium are the same as in Example 2.

Fermentation medium: industrial glucose (Zibo Hongyu Industrial Sugar Co., Ltd.) 150 g/L (initial concentration of fermentation), soybean meal hydrolyzate (preparation method is the same as Example 1) 250 g/L, corn steep liquor (Shanghai Xiwang Starch Sugar Co., Ltd. company) 20 g/L, calcium carbonate 75 g/L. The initial pH of the medium was 6.5. Sterilization was carried out at 115° C. for 20 minutes.

The method for producing L-lactic acid by fermentation method of the present invention comprises the following steps:

(1) inclined plane culture: with embodiment 2;

(2) seed culture: with embodiment 2;

(3) Fermentation culture: Add 10 ml of the seed nutrient solution of step (2) into a 300 ml Erlenmeyer flask equipped with 90 ml of fermentation medium, culture at 40° C. for 50 hours, shake and stir once every 12 hours. Wherein, 8 grams of industrial glucose (Zibo Hongyu Industrial Sugar Co., Ltd.) and 4 grams of calcium carbonate were added in 30 hours. The experiment was repeated 3 times.

At the end of the fermentation, take the fermentation broth, centrifuge at 6,000 rpm for 5 minutes, take the supernatant, and detect the concentration of glucose, L-lactic acid and D-lactic acid in the fermentation broth according to the above detection method, and calculate the optical purity and Conversion rate. The results showed that the concentration of glucose was 2 g/L±0.3 g/L, the concentration of L-lactic acid was 220 g/L±3 g/L, the conversion rate was 96.3%±0.4%, and the optical purity of L-lactic acid was 98.0%±0.2%.

Example 8, Utilizing Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to produce L-lactic acid by fermentation in Erlenmeyer flask

The composition of the medium used in this embodiment is as follows:

The slant medium and seed medium are the same as in Example 2.

Fermentation medium: industrial glucose (Zibo Hongyu Industrial Sugar Co., Ltd.) 150 g/L, soybean meal powder (Beijing Kangmingwei Media Technology Co., Ltd.) 30 g/L, calcium carbonate 75 g/L. The initial pH of the medium was 6.5. Sterilization was carried out at 115° C. for 20 minutes.

The method for producing L-lactic acid by fermentation method of the present invention comprises the following steps:

(1) inclined plane culture: with embodiment 2;

(2) seed culture: with embodiment 2;

(3) Fermentation culture: 10 ml of the seed culture solution of step (2) was inserted into a 300 ml Erlenmeyer flask equipped with 90 ml of fermentation medium, and cultured at 50° C. for 65 hours to finish the fermentation. Wherein, shake and stir once every 12 hours. The experiment was repeated 3 times.

At the end of the fermentation, take the fermentation broth, centrifuge at 6,000 rpm for 5 minutes, take the supernatant, and detect the concentration of glucose, L-lactic acid and D-lactic acid in the fermentation broth according to the above detection method, and calculate the optical purity and Conversion rate. The results showed that the concentration of glucose was 28 g/L±0.8 g/L, the concentration of L-lactic acid was 115 g/L±2 g/L, the conversion rate was 95.2%±0.5%, and the optical purity of L-lactic acid was 98.0%±0.3%.

Example 9, Utilizing Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to produce L-lactic acid by fermentation in Erlenmeyer flask

The composition of the medium used in this embodiment is as follows:

The slant medium and seed medium are the same as in Example 2.

Fermentation medium: industrial glucose (Zibo Hongyu Industrial Sugar Co., Ltd.) 180 g/L, soybean meal powder (Beijing Kangmingwei Medium Technology Co., Ltd.) 30 g/L, protease (Novozyme flavor protease, Flavourzyme 500MG) 0.05 g/L, calcium carbonate 90 g/L. The initial pH of the medium was 7. Sterilization was carried out at 115° C. for 20 minutes. Wherein, protease is added after sterilization.

The method for producing L-lactic acid by fermentation method of the present invention comprises the following steps:

(1) inclined plane culture: with embodiment 2;

(2) seed culture: with embodiment 2;

(3) Fermentation culture: Insert 10 ml of the seed culture solution of step (2) into a 300 ml Erlenmeyer flask equipped with 90 ml of fermentation medium, and culture at 42° C. for 72 hours, and finish the fermentation. Wherein, shake and stir once every 12 hours. The experiment was repeated 3 times.

At the end of the fermentation, take the fermentation broth, centrifuge at 6,000 rpm for 5 minutes, take the supernatant, and detect the concentration of glucose, L-lactic acid and D-lactic acid in the fermentation broth according to the above detection method, and calculate the optical purity and Conversion rate. The results showed that the concentration of glucose was 28 g/L±0.7 g/L, the concentration of L-lactic acid was 145 g/L±3 g/L, the conversion rate was 95.5%±0.3%, and the optical purity of L-lactic acid was 98.5%±0.2%.

Example 10, Utilizing Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to produce L-lactic acid by fermentation in Erlenmeyer flask

The composition of the medium used in this embodiment is as follows:

The slant medium and seed medium are the same as in Example 2.

Fermentation medium: industrial glucose (Zibo Hongyu Industrial Sugar Co., Ltd.) 180 g/L, soybean meal powder (Beijing Kangmingwei Medium Technology Co., Ltd.) 20 g/L, protease (Novozyme flavor protease, Flavourzyme 500MG) 0.1 g/L, calcium carbonate 90 g/L. The initial pH of the medium was 6.5. Sterilization was carried out at 115° C. for 20 minutes. Wherein, protease is added after sterilization.

The method for producing L-lactic acid by fermentation method of the present invention comprises the following steps:

(1) inclined plane culture: with embodiment 2;

(2) seed culture: with embodiment 2;

(3) Fermentation culture: 10 ml of the seed nutrient solution of step (2) was inserted into a 300 ml Erlenmeyer flask equipped with 90 ml of fermentation medium, and cultured at 42° C. for 80 hours to finish the fermentation. Wherein, shake and stir once every 12 hours. The experiment was repeated 3 times.

At the end of the fermentation, take the fermentation broth, centrifuge at 6,000 rpm for 5 minutes, take the supernatant, and detect the concentration of glucose, L-lactic acid and D-lactic acid in the fermentation broth according to the above detection method, and calculate the optical purity and Conversion rate. The results showed that the concentration of glucose was 17 g/L±0.5 g/L, the concentration of L-lactic acid was 156 g/L±5 g/L, the conversion rate was 96.0%±0.3%, and the optical purity of L-lactic acid was 98.2%±0.7%.

Example 11. Production of L-lactic acid by fermentation in a 5-liter fermenter using Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183

The composition of the medium used in this embodiment is as follows:

The slant medium and seed medium are the same as in Example 2.

Fermentation medium: industrial glucose (Zibo Hongyu Industrial Sugar Co., Ltd.) 180 g/L, soybean meal powder (Beijing Kangmingwei Medium Technology Co., Ltd.) 28 g/L, protease (Novozyme flavor protease, Flavourzyme 500MG) 0.08 g/L, calcium carbonate 90 g/L. The initial pH of the medium was 6.5. Sterilization was carried out at 115° C. for 20 minutes. Wherein, protease is added after sterilization.

The method for producing L-lactic acid by fermentation method of the present invention comprises the following steps:

(1) inclined plane culture: with embodiment 2;

(2) Seed culture: the bacterial strain cultivated in step (1) is placed in a 100ml Erlenmeyer flask with 30ml seed culture medium under aseptic conditions with an inoculation loop, and cultured at 37°C for 20 hours; Add 400ml of seed culture medium into the bottle, add 30ml of seed culture solution to 400ml of seed culture medium, and culture at 37°C for 20 hours to obtain the seed culture solution;

(3) Fermentation culture: Add 3.6 liters of fermentation medium into a 5-liter fermentation tank of Shanghai Baoxing BIOTECH, add 400ml of seed culture solution into the fermentation medium, and culture at 45°C for 72 hours to end the fermentation. Wherein, stirring once every 12 hours. The stirring speed is 30 rpm, the radius of rotation is 33 mm, and the stirring time is 40 minutes. The experiment was repeated 3 times.

At the end of the fermentation, take the fermentation broth, centrifuge at 6,000 rpm for 5 minutes, take the supernatant, and detect the concentration of glucose, L-lactic acid and D-lactic acid in the fermentation broth according to the above detection method, and calculate the optical purity and Conversion rate. The results showed that the concentration of glucose was 10 g/L±0.6 g/L, the concentration of L-lactic acid was 160 g/L±4 g/L, the conversion rate was 94.8%±0.4%, and the optical purity of L-lactic acid was 98.7%±0.3%.

Example 12, Utilizing Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to produce L-lactic acid by fermentation in a 5-liter fermenter

The composition of the medium used in this embodiment is as follows:

The slant medium and seed medium are the same as in Example 2.

Fermentation medium: industrial glucose (Zibo Hongyu Industrial Sugar Co., Ltd.) 200 grams/liter (initial concentration of fermentation), soybean meal hydrolyzate (preparation method is the same as Example 1) 230 grams/liter, corn steep liquor (Shanghai Xiwang Starch Sugar Co., Ltd. company) 22 g/L, calcium carbonate 100 g/L. The initial pH of the medium was 6.5. Sterilization was carried out at 115° C. for 20 minutes.

The method for producing L-lactic acid by fermentation method of the present invention comprises the following steps:

(1) inclined plane culture: with embodiment 2;

(2) seed culture: with embodiment 11;

(3) Fermentation culture: Add 3.6 liters of fermentation medium into a 5-liter fermentation tank of Shanghai Baoxing BIOTECH, add 400ml of seed culture solution into the fermentation medium, and culture at 40°C for 60 hours. Wherein, stirring once every 12 hours. The stirring speed is 50 rpm, the radius of rotation is 33 mm, and the stirring time is 30 minutes. Wherein, 200 grams of industrial glucose (Zibo Hongyu Industrial Sugar Co., Ltd.) and 100 grams of calcium carbonate were added in 40 hours. The experiment was repeated 3 times.

At the end of the fermentation, take the fermentation broth, centrifuge at 6,000 rpm for 5 minutes, take the supernatant, and detect the concentration of glucose, L-lactic acid and D-lactic acid in the fermentation broth according to the above detection method, and calculate the optical purity and Conversion rate. The results showed that the concentration of glucose was 3 g/L±0.4 g/L, the concentration of L-lactic acid was 235 g/L±3 g/L, the conversion rate was 95.2%±0.3%, and the optical purity of L-lactic acid was 97.9%±0.5%.

See accompanying drawing 1 for the detection result of fermentation broth high performance liquid chromatography. Wherein C is the high-performance liquid chromatogram of L-lactic acid fermentation broth produced by Lactobacillus rhamnosus (Lactobacillusrhamnosus) CASL CGMCC № 2183 fermentation.

The fermentation process curve of Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 is shown in Figure 2.

Example 13, Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 was used to produce L-lactic acid by fermentation in a 30-liter fermenter

The composition of the medium used in this embodiment is as follows:

The slant medium and seed medium are the same as in Example 2.

Fermentation medium: industrial glucose (Zibo Hongyu Industrial Sugar Co., Ltd.) 200 grams/liter, soybean meal hydrolyzate (preparation method is the same as Example 1) 200 grams/liter, corn steep liquor (Shanghai Xiwang Starch Sugar Co., Ltd.) 30 grams/liter Liter, calcium carbonate 100 g/liter. The initial pH of the medium was 6.5. Sterilization was carried out at 115° C. for 20 minutes.

The method for producing L-lactic acid by fermentation method of the present invention comprises the following steps:

(1) inclined plane culture: with embodiment 2;

(2) Seed culture: the bacterial strain cultivated in step (1) is placed in a 100ml Erlenmeyer flask with 30ml seed culture medium under aseptic conditions with an inoculation loop, and cultured at 37°C for 20 hours; Add 400ml of seed culture medium to the bottle, insert 30ml of seed culture solution into 400ml of seed culture medium, and culture at 37°C for 20 hours; basal, 37 ° C static culture for 20 hours to prepare the seed culture solution;

(3) Fermentation culture: add 18 liters of fermentation medium to Shanghai Baoxing BIOTECH 30 liters fermenter, add 2 liters of seed culture solution to the fermentation medium, and culture at 42°C for 48 hours, and stir every 12 hours Once, the stirring speed is 50 rpm, the radius of rotation is 42 mm, and the stirring time is 30 minutes. The experiment was repeated 3 times.

At the end of the fermentation, take the fermentation broth, centrifuge at 6,000 rpm for 5 minutes, take the supernatant, and detect the concentration of glucose, L-lactic acid and D-lactic acid in the fermentation broth according to the above detection method, and calculate the optical purity and Conversion rate. The results showed that the concentration of glucose was 9 g/L±0.3 g/L, the concentration of L-lactic acid was 182 g/L±4 g/L, the conversion rate was 95.3%±0.4%, and the optical purity of L-lactic acid was 98.3%±0.3%.

Example 14, Utilizing Lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL CGMCC № 2183 to produce L-lactic acid by fermentation in a 30-liter fermenter

The composition of the medium used in this embodiment is as follows:

The slant medium and seed medium are the same as in Example 2.

Fermentation medium: industrial glucose (Zibo Hongyu Industrial Sugar Co., Ltd.) 200 grams/liter (initial concentration of fermentation), soybean meal hydrolyzate (preparation method is the same as Example 1) 230 grams/liter, corn steep liquor (Shanghai Xiwang Starch Sugar Co., Ltd. company) 25 g/L, calcium carbonate 100 g/L. The initial pH of the medium was 6.5. Sterilization was carried out at 115° C. for 20 minutes.

The method for producing L-lactic acid by fermentation method of the present invention comprises the following steps:

(1) inclined plane culture: with embodiment 2;

(2) seed culture: with embodiment 13;

(3) Fermentation culture: add 18 liters of fermentation medium to Shanghai Baoxing BIOTECH 30 liters fermenter, add 2 liters of seed culture solution to the fermentation medium, and culture at 37°C for 68 hours, and stir every 12 hours Once, the stirring speed is 100 rpm, the radius of rotation is 42 mm, and the stirring time is 20 minutes. Wherein, 1000 grams of industrial glucose (Zibo Hongyu Industrial Sugar Co., Ltd.) and 500 grams of calcium carbonate were added in 48 hours. The experiment was repeated 3 times.

At the end of the fermentation, take the fermentation broth, centrifuge at 6,000 rpm for 5 minutes, take the supernatant, and detect the concentration of glucose, L-lactic acid and D-lactic acid in the fermentation broth according to the above detection method, and calculate the optical purity and Conversion rate. The results showed that the concentration of glucose was 15 g/L±0.6 g/L, the concentration of L-lactic acid was 226 g/L±3 g/L, the conversion rate was 96.3%±0.5%, and the optical purity of L-lactic acid was 98.0%±0.3%.

Claims (12)

1, lactobacillus rhamnosus (Lactobacillus rhamnosus) CASL, its preserving number is CGMCC № 2183.

2, a kind of method of producing L-lactic acid is that fermentation lactobacillus rhamnosus (Lactobacillus rhamnosus) CASLCGMCC № 2183 obtains L-lactic acid.

3, method according to claim 2 is characterized in that: contain carbon source, nitrogenous source in the fermention medium of described lactobacillus rhamnosus (Lactobacillusrhamnosus) CASL CGMCC № 2183 and be used to control the neutralizing agent of fermented liquid pH; Carbon source in the described fermention medium is a glucose 150-200 grams per liter; Nitrogenous source in the described fermention medium is following 1) to 3) in any one:

1) soybean meal hydrolysate 100-300 grams per liter;

2) bean cake powder 20-30 grams per liter;

3) soybean meal hydrolysate 100-300 grams per liter, corn steep liquor 10-30 grams per liter.

4, method according to claim 3 is characterized in that: when the nitrogenous source in the described fermention medium was soybean meal hydrolysate, its consumption was 220 grams per liters.

5, method according to claim 3 is characterized in that: when the nitrogenous source in the described fermention medium is bean cake powder, also contain the proteolytic enzyme of 0.05-0.1 grams per liter in the described fermention medium.

6, method according to claim 3 is characterized in that: the neutralizing agent in the described fermention medium is a lime carbonate, and the concentration of described lime carbonate is the 75-100 grams per liter.

7, method according to claim 6 is characterized in that: the concentration of described lime carbonate is 90 grams per liters.

8, according to claim 2 or 3 described methods, it is characterized in that: the pH of described fermention medium is 5.5-7.

9, according to claim 2 or 3 described methods, it is characterized in that: described fermentation condition is 35-50 ℃ and cultivated 48-80 hour.

10, method according to claim 9 is characterized in that: described fermentation condition is 35-45 ℃ and cultivated 50-68 hour.

11, method according to claim 2, it is characterized in that: the training method of described fermentation adopts intermittent control shaking to cultivate in triangular flask, adopt intermittently stir culture in the fermentor tank, be 12 hours pitch time, the rotating speed of described stirring is 30-100 rev/min, rotation radius is 33 or 42mm, and churning time is 20-40 minute.

12, method according to claim 11 is characterized in that: described mixing speed is 50 rev/mins; Churning time is 30 minutes.

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