WO2015161642A1

WO2015161642A1 - Lactobacillus brevis strain producing mannitol and method for producing mannitol

Info

Publication number: WO2015161642A1
Application number: PCT/CN2014/090476
Authority: WO
Inventors: 曹海龙; 杜昱光; 岳敏; 李曙光; 尹恒; 陈玮
Original assignee: 中国科学院大连化学物理研究所
Priority date: 2014-04-22
Filing date: 2014-11-06
Publication date: 2015-10-29
Also published as: CN105002104B; CN105002104A

Abstract

Provided is a Lactobacillus brevis strain producing mannitol named Lactobacillus brevis 3-A20, with deposit number CGMCC8581. The strain is characterized in that it can produce mannitol by fermentation utilizing one or more carbon sources in an hydrolysis liquid containting glucose, fructose, sucrose, oligofructose and fructan. Meanwhile, provided is a method for producing mannitol by fermentation utilizing the strain. The fermentation utilizes one or more carbon sources containing glucose, fructose, sucrose, fructooligosaccharide and fructosan under anaerobic or microaerobic conditions, at pH 3.0-7.0, at a temperature within the range of 25-40 degrees, so as to produce 20-300 g/L of mannitol.

Description

Mannitol-producing Lactobacillus brevis strain and method for producing mannitol

Technical field

The invention relates to a mannitol-producing Lactobacillus brevis 3-A20 strain and a method for fermenting the same to produce mannitol, belonging to the field of food biotechnology.

Background technique

Mannitol is a naturally occurring hexavalent sugar alcohol that is widely used in food, pharmaceutical, chemical and light industry applications. Currently, mannitol is mainly produced by chemical methods or kelp extraction methods. Because of the high cost and energy consumption of existing mannitol production methods. In recent years, methods for the production of mannitol by microbial fermentation with high selectivity and low energy consumption have attracted much attention (Reference [1] Ghoreishi SM, Shahrestani RG. Innovative strategies for engineering mannitol production. Trends in Food Science & Technology, 2009, 20: 263–270).

Many microorganisms in nature can use carbohydrates to synthesize mannitol, such as Torulopsis, Candida, Zygosaccharomyces, Aspergillus candidus, and yellow-yellow Penicillium chrysogenum, Penicillium decumbens, Penicillium scabrosum, Aspergills niger, Lactococcus lactis, Lactobacillus buchneri, Lactobacillus fermentum (Lactobacillus fermentum), Lactobacillus sanfranciscensis, Lactobacillus intermedius, Leuconostoc mesenteroides and Leuconostoc pseudomesenteroides (Ref. [2], Saha BC, Racine FM. Biotechnological production of mannitol and its applications. Applied Microbiology and Biotechnology. 2011, 89(4): 879-91.).

At present, studies on mannitol using Lactobacillus brevis fermentation are rare. Zhu Yu et al found that a strain of Lactobacillus brevis strain M-1 has the ability to ferment mannitol by using Jerusalem artichoke saccharification juice (Reference [3] Zhu Yu, Cao Hailong, Yue Min, Li Shuguang, Bai Xuefang, Zhao Xiaoming, Du Shuguang, with Jerusalem artichoke Study on the fermentation of raw materials to produce mannitol, Northwest Agricultural Journal, 2009, No. 5, 137-141.). Yue Min et al. obtained a strain of Lactobacillus brevis 3-A5 by using atmospheric pressure plasma mutagenesis breeding technology. The fermentation process of this strain was preliminarily studied by pH control technology (Reference [4]Yue Min,Cao Hailong,Zhang Jianping, Li Shuguang, Meng Yanyu, Chen Wei, Huang Lishuxin, Du Yuguang.Improvement of mannitol production by Lactobacillus brevis mutant 3-A5based on dual-stage pH control and fed-batch fermentations, World Journal of Microbiology & Biotechnology, 2013, Vol 29 (10): 1923-1930). However, under high-concentration substrate conditions (above 180g/L total sugar concentration), the growth of the cells is inhibited, and it is impossible to effectively convert all or most of the fructose into mannitol, resulting in a large amount of sugar remaining, which cannot satisfy the industry. The need to zoom in. Therefore, a strain capable of efficiently converting fructose to mannitol under a high concentration substrate is the key to the production of mannitol by fermentation.

Summary of the invention

The object of the present invention is to provide a mannitol-producing Lactobacillus brevis 3-A20. The strain belongs to the genus Lactobacillus, and was deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee on December 13, 2013. The deposit address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China. The deposit number is CGMCC8581.

The strain obtained by the present invention was described in accordance with the ninth edition of the Berger's Bacterial Identification Manual and was analyzed based on 16S rDNA sequence to determine that the strain belongs to Lactobacillus brevis. Lactobacillus brevis 3-A20 is short Lactobacillus brevis 3-A5 was obtained by mutagenesis and acclimation culture under high glucose concentration medium. Compared with Lactobacillus brevis 3-A5, the initial total glucose tolerance range of Lactobacillus brevis 3-A20 was expanded from 0-180 g/L to 0-260 g/L; under batch culture conditions, Lactobacillus brevis 3-A20 mannitol yielded Lactobacillus The yield of brevis 3-A5 mannitol is increased by 5%-50%.

Another object of the present invention is to provide a method for producing mannitol by fermentation using Lactobacillus brevis 3-A20. The steps are as follows: Lactobacillus brevis 3-A20 is inoculated into the fermentation medium at 25-40 degrees, activated by MRS medium, and inoculated at a dose of 1%-20%, in the range of 25-40 degrees, at 0- At 100 rpm, the initial pH is 4.0-7.0, and the fermentation is carried out until 12-16 h, and the pH of the fermentation broth is controlled between 4.0 and 5.0 until the end of the fermentation process.

The composition of the fermentation medium is as follows: the total concentration of glucose and fructose is: 100-260 g/L, the mass ratio of glucose to fructose is 1:2-1:3, the concentration of dry powder of corn syrup is 10-50 g/L, the concentration of manganese sulfate monohydrate. At 10-50 mg / L.

The beneficial effects of the invention are:

1) The strain obtained by the present invention is Lactobacillus brevis 3-A20, and the strain collection number is CGMCC8581. The initial total glucose tolerance concentration is up to 260g/L, and in batch culture conditions, high sugar substrate concentration (total sugar concentration greater than 200g / L), Lactobacillus brevis 3-A20 mannitol yield than the parent strain Lactobacillus brevis 3 -A5 mannitol production increased by 10% - 100%.

2) The strain obtained by the present invention is Lactobacillus brevis 3-A20 at a temperature of 25-40 degrees, an initial pH of 4.0-7.0, a rotational speed of 0-100 rpm, and when the fermentation is carried out for 12-16 hours. When the pH of the fermentation broth is controlled between 4.0 and 5.0, the total sugar substrate at a concentration of 0-260 g/L can be utilized, and the production of mannitol can be carried out efficiently. The concentration of mannitol in the fermentation broth can reach 100-260 g. /L, the fermentation cycle is 2-4 days, which has important industrial application value.

DRAWINGS

Figure 1 shows the yield of the fermentation product and the sugar consumption curve at a high sugar concentration (the ratio of glucose to fructose is 1:2.17);

Figure 2 shows the yield of the fermentation product and the sugar consumption curve at a high sugar concentration (the ratio of glucose to fructose is 1:2.88);

Figure 3 shows the fermentation product formation and sugar consumption curve of Lactobacillus brevis 3-A20 in a 1 ton fermenter.

detailed description

The invention will now be further described in conjunction with the examples. The following examples are illustrative and not limiting, and the scope of the invention is not limited by the following examples.

Example 1

The fermentation steps were as follows: single colonies of Lactobacillus brevis 3-A5 and Lactobacillus brevis 3-A20 were picked from the MRS plate, respectively, and inserted into 20 mL of MRS medium, cultured at 30 degrees for 24 hours; and the bacterial solution was 2% (v/). v) Inoculation amount, which was placed in 300 mL of MRS medium and cultured at 30 degrees for 24 hours as a seed liquid.

A mixed carbon source of glucose and fructose with a total sugar concentration of 180 g/L and 260 g/L (mass ratio of glucose to fructose of 1:2.17) was used as a carbon source for fermentation to produce mannitol at a concentration of 42 g/L of corn. The pulp powder is a nitrogen source, the concentration of manganese sulfate monohydrate is 0.04 g/L, the initial pH is 5.5, the temperature is 37 degrees, and the stirring speed is 100 rpm, 10% (v/v) seed liquid is added, and the fermentation is carried out. When cultured to 16 h, the pH condition was changed to 4.5. The content of glucose, fructose and mannitol in the fermentation supernatant was determined by ion chromatography. The results are shown in Table 1. It is shown that Lactobacillus brevis 3-A20 has better mannitol production performance than Lactobacillus brevis3-A5 at high sugar concentration, and mannitol production of Lactobacillus brevis 3-A20 after 96 hours of fermentation at 260 g/L total sugar concentration. The yield is about 80% higher than that of Lactobacillus brevis 3-A5.

Comparison of Lactobacillus brevis 3-A20 and Lactobacillus brevis 3-A5 mannitol production characteristics:

Table 1 Production characteristics of Lactobacillus brevis 3-A20 and Lactobacillus brevis 3-A5 mannitol at different sugar concentrations

* The growth of the cells was inhibited, resulting in significant effects on the production of mannitol.

Example 2

A single colony of Lactobacillus brevis 3-A20 from the MRS plate was picked out and inserted into 20 mL of MRS medium and cultured at 30 degrees for 24 hours. The inoculum was inoculated at 2% (v/v) and added to 300 mL of MRS medium. It was cultured at 30 degrees for 24 hours as a seed liquid.

A mixed carbon source of glucose and fructose (glucose to fructose mass ratio of 1:2.17) with a total sugar concentration of 258 g/L was used as a carbon source for fermentation to produce mannitol, and a corn syrup having a concentration of 42 g/L was used as a nitrogen source. , the concentration of manganese sulfate monohydrate is 0.04g / L, the initial pH is 5.5, the temperature is 37 degrees, the stirring speed is 100 rpm, 10% (v / v) of the seed liquid is added, fermentation culture, when cultured to 16h, The pH condition was changed to 4.5. The content of glucose, fructose and mannitol in the fermentation supernatant was determined by ion chromatography. The changes in glucose, fructose and mannitol concentrations during fermentation are shown in Figure 1.

The results showed that when the fermentation was carried out for 60 h, the concentration of mannitol in the fermentation broth reached the highest, which was 168.82 g/L. The volumetric production intensity reached 2.81 g/L/h.

Example 3

A mixed carbon source of glucose and fructose with a total sugar concentration of 260g/L (mass ratio of glucose to fructose of 1:2.88) was used as a carbon source for fermentation to produce mannitol, and corn syrup with a concentration of 42g/L was used as a nitrogen source. , the concentration of manganese sulfate monohydrate is 0.04g / L, the initial pH is 5.5, the temperature is 37 degrees, the stirring speed is 100 rpm, 10% (v / v) of the seed liquid is added, fermentation culture, when cultured to 16h, The pH condition was changed to 4.5. The content of glucose, fructose and mannitol in the fermentation supernatant was determined by ion chromatography. The changes in glucose, fructose and mannitol concentrations during fermentation are shown in Figure 2.

The results showed that when the fermentation was carried out for 96 hours, the concentration of mannitol in the fermentation broth reached the highest, which was 172.74 g/L.

Example 4

A single colony of Lactobacillus brevis 3-A20 was picked from MRS plate and inserted into 30 mL of MRS medium and cultured at 30 degrees for 24 hours. The inoculum was inoculated at 2% (v/v) and connected to 1200 mL of MRS medium. The cells were cultured at 30 degrees for 24 hours; the inoculum was inoculated at 5% (v/v) and added to 60 L of MRS medium.

In a 1 ton fermenter, a mixed carbon source of glucose and fructose (glucose to fructose mass ratio of 1:2.88) with a total sugar concentration of 170 g/L was used as a carbon source for fermentation to produce mannitol at a concentration of 42 g/L. The corn syrup powder is a nitrogen source, the concentration of manganese sulfate monohydrate is 0.04 g/L, the initial pH is 5.5, the temperature is 37 degrees, and the stirring speed is 60 rpm. Under the condition, 10% (v/v) seed liquid was added to carry out fermentation culture. The content of glucose, fructose and mannitol in the fermentation supernatant was determined by ion chromatography. The changes in glucose, fructose and mannitol concentrations during fermentation are shown in Figure 3.

The results showed that when the fermentation was carried out for 40h, the concentration of mannitol in the fermentation broth reached the highest, which was 108.86g/L, and the volume production intensity reached 2.72g/L/h.

Claims

A strain of Lactobacillus brevis which is used for the production of mannitol. The strain is named Lactobacillus brevis 3-A20 and the strain is deposited as CGMCC8581. It was deposited on December 13, 2013 at the General Microbiology Center of the China Microbial Culture Collection Management Committee. The address is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.
A method for producing mannitol by using the mannitol-producing mannitol produced by claim 1, characterized in that Lactobacillus brevis 3-A20 has the functions of using glucose, fructose, sucrose, oligofructose and fruit. The ability to ferment mannitol by one or more carbon sources in a sugar hydrolysate.
The method according to claim 2, wherein said Lactobacillus brevis 3-A20 is used as a production strain, and one of glucose, fructose, sucrose, oligofructose and fructan hydrolyzate is used. Or two or more carbon sources are substrates, and the fermentation production of mannitol is carried out under the condition of an oxygen concentration of 0-1 mg/L, a pH of 3.0-7.0, and a temperature of 25-40 degrees.
The method according to claim 3, wherein the mass concentration of the carbon source in the substrate ranges from 5 to 40%, and the mass concentration of glucose, fructose, sucrose and oligofructose ranges from 0 to 40%.