JPH03123482A - Preparation of breeding stock having excellent osmotic pressure resistance - Google Patents

Abstract

PURPOSE:To obtain the subject breeding stock having excellent osmotic pressure resistance and capable of stably performing continuous fermentation of a charged raw material having high concentration and high salt content such as black strap molasses over a long period by applying electric shock to a microorganism, thereby imparting osmotic pressure resistance to the microorganism. CONSTITUTION:The objective breeding stock can be produced by applying electric shock to a microorganism, thereby imparting osmotic pressure resistance to the microorganism. Concretely, two kinds of microorganisms, one of which has alcohol resistance and salt resistance, are converted to protoplast, two kinds of the resultant spheroplasts are mixed with each other, the mixture is subjected to centrifugal precipitation, high-voltage DC pulse is applied and preferably further the treated spheroplasts are regenerated to bacilli.

Description

[Detailed Description of the Invention] [Industrial Application Field] This invention relates to microorganisms used in the fermentation industry and other fields, and more specifically to microorganisms used in fermentation processes using fermentation raw materials with high sugar or salt concentrations. This invention relates to a breeding method for imparting osmotic resistance to.

[Prior Art and its Problems] Currently, in industrial fields such as alcohol fermentation and amino acid fermentation, blackstrap molasses is generally used as a fermentation raw material, and yeast and Nocutilia are used as fermentation microorganisms. And some of these microorganisms, especially bacteria,
Some have low alcohol tolerance and low salt tolerance, making it difficult to operate stable continuous fermentation for a long period of time, which poses a practical problem. However, a breeding method that can efficiently impart alcohol tolerance or salt tolerance to such microorganisms in a short period of time has not yet been established.

In general, methods for breeding microorganisms include mutation treatment, acclimatization, genetic manipulation, and cell fusion, but many genes are involved in osmotic tolerance such as alcohol tolerance and salt tolerance. Therefore, it is difficult to obtain a desired strain by mutation treatment or genetic manipulation that targets a single or a small number of genes.

Additionally, acclimatization has the disadvantage that breeding requires a long time. Furthermore, in eukaryotes such as yeast, molds, and higher plants, cell fusion is a useful means for modifying and improving traits that involve a large number of genes, as shown by many research examples. However, there have been no successful cases of applying this to prokaryotes such as bacteria.

In the process of applying cell fusion to impart alcohol tolerance and salt tolerance to Zymomonas bacteria, which is an alcohol-fermenting bacterium, the present inventors applied electrical stimulation to Zymomonas bacteria to produce the same amount of alcohol. They discovered a phenomenon in which osmotic pressure resistance such as tolerance and salt tolerance increases, and as a result of intensive research, they completed this invention.

[Means for solving the problem] From the above-mentioned viewpoint, the present invention has developed an osmotic pressure resistance that enables stable continuous fermentation operation for a long period of time by charging a raw material with a high salt concentration such as molasses at a high concentration. The purpose of this method is to provide a method for obtaining excellent breeding strains, and the feature is that by applying electrical stimulation to microorganisms, the microorganisms are given osmotic resistance such as alcohol tolerance and salt tolerance. do.

Examples of microorganisms to which the method of the present invention can be applied include bacteria, filamentous fungi, and yeast. A typical example of electrical stimulation given to microorganisms is a high voltage direct current pulse. When using a high-voltage DC pulse, the processing conditions are, for example, pulse voltage: 1-10 kV/cm, processing time: 10-50 μsec, and number of processing times: 1-several times.

However, these values are illustrative and do not limit the invention.

To explain this in more detail, the breeding method according to the present invention first converts microorganisms into spheroplasts, that is, protoplasts, and preferably mixes two types of spheroplasts (one of which has alcohol tolerance or salt tolerance). , preferably by applying a high-voltage direct current pulse to the resulting mixture after centrifugation, and then regenerating the spheroplasts into bacterium (cells).

By subjecting microorganisms to the electrical stimulation treatment described above, breeding strains with significantly improved osmotic resistance such as alcohol tolerance and salt tolerance can be obtained more frequently than untreated strains.

[Effects of the Invention] According to the present invention, by applying electrical stimulation to microorganisms, breeding strains with significantly improved osmotic resistance such as alcohol tolerance and salt tolerance can be obtained more frequently than untreated strains. can. Therefore, by using the thus obtained breeding strains with excellent osmotic pressure tolerance such as alcohol tolerance and salt tolerance as fermentation microorganisms, raw materials with high salt concentrations such as blackstrap molasses can be charged at high concentrations for stable continuous fermentation over a long period of time. It becomes possible to drive.

[Examples] Next, the present invention will be specifically described based on illustrated examples.

a) Obtaining breeding strains Alcohol-fermenting bacteria [Zymomonas mobilis H5
ZM 1010 (Feikoken Bacterium No. 10821)] and [Zymomonas HSZM1087 (Feiken Bacterial Serial No. 10822)] were each cultured at 30°C for 24 hours using RM medium (Note 1), and the culture solution was inoculated into T medium (Note 2) and cultured at 30°C for 15 hours. The culture solution was again inoculated into T medium (Note 2) and cultured at 30°C for 7 hours.

After collecting the bacteria, suspend them in S medium (Note 3) so that the number of bacteria becomes 108 cells/m /m, add penicillin G potassium aqueous solution (400 U/ml), and culture at 30°C for 15 hours. Bacteria were collected. This is suspended in Ef warm solution Note 4),
Two types of spheroplasts were prepared.

The thus prepared HS ZMI 010 spheroplast suspension and the H82M1087 spheroplast suspension were mixed in equal amounts (108 pieces/m/m), centrifuged, and subjected to high-pressure DC pulse treatment (pulse voltage: 3 -7 kV/cm, processing time: 230 μsec, 6 processing times: 2 times).

Each of the above electrical pulse treatment solutions was inoculated into S medium (Note 3), and cultured at 30° C. for 4 days to regenerate spheroplasts into rods (live bacteria).

Thus, the electric pulse treated strain [Zymomonas eH3ZM
1119 (Feikoken Bibori No. 10823) was obtained.

b) Osmotic pressure tolerance test l Ethanol tolerance The electric pulse treatment method regenerated by the above procedure was applied to a sucrose agar medium to which a prescribed concentration of ethanol was added (
Note 5) and cultured at 30°C for 6 days. Colonies generated on the agar medium were isolated, inoculated into RM medium (Note 1), and cultured at 30°C for 24 hours. 5μ/ of each culture solution
Inoculated into RM medium (Note 1) containing V% ethanol,
The cells were cultured at 0°C for 28 hours. The absorbance of this culture solution (OD6
60) to examine the growth rate of this breeding strain and evaluate the alcohol tolerance of the strain.

In addition, the alcohol tolerance of bacterial strains obtained by the same procedure as above except that electric pulse treatment was not performed was similarly evaluated by measuring the absorbance of the culture solution.

Examples of evaluation test results for these strains are shown in Table 1 and Figure 1. As is clear from these results, the electric pulse treatment method showed a significant increase in absorbance, that is, an improvement in growth rate, starting from about 8 hours of culture time. In contrast, the absorbance of the untreated strain increased only slightly over time. From this, it can be seen that the electric pulse treatment method described above allows for growth without any problem even if the culture solution contains ethanol, and has excellent alcohol tolerance.

11 Salt tolerance In addition, the isolated electric pulse treatment method was applied to RM medium (
Note 1) and cultured at 30°C for 24 hours. Each culture solution was inoculated into RM medium (Note 1) containing 0.8% sodium chloride, and cultured at 30°C for 32 hours. The growth rate of this breeding strain was examined by determining the absorbance (OD66o) of this culture solution at 7 fgl, and the salt tolerance of the strain was evaluated.

In addition, the salt tolerance of the bacterial strains obtained by the same procedure as above except that the electric pulse treatment was not performed was similarly evaluated by measuring the absorbance of the culture solution.

Examples of evaluation test results for these strains are shown in Table 2 and Figure 2. As is clear from these results, the electric pulse treatment method showed a significant increase in absorbance, that is, an improvement in the growth rate after about 12 hours of culture time. In contrast, the absorbance of the untreated strain did not increase significantly over time. From this, it can be seen that the electric pulse treatment method described above allows for growth without any problem even if the culture solution contains sodium chloride, and has excellent salt tolerance.

Table 1 Yeast Extract Monopotassium Phosphate (Note 2) T Medium Glucose Yeast Extract Monopotassium Phosphate Ammonium Sulfate Magnesium Sulfate 10g // 2 g // Heptahydrate too g // 10g/1 10g/ 1 1g/ 1 0.5 g/ / Table 2 (Note 3) S medium Glucose yeast extract Monopotassium phosphate Magnesium sulfate Sucrose heptahydrate 20 g // log // 2 g // 2 g // 200 g/ / (Note 1) RM medium Glucose 20 g/l (Note 4) Ef medium Sodium chloride Magnesium hydrochloride 4 g/1 1 g // Calcium chloride 0.8 g // Sorbitol 160 g/110 IUM) Liss-HCl buffer (p) 17.2) Growth over time Absorbance (O
D660).

that's all (Note 5) Sucrose agar medium

Claims (1)

[Claims] A method for obtaining a breeding strain with excellent osmotic resistance, which is characterized by imparting osmotic resistance to microorganisms by applying electrical stimulation.