JPH0817696B2 - Method for obtaining breeding strains with excellent osmotic pressure resistance - Google Patents

Description

TECHNICAL FIELD The present invention relates to a microorganism used in the fermentation industry and the like, and more specifically, a microorganism used in a fermentation process using a fermentation raw material having a high sugar concentration or a high salt concentration. The present invention relates to a breeding method for imparting osmotic resistance to.

[Prior Art and Problems Thereof] Currently, in the industrial fields such as alcohol fermentation and amino acid fermentation, molasses is generally used as a fermentation raw material, and yeast and bacteria are used as fermentation microorganisms. Further, some of these microorganisms, particularly bacteria, have low alcohol resistance and salt resistance, and it is difficult to carry out stable continuous fermentation operation for a long period of time, which causes a problem in practical use. However, a breeding method capable of efficiently imparting alcohol tolerance and salt tolerance to such microorganisms in a short period of time has not yet been established.

Generally, as a method for breeding microorganisms, mutation treatment,
Although techniques such as habituation, gene manipulation, and cell fusion have been adopted, many genes are considered to be involved in osmotic resistance such as alcohol resistance and salt tolerance, and a single gene or a small number of genes are It is difficult to obtain a desired strain by targeted mutation treatment or genetic manipulation. Also,
Acclimation has the drawback that breeding takes a long time.
Furthermore, cell fusion is a useful means for modifying / improving traits involving a large number of genes in eukaryotes such as yeasts, molds and higher plants, as many studies show. , There are no successful cases of applying this to prokaryotes such as bacteria.

The present inventors, in the process of applying cell fusion to impart alcohol resistance and salt tolerance to Zymomonas, which is an alcohol-fermenting bacterium, by electrically stimulating Zymomonas bacterium to give alcohol to the bacterium. As a result of intensive research, they found that the osmotic pressure resistance such as tolerance and salt resistance increases, and as a result, they completed the present invention.

[Means for Solving Problems] From the above viewpoints, the present invention provides an osmotic pressure resistance that enables a continuous fermentation operation to be performed for a long period by charging a raw material having a high salt concentration such as molasses at a high concentration. It is intended to provide a method for obtaining an excellent breeding strain.

That is, the present invention, by applying electrical stimulation to bacteria of the genus Zymomonas, which is an alcohol-fermenting bacterium,
A method for obtaining a breeding strain excellent in osmotic pressure resistance, which is characterized by imparting alcohol resistance and salt resistance to the bacterium.

A typical example of the electrical stimulation given to the bacteria is a high voltage DC pulse. When a high-voltage DC pulse is used, the processing conditions are, for example, pulse voltage: 1-10 kV / cm, processing time: 10-50 μsec. Number of processing times: 1 to several times. However,
These values are examples and do not limit the present invention.

The breeding method according to the present invention will be described in more detail. First, bacteria of the genus Zymomonas are converted into spheroplasts, that is, protoplasts, and preferably two types (one of which has alcohol resistance and salt tolerance) are spheroplasts. Is mixed, and a high-voltage direct current pulse is applied to the resulting mixture, preferably after centrifugation, and then spheroplasts are regenerated into bacilli (cells).

In this way, by applying the electrical stimulation treatment as described above to the bacteria of the genus Zymomonas, at a higher frequency than the untreated strain,
A breeding strain having markedly improved osmotic pressure resistance such as alcohol resistance and salt resistance can be obtained.

EFFECTS OF THE INVENTION According to the present invention, a breeding strain whose osmotic resistance such as alcohol tolerance and salt tolerance is markedly improved by applying electrical stimulation to Zymomonas bacteria is more frequently compared to the untreated strain. Obtainable. Therefore, by using the breeding strain thus obtained having excellent osmotic resistance such as alcohol resistance and salt resistance as a microorganism for fermentation, a raw material with a high salt concentration such as molasses can be charged at a high concentration for long-term stable continuous fermentation. It becomes possible to drive.

[Embodiment] Next, the present invention will be specifically described based on the illustrated embodiment.

a) Acquisition of breeding strain Alcoholic fermentative bacteria [Zymomonas mobilis HS
RM for ZM 1010 (Microtechnological Research Institute No. 10821)] and [Zymomonas HSZM1087 (Microtechnical Research Institute No. 10822)]
Incubate for 24 hours at 30 ℃ using medium (Note 1),
After inoculating the medium (Note 2), it was cultured at 30 ° C for 15 hours. The culture solution was inoculated again into the T medium (Note 2), and cultured at 30 for 7 hours.
After collecting the cells, the cells were suspended in S medium (Note 3) so that the number of cells would be 10 ⁸ cells / ml, and penicillin G potassium aqueous solution (400 U / m
l) was added, and the cells were collected after culturing at 30 ° C for 15 hours. This is E
Two types of spheroplasts were prepared by suspending them in the f solution (Note 4).

The thus prepared HSZM1010 spheroplast suspension and HSZM1087 spheroplast suspension were prepared in the same amount (10 ⁸ /
After mixing, the mixture was spun down and subjected to high-voltage DC pulse treatment (pulse voltage: 3-7 kV / cm, treatment time: 30 μs, treatment number: 2 times).

Each of the above electric pulse treatment liquids was added to S medium (Note 3)
And cultivated at 30 ° C. for 4 days to regenerate spheroplasts into bacilli (viable cells). In this way, the electric pulse treatment strain [Zymomonas HSZM 1119
No.)] was obtained.

b) Osmotic pressure tolerance test i Ethanol tolerance The electric pulse treated strain regenerated by the above operation was smeared on a sucrose agar medium (Note 5) supplemented with ethanol at a predetermined concentration and cultured at 30 ° C for 6 days. The colonies formed on the agar medium were isolated, inoculated into RM medium (Note 1), and cultured at 30 ° C for 24 hours. Each culture solution was inoculated into RM medium (Note 1) containing 5 V / V% ethanol and cultured at 30 ° C for 28 hours. By measuring the absorbance (OD ₆₆₀ ) of this culture solution, the growth degree of this breeding strain was examined, and the alcohol tolerance of the strain was evaluated.

The alcohol resistance was also evaluated by measuring the absorbance of the culture medium for the strains obtained by the same procedure as above except that the electric pulse treatment was not performed.

Examples of evaluation test results of these strains are shown in Table 1 and Table 1.
Shown in the figure. As is clear from these results, the electric pulse-treated strain showed a marked increase in absorbance from the culture time of about 8 hours, that is, an improvement in growth rate. On the other hand, the absorbance of the untreated strain increased only slightly with time. From this, it is understood that the above-mentioned electric pulse-treated strains can grow without trouble even if the culture solution contains ethanol and have excellent alcohol tolerance.

ii Salt Tolerance Each of the isolated electric pulse-treated strains was inoculated into an RM medium (Note 1) and cultured at 30 ° C for 24 hours. Each culture
It was inoculated into RM medium (Note 1) containing 0.8% sodium chloride, and cultured at 30 ° C for 32 hours. Absorbance (OD
₆₆₀ ) to measure the degree of growth of this breeding strain and evaluate the salt tolerance of the strain.

Also, the salt resistance of the strain obtained by the same procedure as above except that the electric pulse treatment was not performed was also evaluated by measuring the absorbance of the culture solution.

Examples of evaluation test results of these strains are shown in Table 2 and Table 2.
Shown in the figure. As is clear from this result, the strain treated with electric pulse showed a remarkable increase in the absorbance, that is, an improvement in the growth rate after about 12 hours of culture. In contrast, the absorbance of the untreated strain did not increase so much with time. From this, it can be seen that the above-mentioned electric pulse-treated strain can grow without trouble even if the culture solution contains sodium chloride and has excellent salt tolerance.

(Note 1) RM medium glucose 20g / l yeast extract 10g / l 1 potassium phosphate 2g / l (Note 2) T medium glucose 100g / l yeast extract 10g / l 1 potassium phosphate 10g / l ammonium sulfate 1g / l magnesium sulfate・ Hydrate 0.5g / l (Note 3) S medium Glucose 20g / l Yeast extract 10g / l Monopotassium phosphate 2g / l Magnesium sulfate ・ Hydrate 2g / l Sucrose 200g / l (Note 4) Ef medium Sodium chloride 4g / l Magnesium chloride 1g / l Calcium chloride 0.8g / l Sorbitol 160g / l 10mM Tris-HCl buffer (pH7.2) (Note 5) Sucrose agar sucrose 20g / l Yeast extract 10g / l Phosphorus Acid 1 potassium 2g / l Agar 15g / l

[Brief description of drawings]

FIG. 1 shows the time course of both strains in the presence of 5 V / V% ethanol, in order to compare the alcohol tolerance between the electric pulse treated strain and the electric pulse non-treated strain bred by the method shown in the example of the present invention. 3 is a graph showing the specific growth rate by absorbance (OD ₆₆₀ ). In addition, FIG. 2 shows the comparison of salt tolerance between the electric pulse treated strains and the electric pulse non-treated strains bred by the method shown in the example of the present invention, in the presence of 0.8% sodium chloride. Absorbance over time (OD ₆₆₀ )
It is the graph shown by.

Claims (1)

[Claims] 1. A method for imparting alcohol tolerance and salt tolerance to a bacterium of the genus Zymomonas, which is an alcohol-fermenting bacterium, by imparting electrical resistance to the bacterium.
A method for obtaining a breeding strain having excellent osmotic pressure resistance.