TW202302836A - Medium for culturing methylobacillus and culture method - Google Patents

Abstract

The invention provides a culture medium and a culture method for producing pyrroloquinoline quinone by fermentation of methyl bacteria. The culture medium contains methanol of a specific concentration as the main carbon source, ammonium sulfate of a specific concentration as the main nitrogen source. In the culture method, precisely controls the pH value, rotation speed, temperature, dissolved oxygen, feed timing and replacement timing, composition, ratio and other parameters is conducive to achieve automatic control and continuous fermentation.

Description

Medium and culture method for cultivating methylobacteria

The invention relates to a culture medium component and a culture method, in particular to a culture medium and a culture method for culturing methylobacteria.

Pyrroloquinoline quinone (Pyrroloquinoline quinone, PQQ) is a redox cofactor in bacterial dehydrogenase and an antioxidant, which can avoid cell damage caused by intracellular oxidation reaction and active oxygen produced by biologically active substances in vitro. It provides nutrients and vitamins for the growth and development of the cells, and at the same time makes the cells resistant to oxidation, so PQQ is often used as a component of health food.

At present, PQQ is mainly obtained by chemical synthesis, and natural production by microorganisms has the advantages of clean, low-carbon, mild and controllable, and low cost, and is a production method with great potential. The main factors affecting the large-scale application of microbial fermentation to produce PQQ are: (1) There are not many microorganisms suitable for industrial production; (2) The production cycle of PQQ production strains is long and the yield is not high; Microbial secondary metabolites, whose synthetic regulation is limited by culture conditions and metabolic regulation.

In order to solve the above problems, the present invention provides a culture medium and a culture method capable of increasing the amount of bacteria and the yield of PQQ, so as to achieve the purpose of efficiently producing PQQ by utilizing methylobacteria.

A kind of medium for cultivating methylobacteria of the present invention, its composition comprises: 1~3% Methanol; 0.5~2.5g/L ammonium sulfate; and Wherein the pH value of the culture medium is 7-9.

Wherein, the present invention provides a method for cultivating methylobacteria using the above medium, comprising: inoculating a methylobacterium into a culture vessel containing the culture medium to form a fermentation broth; and The culture container is placed in an environment with a temperature of 30-35° C., and the culture container is shaken at a speed of 100-200 rpm for 10 days.

The present invention also provides a method for cultivating methylobacteria, comprising: Inoculating a methyl bacterium into a culture container containing a medium containing 0.5-2.5 g/L ammonium sulfate and having a pH value of 7-9 to form a fermentation broth; Place the culture container in an environment with a temperature of 30-35°C, and shake the culture container at a speed of 100-200rpm for cultivation; Adding 1-3% methanol to the fermentation broth when culturing for 4-8 days; and Continue to cultivate until 10 days.

The following embodiments are used in conjunction with the drawings to illustrate the spirit of the present invention so that those skilled in the art can clearly understand the technology of the present invention, but are not intended to limit the scope of the present invention. The patent scope of the present invention should be determined by the claims defined. It is emphasized that the drawings are for illustration only, and do not represent the actual size or quantity of components, and some details may not be fully drawn for the sake of simplicity of the drawings.

The invention provides a medium for cultivating methylobacteria, the composition of which comprises 1-3% methanol (Methanol), 0.5-2.5g/L ammonium sulfate ((NH ₄ ) ₂ SO ₄ ), 100-300mg/L magnesium sulfate (MgSO ₄ ), 0.05~2mg/L ferric chloride (FeCl ₃ ) and 1~5g/L yeast extract, the pH of the culture medium is 7~9. In a preferred embodiment, its ingredients include 2~3% methanol, 1~2g/L ammonium sulfate, 200~300mg/L magnesium sulfate, 1~2mg/L ferric chloride and 1~3g/L yeast extract.

In other embodiments, in addition to ammonium sulfate and yeast extract, the nitrogen source of the medium can be selected from one or more of the following combinations, and the concentration is 2g/L: sodium nitrate (NaNO ₃ ), ammonium nitrate ( NH ₄ NO ₃ ), meat peptone, soy peptone, calcium nitrate (CaNO ₃ ), ammonium chloride (NH ₄ Cl) and skim milk. In addition to magnesium sulfate and ferric chloride, the metal ion source of the culture medium can be selected from one or more of the following: 0.05g/L calcium chloride (CaCl ₂ ), 5 μg/L copper sulfate (CuSO ₄ ), 10μg/L manganese sulfate (MnSO ₄ ), 10μg/L sodium molybdate (Na ₂ MoO ₄ ), 10μg/L boric acid (H ₃ BO ₃ ), 70μg/L zinc sulfate (ZnSO ₄ ) and 5μg/L cobalt chloride ( _CoCl2 ).

The present invention provides a method for cultivating methylobacteria using the culture medium of any of the above embodiments, comprising inoculating methylobacteria into a culture container containing the culture medium to form a fermentation broth, and placing the culture container at a temperature of 30-35°C In the environment, shake the culture container at 100~200rpm and cultivate for 10 days.

The invention provides another method for cultivating methylobacteria, comprising inoculating methylobacteria into a culture vessel containing a culture medium containing 0.5-2.5 g/L ammonium sulfate and having a pH value of 7-9 to form a fermentation broth, and cultivating The container is placed in an environment with a temperature of 30-35 ° C, and the culture container is shaken at a speed of 100-200 rpm for cultivation. After 4-8 days of cultivation, 1-3% methanol is added to the fermentation liquid, and the cultivation is continued for 10 days.

Wherein the methyl bacteria used above can be selected from Hyphomicrobium sp. BR-1, Methylobacterium extorquens BR-2, Methylophilus methylotrophus BR-3, Methylobacterium sp. BR-4, Hyphomicrobium methylovorum BR-5, Hyphomicrobium sp. BR-6 or The combination thereof, these bacterial strains are provided by the Bioresources Preservation and Research Center of the Food Industry Development Research Institute of the Foundation.

In a preferred embodiment, alternatively, 0.3~1vvm (air volume/culture volume/min) air can be ventilated into the culture container for cultivation, so that the dissolved oxygen (Dissolved oxygen, DO) of the fermentation broth can be maintained at 20~40%.

After 10 days of culture according to any of the above culture methods, (1) replace 25-75% of the fermentation broth with new medium, and then culture for 6 days; % of the fermentation broth, and then cultivated for 12 days, wherein, optionally, 1~5g/L yeast extract was added to the fermentation broth according to the replacement cycle of 2 days. Through the cultivation method of the above embodiment, the bacterial mass (Biomass) and PQQ yield can be increased rapidly and continuously, which is suitable for large-scale production process.

The following are the experimental results of relevant parameters:

Methanol concentration:

Methylobacterium sp. BR-4 was cultured in 250mL Erlenmeyer flasks to test the yield of PQQ containing 1, 1.5, 2, 2.5, and 3% methanol concentrations. The culture conditions included 10% inoculum and 100 mL of MMS ( Methanol mineral salt) medium, 200 rpm, temperature 30°C and initial pH 7. After 10 days of cultivation, it can be seen from Figure 1-1 and Figure 1-2 that the addition of 1-3% methanol did not inhibit the growth of the bacteria, but the addition of 2-3% methanol obviously helped to increase the production of PQQ.

Ammonium sulfate concentration:

Using a 250mL Erlenmeyer flask to test the PQQ yield of Methylobacterium sp. BR-4 cultured at 0.5, 1, 1.5, 2, 2.5 g/L ammonium sulfate concentration, the culture conditions include 10% inoculation amount, MMS with a working volume of 100 mL Medium, 200 rpm, temperature 30°C and initial pH 7. After culturing for 10 days, it can be seen from Figure 2-1 and Figure 2-2 that as the concentration of ammonium sulfate increases, the growth of the bacteria increases, showing that the addition of ammonium sulfate is helpful for the growth of the bacteria, and the addition of ammonium sulfate is helpful for the growth of the bacteria. 1~2 g/L ammonium sulfate obviously contributed to the increase of PQQ production.

Magnesium Sulfate Concentration:

Using 250mL Erlenmeyer flask to test the PQQ production of Methylobacterium sp. BR-4 cultured at 100, 150, 200, 300 mg/L magnesium sulfate concentration, the culture conditions include 10% inoculum amount, MMS medium with a working volume of 100 mL , 200 rpm, temperature 30°C and initial pH 7. After culturing for 10 days, it can be seen from Figure 3-1 and Figure 3-2 that the addition of 100-300 mg/L magnesium sulfate has no inhibitory effect on the growth of the bacteria, and the addition of 200-300 mg/L magnesium sulfate will help increase the production of PQQ .

Ferric Chloride Concentration:

A 250mL Erlenmeyer flask was used to test the PQQ yield of Methylobacterium sp. BR-4 cultured at 0.05, 0.5, 1, 1.5, and 2 mg/L ferric chloride concentrations. The culture conditions included 10% inoculum and a working volume of 100 mL MMS medium, 200 rpm, temperature 30°C and initial pH 7. After culturing for 10 days, it can be seen from Figure 4-1 and Figure 4-2 that the addition of 0.05-2 mg/L ferric chloride has no inhibitory effect on the growth of the bacteria. When the addition of 1-2 mg/L ferric chloride helps PQQ Yield increased.

Rotating speed:

A 250mL Erlenmeyer flask was used to test the PQQ yield of Methylobacterium sp. BR-4 cultured at 50, 100, 150, and 200 rpm. The culture conditions included 10% inoculum, MMS medium with a working volume of 100 mL, temperature at 30°C and Initial pH 7. After culturing for 10 days, it can be seen from Figure 5-1 and Figure 5-2 that increasing the rotational speed is helpful for cell growth and PQQ production, and when 100~200 rpm is helpful for the increase of PQQ production.

temperature:

A 250mL conical flask was used to test the PQQ yield of Methylobacterium sp. BR-4 cultured at temperatures of 20, 25, 30, and 35°C. The culture conditions included 10% inoculum, MMS medium with a working volume of 100 mL, 200 rpm and Initial pH 7. After 10 days of culture, it can be seen from Figure 6-1 and Figure 6-2 that increasing the culture temperature is beneficial to the growth of bacteria, and 30~35°C is more suitable for the growth of bacteria and the increase of PQQ production.

pH value:

A 250mL Erlenmeyer flask was used to test the PQQ production of Methylobacterium sp. BR-4 cultured in the initial MMS medium pH 5, 7, 9, and 11. The culture conditions included 10% inoculum, 100 mL working volume of MMS medium at 200 rpm and 30°C. After 10 days of culture, it can be seen from Figure 7-1 and Figure 7-2 that pH 7~9 is conducive to the growth of bacteria, and the trend of PQQ production is the same as that of bacteria growth. Both are because pH 7~9 is conducive to the production of PQQ.

According to the above parameter types, the PQQ optimization condition test was carried out, and Methylobacterium sp. BR-4 was cultivated in a 250mL Erlenmeyer flask containing 2.24% methanol, 2.01 g/L ammonium sulfate, 254.8 mg/L magnesium sulfate and 1.56 mg/L chlorine MMS medium with ferric chloride, the culture conditions include 10% inoculum, MMS medium with a working volume of 100 mL, 200 rpm and initial pH 7. After 10 days of cultivation, the maximum fermentable PQQ concentration was 127.7±14 mg/L, and the error with the predicted value of PQQ yield was 5.5%.

Ventilation:

Methylobacterium sp. BR-4 was cultured in a 5L fermenter to test the PQQ yield at 0.3, 0.6, and 1 vvm ventilation. Initial pH 7. It can be seen from Figure 8-1 and Figure 8-2 that as the ventilation rate increases, the growth of the bacteria also increases, and the PQQ production is positively correlated with the growth of the bacteria, and the ventilation rate of 0.6~1 vvm has a higher PQQ production quantity.

Feed composition:

Methylobacterium sp. BR-4 was cultured in a 5L fermenter with different feed ingredients including 0.75% methanol, 0.75% methanol and ammonium sulfate, and 1.5% methanol for PQQ yield test, respectively as Figure 9-1 and Figure 9-2 As shown in Figure 9-3, the culture conditions include 10% inoculum, MMS medium with a working volume of 3 L, 0.6 vvm, 150 rpm, temperature 30°C, initial pH 7, and feed addition on the sixth day of culture. When the feed composition is 0.75% methanol, it can be clearly seen that the growth of the bacteria is lower than that of other feed ingredients, presumably because the accumulation of other metal components in the MMS medium causes the growth of the bacteria to be inhibited; when the feed composition is 0.75% methanol and ammonium sulfate, the cell growth and PQQ production are slightly lower than 1.5% methanol, obviously adding carbon source and nitrogen source are both helpful for cell growth and PQQ production, and 1.5% methanol, which is the easiest to add, is used as the feed ingredient The obtained cell growth and PQQ yield were the best.

Feed time:

Methylobacterium sp. BR-4 was carried out in a 5L fermenter with 1.5% methanol as the feed composition, and the PQQ production test was carried out under different feeding timings including the fourth day, the sixth day and the eighth day, as shown in Figure 10- 1. As shown in Figure 10-2 and Figure 10-3, the culture conditions include 10% inoculum, MMS medium with a working volume of 3 L, 0.6 vvm, 150 rpm, temperature 30°C, and initial pH 7. Regardless of the feeding on the fourth day, the sixth day, and the eighth day, there was no significant difference in the growth of the bacteria. The PQQ production was the best when feeding on the sixth day. Different feeding timings mainly caused the PQQ yield per unit of bacteria , it can be clearly seen that the PQQ production per unit cell obtained on the fourth day and the eighth day feeding timing is significantly lower than that of the sixth day feeding.

Constant pH value:

A 5L fermenter was used to test the PQQ production of Methylobacterium sp. BR-4 cultured without controlling the pH value and maintaining the pH value of 8.5 with sodium hydroxide (NaOH). Culture medium, feed 1.5% methanol, 0.9 vvm, 160 rpm and temperature 30°C on the 6th day of culture. After culturing for 10 days, it can be seen from Figure 11-1 and Figure 11-2 that maintaining the pH value at 8.5 is helpful for cell growth and PQQ production.

According to the above parameter types, a 5L-scale PQQ optimization condition test was carried out, and Methylobacterium sp. BR-4 was cultivated in a 5L fermenter. Feed 1.5% methanol, 0.9 vvm, 160 rpm and initial pH 8.5. After 10 days of cultivation, the maximum fermentable PQQ concentration was 229.5±15.2 mg/L, and the error with the predicted value of PQQ yield was 6%.

Dissolved oxygen:

A 5L fermenter was used to test the PQQ yield of Methylobacterium sp. BR-4 cultured without controlling dissolved oxygen and controlling dissolved oxygen to maintain 20 and 40%. The culture conditions included 10% inoculum, MMS medium with a working volume of 3L, On the sixth day of culture, feed was fed with 1.5% methanol, 0.9 vvm, 160 rpm and temperature 30°C. After culturing for 10 days, it can be seen from Figure 12-1 and Figure 12-2 that maintaining the dissolved oxygen at 20% is helpful for cell growth and PQQ production.

Replacement ingredients:

A 5L fermenter was used to test the PQQ yield of Methylobacterium sp. BR-4 cultured without replacing the medium and after 10 days of culture with 50% medium replaced. Daily feed 1.5% methanol, 0.9 vvm, 160 rpm, pH 8.5 and temperature 30°C. From Figure 13-1 and Figure 13-2, it can be seen that after replacing 50% of the culture medium and culturing for 6 days, the bacterial count and PQQ production before replacement can be achieved.

Replacement ratio:

A 5L fermenter was used to test the PQQ yield of Methylobacterium sp. BR-4 cultured without replacing the medium, and after 10 days of culture with 25%, 50% and 75% medium replaced. The culture conditions included 10% inoculum and a working volume of 3L MMS medium, 1.5% methanol, 0.9 vvm, 160 rpm, pH 8.5 and temperature 30°C on the 6th day of culture. From Figure 14-1 and Figure 14-2, it can be seen that after replacing 50% of the culture medium and culturing for 6 days, the bacteria amount and PQQ production before replacement can be achieved.

Replacement cycle:

Methylobacterium sp. BR-4 was cultured in a 5L fermenter. After replacing 50% of the medium on the 10th day of culture, the PQQ production test was performed by replacing 50% of the medium every 2, 4, and 6 days. The culture conditions included 10% inoculum , MMS medium with a working volume of 3L, feed 1.5% methanol, 0.9 vvm, 160 rpm, pH 8.5 and temperature 30°C on the 6th day of culture. After cultivating for 12 days according to each replacement cycle, it can be seen from Figure 15-1, Figure 15-2 and Figure 15-3 that the fewer days in the replacement cycle, the higher the bacterial count and PQQ production, and the 6-day replacement cycle can The maximum bacterial unit PQQ production was 41.8 mg PQQ/g.

Yeast Extract:

Using a 250mL Erlenmeyer flask to test the PQQ yield of Methylobacterium sp. BR-4 cultured at 0, 1, 3, 5 g/L yeast extract concentration, the culture conditions include 10% inoculation amount, MMS medium with a working volume of 100 mL , 200rpm and a temperature of 30°C. After 10 days of culture, it can be seen from Figure 16-1 and Figure 16-2 that the addition of 0-5 g/L yeast extract slightly increases the growth of the bacteria, and the addition of 1-3 g/L yeast extract helps to increase the production of PQQ.

Replacement timing, composition, ratio and cycle:

Methylobacterium sp. BR-4 was cultured in a 5L fermenter. After replacing 50% of the medium on the 10th day of culture, the PQQ production test was performed by replacing 50% of the medium every 6 days and adding 2 g/L yeast extract every 2 days. The culture conditions included 10% inoculum, MMS medium with a working volume of 3L, feeding 1.5% methanol on the 6th day of culture, 0.9 vvm, 160 rpm, constant pH8.5 and temperature 30℃. After culturing for 12 days according to the above replacement cycle, it can be seen from Figure 17 that feeding the yeast extract every 2 days and replacing 50% of the fermentation broth with the medium every 6 days can produce the maximum amount of 315.5 mg/L PQQ.

The medium components and their concentrations provided by the present invention can significantly increase the growth of methylobacteria and the production of PQQ. The present invention also provides a variety of parameter conditions to optimize and maximize the increase in the amount of bacteria and PQQ production, and can be applied to large-scale mass production processes .

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Figure 1-1 and Figure 1-2 are bar graphs showing the effect of methanol on bacterial growth and PQQ production.

Figure 2-1 and Figure 2-2 are bar graphs showing the effect of ammonium sulfate on bacterial growth and PQQ production.

Figure 3-1 and Figure 3-2 are bar graphs showing the effect of magnesium sulfate on bacterial growth and PQQ production.

Figure 4-1 and Figure 4-2 are bar graphs showing the effect of ferric chloride on bacterial growth and PQQ production.

Figure 5-1 and Figure 5-2 are bar graphs showing the effect of rotational speed on bacterial growth and PQQ yield.

Figure 6-1 and Figure 6-2 are bar graphs showing the effect of temperature on bacterial growth and PQQ production.

Figure 7-1 and Figure 7-2 are bar graphs showing the effect of pH on bacterial growth and PQQ production.

Figure 8-1 and Figure 8-2 are graphs showing the effect of air flow on bacterial growth and PQQ production.

Fig. 9-1, Fig. 9-2 and Fig. 9-3 are graphs showing the influence of feed ingredients on bacterial growth and PQQ yield.

Fig. 10-1, Fig. 10-2 and Fig. 10-3 are graphs showing the influence of feeding timing on bacterial growth and PQQ yield.

Figure 11-1 and Figure 11-2 are bar graphs showing the effect of a constant pH value on bacterial growth and PQQ production.

Figure 12-1 and Figure 12-2 are bar graphs showing the effect of dissolved oxygen on bacterial growth and PQQ production.

Fig. 13-1 and Fig. 13-2 are graphs showing the effect of replacement components on bacterial cell growth and PQQ yield.

Figure 14-1 and Figure 14-2 are graphs showing the effect of the replacement ratio on the growth of the bacteria and the yield of PQQ.

Fig. 15-1, Fig. 15-2 and Fig. 15-3 are graphs showing the effect of the replacement cycle on the growth of bacteria and the yield of PQQ.

Figure 16-1 and Figure 16-2 are bar graphs showing the effect of yeast extract on cell growth and PQQ production.

Fig. 17 is a graph showing the influence of comprehensive replacement timing, components, proportions and periods on bacterial growth and PQQ yield.

Claims (12)

A medium for cultivating methylobacteria, the composition of which comprises: 1~3% Methanol; 0.5~2.5g/L ammonium sulfate; and Wherein the pH value of the culture medium is 7-9. The culture medium as described in claim item 1, wherein the concentration of methanol is 2-3% and the concentration of ammonium sulfate is 1-2g/L. The culture medium as described in Claim 1, further comprising metal ion sources selected from one or more of the following combinations: 100-300 mg/L magnesium sulfate, 0.05-2 mg/L ferric chloride, 0.05 g/L chloride Calcium, 5 μg/L copper sulfate, 10 μg/L manganese sulfate, 10 μg/L sodium molybdate, 10 μg/L boric acid, 70 μg/L zinc sulfate and 5 μg/L cobalt chloride. The culture medium as described in Claim 1, further comprising nitrogen sources selected from one or more of the following combinations: 1~5g/L yeast extract, 2g/L sodium nitrate, 2g/L ammonium nitrate, 2g/L Meat protein, 2g/L soy protein, 2g/L calcium nitrate, 2g/L ammonium chloride and 2g/L skimmed milk. The medium as described in claim item 3 or 4, wherein the concentration of magnesium sulfate is 200-300 mg/L, the concentration of ferric chloride is 1-2 mg/L and the concentration of yeast extract is 1-3 g/L. A method utilizing the culture medium as described in claim 1 to cultivate methylobacteria, comprising: inoculating a methylobacterium into a culture vessel containing the culture medium to form a fermentation broth; and The culture container is placed in an environment with a temperature of 30-35° C., and the culture container is shaken at a speed of 100-200 rpm for 10 days. A method for cultivating methylobacteria, comprising: Inoculating a methyl bacterium into a culture container containing a medium containing 0.5-2.5 g/L ammonium sulfate and having a pH value of 7-9 to form a fermentation broth; Place the culture container in an environment with a temperature of 30-35°C, and shake the culture container at a speed of 100-200rpm for cultivation; Adding 1-3% methanol to the fermentation broth when culturing for 4-8 days; and Continue to cultivate until 10 days. The method as described in Claim 6 or 7, further comprising ventilating 0.3-1vvm of air into the culture container. The method as described in claim 8, wherein the dissolved oxygen of the fermented liquid is 20-40%. The method as described in claim 6 or 7, further comprising replacing 25-75% of the fermentation broth with a new medium after culturing for 10 days, and culturing for another 6 days. The method as described in claim 6 or 7, further comprising replacing 25-75% of the fermentation broth with a new medium according to a replacement cycle of 2, 4 or 6 days after culturing for 10 days, and culturing for another 12 days. The method as described in Claim 11, further comprising adding 1-5 g/L yeast extract to the fermentation broth every 2 days.

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