阅读说明：本技术 一种利用超声波提高细菌胞外多糖产量的方法 (Method for improving yield of extracellular polysaccharide of bacteria by using ultrasonic waves ) 是由 周文文 竺莹莹 钱超 于 2020-12-10 设计创作，主要内容包括：本发明公开了一种利用超声波处理提高细菌胞外多糖产量的方法,以多粘类芽孢杆菌PYQ1为对象,将超声波技术引入微生物发酵过程中。本发明通过研究不同生长阶段超声处理、不同超声时间和不同超声功率对产糖菌生物量和多糖产量的影响,证明使用低频超声波辅助多粘类芽孢杆菌PYQ1多糖发酵具有可行性,同时验证得到最适合多粘类芽孢杆菌PYQ1发酵的超声参数,可以有效提高其胞外多糖产量。本发明公开的超声波方法可应用于相关多糖工业生产中,具有节约生产成本,缩短生产周期,提高经济效益等优势,有良好的市场应用前景。(The invention discloses a method for improving the yield of extracellular polysaccharide of bacteria by utilizing ultrasonic treatment, which takes Paenibacillus polymyxa PYQ1 as an object and introduces an ultrasonic technology into a microbial fermentation process. According to the invention, the influence of ultrasonic treatment at different growth stages, different ultrasonic time and different ultrasonic power on the biomass of the sugar-producing bacteria and the polysaccharide yield is researched, so that the low-frequency ultrasonic wave assisted fermentation of the polysaccharide of the paenibacillus polymyxa PYQ1 is proved to have feasibility, and meanwhile, the ultrasonic parameters most suitable for fermentation of the paenibacillus polymyxa PYQ1 are verified, so that the extracellular polysaccharide yield can be effectively improved. The ultrasonic method disclosed by the invention can be applied to the industrial production of related polysaccharides, has the advantages of saving the production cost, shortening the production period, improving the economic benefit and the like, and has good market application prospect.)

1. A method for improving the yield of extracellular polysaccharide of bacteria by using ultrasonic waves is characterized by comprising the following steps:

(1) activating a paenibacillus polymyxa PYQ1 strain on a plate culture medium, selecting a viscous single colony with a wire drawing, inoculating the single colony into a seed culture medium, and performing shake-flask culture at 30-35 ℃ for 12-24 h to obtain a paenibacillus polymyxa seed solution;

(2) inoculating the seed solution obtained in the step (1) into a fermentation culture medium in an inoculation amount of 3-10% in percentage by volume, carrying out shake culture in a shaking table at the temperature of 30-37 ℃ and the rotating speed of 180-250 rpm for 2-14 h, and then carrying out ultrasonic treatment on the bacterial solution; ultrasonic treatment of fermentation medium with intensity of 30W-150W and frequency of 25kHz-50kHz is carried out at an interval of 1s-2s every 1s-3s, an ultrasonic probe is placed at a position 1cm-2cm below the liquid level to obtain stable ultrasonic radiation intensity, the ultrasonic treatment time is 15s-120s, and after treatment, the fermentation medium is continuously cultured for 36h-48h by oscillation in a shaking table.

2. The method for increasing the yield of extracellular polysaccharide of bacteria by using ultrasonic waves as claimed in claim 1, wherein the seed culture medium comprises 20g to 35g of sucrose, 5g to 10g of yeast extract, 5g to 10g of tryptone and K₂HP0₄3g to 5g, 1000mL of distilled water, and sterilizing for 15min to 20min at the temperature of 115 ℃ to 121 ℃.

3. The method for increasing the yield of extracellular polysaccharide of bacteria by using ultrasonic waves as claimed in claim 1, wherein the fermentation medium comprises sucrose 40g-60g, yeast extract 5g-8g, tryptone 5g-8g, K₂HP0₄2g to 5g, 1000mL of distilled water, and sterilizing for 15min to 20min at the temperature of 115 ℃ to 121 ℃.

4. The method for improving exopolysaccharide production by ultrasound according to claim 1, wherein the plate medium in step (1) is LB solid medium.

5. The method for improving the yield of extracellular polysaccharide by using ultrasonic waves as claimed in claim 1, wherein the ultrasonic conditions are ultrasonic frequency of 25kHz, power of 90W and ultrasonic treatment time of 45 s.

Technical Field

The invention belongs to the technical field of microbial technology and polysaccharide production, and particularly relates to a method for improving extracellular polysaccharide of bacteria by using an ultrasonic technology.

Background

Ultrasonic waves are sound waves with a frequency above 20000 hz and above the human hearing threshold, which, when propagating in a medium, produce a series of physical and chemical effects by interaction with the medium, the most important of which are mechanical effects and cavitation. Ultrasonic waves have been used primarily for cell disruption in the biological field for the first time, but with the progress of research in life sciences by researchers, in recent years, ultrasonic waves, a novel technology, has been widely used in the fields of food and biological processing at home and abroad.

In the microbial fermentation process, the fluid pressure on the surface of the microbial cell membrane can be improved by means of ultrasonic stimulation, so that the permeability of the cell membrane is increased, the secretion rate of metabolites is accelerated, the fermentation time is effectively shortened, and the yield of beneficial metabolites is improved. In traditional microbial fermentation, high-power ultrasound has been used to destroy microbial cells to release intracellular substances, and is considered as a means for killing microbes, and the existing research shows that low-frequency ultrasound has no destructive stimulation to some microbial cells, effectively improves the permeability of bacterial cell membranes and the speed of cell metabolism, and when applied to microbial liquid fermentation, is more beneficial to improving the fermentation efficiency and the yield of fermentation products. The research of Yadav and the like finds that the enzyme activities of pectinase, cellulase and xylanase are respectively improved by about 38.15%, 53.77% and 24.59% compared with the enzyme activities of a non-ultrasonic group by using low-intensity (25kHz) ultrasonic to assist the fermentation of bacillus subtilis ABDR01, and meanwhile, compared with low-frequency ultrasonic, the higher-frequency (40kHz) ultrasonic is pointed out to be not beneficial to generating cavitation bubbles, but also can cause bacterial cell rupture and improve the enzyme activity [1 ]; similar conclusions were also drawn by Roden et al, and experiments found that sonication at 40kHz and 59kHz inhibited the activity of inulinase produced by Aspergillus niger, with the higher the frequency, the more pronounced the inhibition effect [2 ]. Sulaiman et al applied 20kHz ultrasonic wave with 20% intermittent ratio during Kluyveromyces marxianus yeast fermentation, and the final fermentation obtained ethanol was 3.5 times of that of the control group, and the biomass and cell viability of the yeast were improved to some extent [3 ].

The ultrasonic-assisted microbial fermentation with low cost, easy treatment and remarkable effect has been researched for improving the yield of alcohol, biological enzyme, lactose and the like, but research objects mainly focus on yeast, mould, algae and the like, and on the premise of carrying out massive optimization on ultrasonic conditions and a culture medium, the fermentation process of a large number of microorganisms belonging to the three types still shows that the microorganisms are insensitive to ultrasonic waves, or the ultrasonic waves show that the microorganisms have an inhibition effect on the fermentation process, or the ultrasonic waves still show that the microorganisms still have an obvious killing and damaging effect on microbial cells under low frequency. Therefore, the ultrasonic treatment method, treatment intensity, treatment effect, mechanism and the like of different kinds of microorganisms are different. The research of improving the yield of the extracellular polysaccharide of the paenibacillus polymyxa by using low-frequency ultrasonic treatment is not available, and the intensive research of researchers needs to be urgently awaited, so that the ultrasonic technology is better applied to the fermentation production of the sugar-producing bacteria.

Reference documents:

[1]Yadav A,Ali AAM,Ingawale M,et al.Enhanced co-production of pectinase,cellulase and xylanase enzymes from Bacillus subtilis ABDR01 upon ultrasonic irradiation[J].Process Biochemistry,2020,92:197-201.

[2] influence of ultrasound on inulinase production by aspergillus niger [ J ] acoustic techniques, 2012,31: 502-.

[3]Sulaiman AZ,Ajit A,Yunus RM,et al.Ultrasound-assisted fermentation enhances bioethanol productivity[J].Biochemical Engineering Journal,2011,54:141-150.

Disclosure of Invention

The invention aims to provide a method for improving yield of Paenibacillus polymyxa exopolysaccharide by utilizing ultrasonic waves, which can efficiently produce and obtain exopolysaccharide with wide application prospect and play a reference role in actual production and application of Paenibacillus polymyxa polysaccharide in future.

The invention is realized by the following technical scheme.

The Paenibacillus polymyxa strain is preserved in China general microbiological culture collection center with the preservation number of CGMCC No.16444 and the preservation date of 2018, 9 months and 10 days, and has the preservation name of Paenibacillus polymyxa PYQ 1. Said paenibacillus polymyxa strain PYQ1 is disclosed in patent ZL 201811216217.0.

A method for improving the yield of extracellular polysaccharide of bacteria by using ultrasonic waves comprises the following steps:

(1) activating a paenibacillus polymyxa strain on a plate culture medium, then inoculating the activated strain into a seed culture medium, and performing shake-flask culture at 30-35 ℃ for 12-24 h to obtain a paenibacillus polymyxa seed solution;

(2) inoculating the seed solution obtained in step (1) into a fermentation culture medium at an inoculation amount of 3-10% by volume, performing shaking culture in a shaking table at 30-37 deg.C and rotation speed of 180-250 rpm for 2-14 h, and performing ultrasonic radiation treatment on the bacterial solution (for example, an ultrasonic cell crusher can be used). Treating the fermentation medium with intensity of 30-150W and frequency of 25-50 kHz, with ultrasonic period of 1.0-3.0s-ON and 1.0-2.0 s-OFF. The ultrasonic probe is placed at a distance of 1-2 cm below the liquid level to obtain stable ultrasonic radiation intensity, and the ultrasonic treatment time is 15-120 s. After treatment, the shaking culture is continued in the shaking table for 36h-48 h.

Preferably, the seed culture medium comprises 20g to 35g of sucrose, 5g to 10g of yeast extract, 5g to 10g of tryptone and K₂HP0₄3g to 5g, 1000mL of distilled water, and sterilizing for 15min to 20min at the temperature of 115 ℃ to 121 ℃.

Preferably, the fermentation medium comprises 40g to 60g of sucrose, 5g to 8g of yeast extract powder, 5g to 8g of tryptone and K₂HP0₄2g to 5g, 1000mL of distilled water, and sterilizing for 15min to 20min at the temperature of 115 ℃ to 121 ℃.

The fermentation broth can be used for polysaccharide extraction in the following way: centrifuging the fermentation liquor to remove thallus to obtain supernatant, deproteinizing the supernatant for 4-6 times, and adding absolute ethanol to precipitate polysaccharide; and (3) redissolving the precipitated polysaccharide, dialyzing to remove small molecular substances, and freeze-drying to obtain the polysaccharide. The dialysis step in the above process can adopt 3500D dialysis bag, firstly dialyzing with running water for 24h, then dialyzing with distilled water for 48h, and changing distilled water every 8 h. The invention improves the yield of the extracellular polysaccharide by a proper ultrasonic means without increasing the extraction difficulty and the extraction cost of the polysaccharide; the extracellular polysaccharide with high purity can still be obtained by adopting the extraction method.

The invention has the beneficial effects that:

the invention creatively improves the yield of the paenibacillus polymyxa exopolysaccharide with the assistance of low-frequency ultrasonic waves, finally obtains the ultrasonic parameters (ultrasonic treatment growth stage, treatment time and treatment power) most suitable for the production of the paenibacillus polymyxa exopolysaccharide through optimizing a single-factor experiment one by one, and better understands the effect of the ultrasonic waves in the microbial fermentation process. The low-frequency ultrasonic wave assisted fermentation of the bacillus polysaccharide is proved to be feasible, and the ultrasonic wave with proper intensity can well enhance the permeability of cell membranes and absorb nutrients for a certain time, thereby effectively improving the biomass of thalli and the yield of fermentation products, regulating the carbon metabolic flow and inhibiting the generation of impurities. The invention is applied to the industrial production of related bacterial polysaccharides, and has the advantages of saving production cost, shortening production period, improving economic benefit and the like.

Drawings

FIG. 1 the results of the present invention using ultrasound on the biomass and polysaccharide production of P.polymyxa PYQ1 at different growth stages;

FIG. 2 shows the effect of different sonication times on P.polymyxa PYQ1 biomass and polysaccharide yield;

fig. 3 the results of the effect of different ultrasonic powers used in the present invention on the biomass of p.polymyxa PYQ1 and the yield of polysaccharide;

FIG. 4 is a graph showing the effect of P.polymyxa PYQ1 on growth and fermentation for sugar production under optimal ultrasonic parameters;

FIG. 5 Effect of sonication on exopolysaccharide production by different Bacillus strains.

Detailed Description

The following examples of the present invention are described in detail, but the following examples are not intended to limit the scope of the present invention.

Before polysaccharide fermentation, a culture medium is prepared, and a seed solution is prepared. The culture medium used includes LB solid medium, seed medium and fermentation medium.

LB solid medium: 5g of yeast extract powder, 10g of tryptone, 10g of sodium chloride, 18g of agar and 1000mL of distilled water, sterilizing at 121 ℃ for 15min, and pouring the mixture into a flat plate.

Seed culture medium: 30g of cane sugar, 10g of yeast extract powder, 5g of tryptone and K₂HP0₄3g, distilled water 1000mL, sterilizing at 121 ℃ for 15 min.

Fermentation medium: 50g of cane sugar, 5g of yeast extract powder, 5g of tryptone and K₂HP0₄3g, distilled water 1000mL, sterilizing at 121 ℃ for 15 min.

Activating the strain of Paenibacillus polymyxa on a plate culture medium, inoculating the activated strain in a seed culture medium, and performing shake-flask culture at 30 ℃ for 24h to obtain a Paenibacillus polymyxa seed solution.

Example 1: ultrasonic assisted fermentation experiment

6% seed bacterial liquid was added to 25mL of a fermentation medium (initial sucrose addition amount: 50g/L), and the bacterial liquid was subjected to ultrasonic irradiation treatment using an ultrasonic cell disruptor. The fermentation medium was irradiated with intermittent (periodic ultrasonic irradiation) conditions of intensity and frequency 25kHz for periods of 1.0s-ON and 1.0 s-OFF. The ultrasound probe was placed at a distance of 1cm from the surface of the medium to obtain a stable ultrasound radiation intensity and a temperature of 28 ℃ was maintained in all experiments. And evaluating the influence of three treatments of ultrasound, ultrasound time and ultrasound power on the fermentation of the paenibacillus polymyxa PYQ1 in different growth stages by adopting a single-factor method.

Measurement of cell biomass: the OD value of the bacterial suspension is measured by a microplate reader according to the method of the existing literature.

Determination of extracellular polysaccharide content: and (3) determining the sugar content of the fermentation liquor by using a phenol-sulfuric acid colorimetric method.

1) Ultrasonic treatment at different growth stages

According to the growth curve of the Paenibacillus polymyxa PYQ1, the bacteria liquid grown to the early stage (1h) of the lag phase, the late stage (9h) of the lag phase, the early stage (14h) of the logarithmic phase, the middle stage (24h) of the logarithmic phase and the late stage (32h) of the logarithmic phase are respectively subjected to ultrasonic treatment with the power of 75W for 60s, and two groups of parallel treatment are set. Then cultured for 36h in a shaker at 28 ℃ and 180rpm, and the polysaccharide content and the OD value of the bacterial suspension are measured to obtain the result shown in figure 1. The figure shows that after the thalli at different growth stages are subjected to ultrasonic treatment, the extracellular polysaccharide yield of the thalli is improved to a certain extent, wherein the optimization effect at the late lag phase is optimal, the final biomass of the thalli is 2.56g/L, the extracellular polysaccharide yield is 5.23g/L, and the extracellular polysaccharide yield is improved by 23.98% compared with that of a non-ultrasonic group. Therefore, the fermentation liquor growing to the late stage of the lag phase is selected for subsequent experiments.

2) Different duration ultrasonic treatment

Selecting the bacteria liquid which grows to the optimal ultrasonic treatment growth stage, carrying out ultrasonic treatment for 15s, 30s, 45s, 60s, 75s, 90s and 120s respectively, setting the ultrasonic power at 75W, and arranging two groups in parallel. After treatment, the cells were cultured for 36 hours at 28 ℃ on a shaker at 180rpm, and the polysaccharide content and OD of the bacterial suspension were measured, and the results are shown in FIG. 2. As can be seen from FIG. 2, when the ultrasonic treatment time is within the range of 15-60s, the growth and fermentation of Paenibacillus polymyxa PYQ1 are promoted to a certain extent, the promotion effect shows a trend of ascending first and then descending, the ultrasonic treatment time of 45s has the optimal fermentation effect on Paenibacillus polymyxa PYQ1, the final yield of the treated extracellular polysaccharide is 4.92g/L, and the yield is increased by 19.31% compared with that of a control group.

3) Different intensity sonication

Selecting the bacteria liquid which grows to the optimal ultrasonic treatment growth stage, carrying out ultrasonic treatment for 60s with the power of 30W, 60W, 90W, 120W and 150W respectively, and arranging two groups of parallel bacteria liquid. After treatment, the cells were cultured for 36 hours at 28 ℃ on a shaker at 180rpm, and the polysaccharide content and OD of the bacterial suspension were measured, and the results are shown in FIG. 3. From the results, 30W to 150W of ultrasonic waves promoted the growth and exopolysaccharide synthesis of Paenibacillus polymyxa PYQ1 to various degrees. When the ultrasonic power is increased, the biomass and extracellular polysaccharide yield trend of the paenibacillus polymyxa is basically increased and then decreased, wherein the extracellular polysaccharide yield is the highest and is 5.34g/L when the ultrasonic power is 90W, and is increased by 26.79 percent compared with the non-ultrasonic group.

4) Total sugar change in the media after sonication

The initial total sugar concentration of the fermentation medium used in the invention is 61.1 +/-1.4 g/L, the residual sugar concentration of the medium after fermentation is 6.7 +/-0.4 g/L in a control group, and the residual sugar content of the medium after ultrasonic treatment (25kHz, 90W and 45s) is 6.5 +/-0.9 g/L, and has no significant difference. The total energy of the fermentation process system is conserved, the residual sugar (residual carbon source) of the culture medium is not changed too much after the fermentation is finished, but the amount of the extracellular polysaccharide synthesized by the fermentation is increased, the cell amount is also increased (figure 3), which shows that the carbon source metabolism for other branches in the cell is reduced after the ultrasonic treatment, and the amount of the synthesized impurities is also reduced.

Example 2: verification of ultrasonic wave treatment effect under optimal ultrasonic parameters

According to the results of example 1, fermentation production was performed by selecting a fermentation medium with sucrose addition of 170g/L (other fermentation medium conditions were not changed), and the optimal ultrasonic conditions were used to assist fermentation, and the control group was not subjected to ultrasonic treatment. Then culturing the strain in a shaker at 28 ℃ and 180rpm for 36h, and determining the polysaccharide content and the OD value of the bacterial suspension, wherein the experimental result is shown in figure 4. After ultrasonic treatment with the frequency of 25kHz and the power of 90W for 45s, the yield of the extracellular polysaccharide of the paenibacillus polymyxa PYQ1 is increased from 11.20g/L to 14.99g/L, the increasing rate is 33.79%, meanwhile, the biomass of the bacteria is also obviously increased, and a new way is opened up for improving the fermentation production capacity of the strain PYQ 1.

Example 3: sugar production effect of ultrasonic wave treatment of different bacillus strains under optimal ultrasonic parameters

According to the results of example 1, the fermentation production is carried out by selecting sucrose addition amount of 170g/L (other fermentation medium conditions are not changed), and the optimal ultrasonic conditions are adopted to assist the fermentation, and then the fermentation is carried out in a shaker at 28 ℃ and 180rpm for 36 h. Bacillus strains, namely Paenibacillus PYQ1, Paenibacillus HT16, Bacillus PYQ12 and Bacillus 50-3 are selected as starting strains, and the sugar yield of ultrasonic treatment and normal fermentation conditions is tested. The results of the polysaccharide content test are shown in FIG. 5. After ultrasonic treatment with the frequency of 25kHz and the power of 90W for 45s, the extracellular polysaccharide yield of the strain PYQ1 is increased to 14.99g/L, which is increased by 33.79% compared with an untreated group; after sonication, exopolysaccharide production by strains HT16 and PYQ12 did not change significantly relative to the untreated group; exopolysaccharide production by strain 50-3 was reduced 80.63% relative to the untreated group. It can be seen that different bacillus strains differ in their tolerance to sonication. Under the same ultrasonic treatment condition, the strain can generate different stress reactions, and different influences and effects can be generated on the yield of the exopolysaccharide.