Method for screening aspergillus terreus strains by combining ultrasound with xylose pressure

Abstract

The invention belongs to the technical field of aspergillus terreus strain screening, and particularly discloses a method for screening aspergillus terreus strains by combining ultrasound with xylose pressure. The method is used for solving the problems that a traditional aspergillus terreus screening method is low in efficiency, and high-yield strains are difficult to obtain. Comprising the following steps: activating an aspergillus terreus strain to prepare a spore suspension; placing the spore suspension in an ultrasonic cleaning machine for ultrasonic treatment; then uniformly coating a xylose pressure plate with the concentration of 40g / L-60g / L with the spore suspension, and culturing until single colonies grow; picking single colonies, inoculating the single colonies into a bromomethyl green indicator culture medium, and culturing; selecting strains with obvious discoloration degree and inoculating the strains on a test tube slope to obtain mature spores. The method is beneficial to obtaining strains with higher production performance, and is simple to operate, low in cost and high in screening efficiency; after primary screening and secondary screening, the acid yield is stable, hyphae are thick and strong and are not easy to break, and subsequent production of the hyphae as dominant strains is facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of Aspergillus terreus strain screening, and in particular relates to a method for screening Aspergillus terreus strains by combining ultrasound with xylose pressure. Background Art

[0002] Aspergillus terreus is an important industrial microorganism, widely used in the production of enzymes, organic acids, antibiotics and other biological products. As an industrial strain, Aspergillus terreus has excellent performance and is not prone to mutation after multiple generations. As the market demand for itaconic acid increases year by year, using Aspergillus terreus as the starting strain for fermentation and increasing the yield of itaconic acid has become a topic of great concern. Traditional screening methods for Aspergillus terreus are inefficient, and it is difficult to obtain high-yield strains. Therefore, the development of an efficient screening method for Aspergillus terreus strains is of great significance for improving the acid production performance of Aspergillus terreus. Summary of the invention

[0003] The purpose of the present invention is to provide a method for screening Aspergillus terreus strains by combining ultrasound with xylose pressure, which effectively solves the problem that the traditional screening method for Aspergillus terreus is low in efficiency and difficult to obtain high-yield strains.

[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is:

[0005] A method for screening Aspergillus terreus strains by combining ultrasound with xylose pressure comprises the following steps: S1, activating the Aspergillus terreus strains and preparing a spore suspension.

[0006] S2. Place the spore suspension in an ultrasonic cleaning machine with an ultrasonic power of 400 W, an ultrasonic frequency of 40 kHz, and an ultrasonic action time of 10 to 30 min.

[0007] S3. After the ultrasound is completed, the spore suspension is evenly spread on a xylose pressure plate with a concentration of 40g / L to 60g / L. The culture condition is pH 5.5 and placed in a constant temperature incubator at 30°C until a single colony grows.

[0008] S4. Pick a single colony and inoculate it into bromomethyl green indicator medium and culture it at 30℃ for 6 days.

[0009] S5. Select the strain with obvious color change and inoculate it on the slant of the test tube. Culture it at 30℃ for 6 days to obtain mature spores.

[0010] S6. Inoculate the mature spores into the fermentation medium for shaking fermentation at 36°C and 220 r / min for 3 days.

[0011] S7. After the fermentation is completed, filter and take the supernatant to determine the acid production, sugar consumption and sugar-acid conversion rate.

[0012] Furthermore, in step S2, the ultrasonic action time is 20 minutes.

[0013] Further, in step S3, the concentration of the xylose pressure plate is 50 g / L.

[0014] Compared with the prior art, the beneficial technical effects of the present invention are:

[0015] (1) The method of screening Aspergillus terreus strains by ultrasound combined with xylose pressure is beneficial to obtain strains with higher production performance, improve the production efficiency of itaconic acid, and bring higher economic benefits. At the same time, the method is simple to operate, low in cost, and high in screening efficiency.

[0016] (2) Under the condition of ultrasonic treatment time of 20 minutes and xylose pressure plate concentration of 50 g / L, the acid production of the screened Aspergillus terreus strain was 89.81 g / L, which was significantly increased by 10.49% compared with the control; the sugar consumption was reduced by 5.88%; the sugar-acid conversion rate was 67.76%, which was significantly increased by 10.04% compared with the control. Moreover, after primary screening and secondary screening, the acid production was stable, and the mycelium was thick and not easy to break, which was conducive to subsequent production as a dominant strain. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 These are the morphological pictures of mycelium in the experimental group and the control group under a microscope. DETAILED DESCRIPTION

[0018] In the following examples, itaconic acid was determined using high performance liquid chromatography, and sugar consumption was determined using Fehling titration. The sugar-acid conversion rate formula is: Sugar-acid conversion rate = (itaconic acid concentration / consumed sugar concentration) × 100%.

[0019] Example 1: The method for screening Aspergillus terreus by ultrasound combined with xylose pressure comprises the following steps: (1) After the Aspergillus terreus strains are activated, a spore suspension is prepared.

[0020] (2) The spore suspension was placed in an ultrasonic cleaning machine with an ultrasonic power of 400 W, an ultrasonic frequency of 40 kHz, and an ultrasonic action time of 10 min.

[0021] (3) After the ultrasound, the spore suspension was evenly spread on a xylose pressure plate with a concentration of 40 g / L, the culture condition was pH 5.5 and placed in a constant temperature incubator at 30°C until a single colony grew.

[0022] (4) Pick a single colony and inoculate it into bromomethyl green indicator medium and culture it at 30°C for 6 days.

[0023] (5) Select strains with obvious color change and inoculate them on the slant of a test tube. Culture them at 30°C for 6 days to obtain mature spores.

[0024] (6) The mature spores were inoculated into the fermentation medium for shaking fermentation at 36°C and 220 r / min for 3 days. After the fermentation was completed, the supernatant was filtered and the acid production, sugar consumption and sugar-acid conversion rate were determined.

[0025] Control Example: (1) After Aspergillus terreus was activated, a spore suspension was prepared.

[0026] (2) Dilute the spore suspension appropriately and spread it evenly on the plate. Incubate at 30°C for 3 days.

[0027] (3) After the incubation period, pick a single colony and inoculate it onto the slant of a test tube and incubate at 30°C for 6 days.

[0028] (4) After the culture is completed, the mixture is inoculated into the fermentation medium for shaking fermentation at 36°C and 220 r / min for 3 days.

[0029] (5) After fermentation, filter and take the supernatant to determine the acid production, sugar consumption and sugar-acid conversion rate.

[0030] Example 2: The difference from Example 1 is that the ultrasonic treatment time is 10 min and the concentration of the xylose pressure plate is 60 g / L.

[0031] Example 3: The difference from Example 1 is that the ultrasonic treatment time is 20 min and the concentration of the xylose pressure plate is 50 g / L.

[0032] Example 4: The difference from Example 1 is that the ultrasonic treatment time is 20 min and the concentration of the xylose pressure plate is 60 g / L.

[0033] Example 5: The difference from Example 1 is that the ultrasonic treatment time is 30 min and the concentration of the xylose pressure plate is 40 g / L.

[0034] Example 6: The difference from Example 1 is that the ultrasonic treatment time is 30 min and the concentration of the xylose pressure plate is 60 g / L.

[0035] Table 1 shows the acid production, sugar consumption and sugar-acid conversion rate of each embodiment and control example. It can be seen from Table 1 that the acid production, sugar consumption and sugar-acid conversion rate of the Aspergillus terreus strains were significantly changed in the shaker fermentation test after ultrasound combined with xylose pressure screening. When the ultrasonic treatment time was 20min and the xylose pressure concentration was 50g / L, the acid production of the screened strains in the shaker fermentation was the highest, significantly increased by 10.49% compared with the control group, the sugar consumption was reduced by 5.88%, and the sugar-acid conversion rate was increased by 10.04%.

[0036] Table 1 Acid production, sugar consumption and sugar-acid conversion rate of Aspergillus terreus strains treated with different methods

[0037] Processing Acid production (g / L) Sugar consumption (g / L) Sugar-acid conversion rate (%) Comparison Example 81.28±0.15c 140.82±0.74a 57.72±0.29f Example 1 76.36±0.78d 128.20±0.81e 59.56±0.48e Example 2 83.62±0.64f 130.88±0.53c 63.89±0.15c Example 3 89.81±0.18g 132.54±0.21d 67.76±0.83a Example 4 82.19±0.23a 127.56±0.45d 64.43±0.77b Example 5 80.85±0.25b 132.74±0.59b 60.91±0.90d Example 6 78.44±0.39e 133.22±0.36b 58.88±0.67e

[0038] Note: Different lowercase letters in Table 1 represent significant differences among treatments (P<0.05).

[0039] In order to solve the problem of unstable acid production of strains after screening, the strains of Example 3 obtained by screening were subjected to multiple primary screenings, rescreenings, and genetic stability tests. The strains were subcultured 10 times, inoculated into fermentation medium, and cultured in a shaking incubator at 36°C and 220 r / min for 3 days, and the acid production of each generation was measured.

[0040] Table 2 Acid production of strains in Example 3 at different passage times

[0041] Number of passages Acid production (g / L) 1 88.28±0.11a 2 89.06±0.26b 3 89.49±0.33b 4 88.96±0.35a 5 89.63±0.18b 6 90.58±0.31c 7 89.79±0.20b 8 91.19±0.10d 9 90.52±0.23c 10 89.45±0.24b

[0042] Note: Different lowercase letters in Table 2 represent significant differences among treatments (P<0.05).

[0043] The results are shown in Table 2. After 10 consecutive passages, the itaconic acid production of the mutant strain was basically stable at 89.70 g / L, and the genetic stability was good, which is conducive to bringing higher value to the subsequent selection and breeding of itaconic acid fermentation strains.

[0044] The strain of Example 3 was selected as the test group and the strain of the control example was selected as the control group for shaker fermentation culture. After 18 hours of culture, the mycelial morphology was observed under a microscope. The results were as follows: Figure 1 As shown. The results showed that the mycelia in the control group were thinner and dispersed, with more breakages; while the mycelia in the test group screened by ultrasonic treatment were thicker, aggregated, and less broken. The above results show that it is feasible to solve the problem of easy breakage of mycelia by improving the antagonistic ability of Aspergillus terreus through ultrasonic treatment.

[0045] The parts not described in the present invention can be realized by adopting or drawing on the existing technology.

[0046] Of course, the above description is not a limitation of the present invention, and the present invention is not limited to the above examples. Changes, modifications, additions or substitutions made by technicians in this technical field within the essential scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A method for screening Aspergillus terreus strains by combining ultrasound with xylose pressure, characterized in that: The following steps are involved: S1. activating the Aspergillus terreus strain and preparing a spore suspension; S2, placing the spore suspension in an ultrasonic cleaning machine, with an ultrasonic power of 400 W, an ultrasonic frequency of 40 kHz, and an ultrasonic action time of 10 to 30 min; S3. After the ultrasound is finished, the spore suspension is evenly spread on a xylose pressure plate with a concentration of 40 g / L to 60 g / L, the culture condition is pH 5.5 and placed in a constant temperature incubator at 30°C until a single colony grows; S4, pick a single colony and inoculate it into bromomethyl green indicator medium, and culture it at 30℃ for 6 days; S5. Select the strain with obvious color change and inoculate it on the slant of the test tube, and culture it at 30℃ for 6 days to obtain mature spores; S6, inoculating the mature spores into the fermentation medium for shaking fermentation, and culturing in a shaking incubator at 36°C and 220 r / min for 3 days; S7. After the fermentation is completed, filter and take the supernatant to determine the acid production, sugar consumption and sugar-acid conversion rate.

2. The method for screening Aspergillus terreus by ultrasound combined with xylose pressure according to claim 1, characterized in that: In step S2, the ultrasonic action time is 20 minutes.

3. The method for screening Aspergillus terreus by ultrasound combined with xylose pressure according to claim 2, characterized in that: In step S3, the concentration of the xylose pressure plate is 50 g / L.

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