阅读说明：本技术 Fe3O4纳米粒子提升酿酒酵母发酵性能的用途 (Fe3O4Application of nano particles in improving fermentation performance of saccharomyces cerevisiae ) 是由 杨粟平 李勇 侯长军 霍丹群 张宿义 杨平 周军 黄锐 于 2021-09-14 设计创作，主要内容包括：本发明公开了一种Fe-(3)O-(4)纳米粒子提升酿酒酵母发酵性能的用途,属于生物工程领域、食品微生物领域和工业微生物领域。提升酿酒酵母发酵性能的用途主要为提升酿酒酵母对乙醇耐受性的用途和提升酿酒酵母的生长率的用途。所述Fe-(3)O-(4)纳米粒子为：粒径为10-50nm的Small-Fe-(3)O-(4)、粒径为250-450nm的Big-Fe-(3)O-(4)、粒径为50-100nm的组氨酸修饰His-Fe-(3)O-(4)中的任意一种。本发明的Fe-(3)O-(4)纳米粒子提升酿酒酵母发酵性能的用途,可有效解决酿酒酵母对乙醇耐受性较差的问题。(The invention discloses Fe 3 O 4 The application of nano particles in improving the fermentation performance of saccharomyces cerevisiae belongs to the fields of bioengineering, food microorganisms and industrial microorganisms. The application of improving the fermentation performance of the saccharomyces cerevisiae is mainly the application of improving the ethanol tolerance of the saccharomyces cerevisiae and the application of improving the growth rate of the saccharomyces cerevisiae. Said Fe 3 O 4 The nano particles are: Small-Fe with grain diameter of 10-50nm 3 O 4 Big-Fe with particle size of 250-450nm 3 O 4 Histidine modified His-Fe with particle size of 50-100nm 3 O 4 Any one of them. Fe of the invention 3 O 4 The application of the nanoparticles in improving the fermentation performance of the saccharomyces cerevisiae can effectively solve the problem of poor ethanol tolerance of the saccharomyces cerevisiae.)

1.Fe₃O₄The application of the nano particles in improving the fermentation performance of the saccharomyces cerevisiae.

2. Fe of claim 1₃O₄Nanoparticle enhanced saccharomyces cerevisiaeUse of fermentation performance, characterized in that: said Fe₃O₄The nano particles are: Small-Fe with grain diameter of 10-50nm₃O₄Big-Fe with particle size of 250-450nm₃O₄Histidine modified His-Fe with particle size of 50-100nm₃O₄Any one of them.

3. Fe according to claim 1 or 2₃O₄The application of the nanoparticles in improving the fermentation performance of the saccharomyces cerevisiae is characterized in that: the application of improving the fermentation performance of the saccharomyces cerevisiae is the application of improving the ethanol tolerance of the saccharomyces cerevisiae.

4. Fe according to claim 1 or 2₃O₄The application of the nanoparticles in improving the fermentation performance of the saccharomyces cerevisiae is characterized in that: the purpose of improving the fermentation performance of the saccharomyces cerevisiae is to improve the growth rate of the saccharomyces cerevisiae.

5. Fe according to claim 2₃O₄The application of the nanoparticles in improving the fermentation performance of the saccharomyces cerevisiae is characterized in that: when said Fe is present₃O₄The nano particles are His-Fe₃O₄And/or Big-Fe₃O₄The purpose of improving the fermentation performance of the saccharomyces cerevisiae is to improve the electron transfer rate in a system and reduce the oxidation-reduction reaction potential.

6. Fe according to claim 2₃O₄The application of the nanoparticles in improving the fermentation performance of the saccharomyces cerevisiae is characterized in that: when said Fe is present₃O₄The nano particles are His-Fe₃O₄The purpose of improving the fermentation performance of the saccharomyces cerevisiae is to improve the yield of the ethanol acetic acid.

7. Big-Fe according to claim 2₃O₄The preparation method is characterized by comprising the following steps: mixing ferric chloride, trisodium citrate, glycol and sodium acetate in the weight ratio of 13 to 4 to 440 to 24After trisodium citrate is completely dissolved in ethylene glycol, adding sodium acetate, violently stirring until the trisodium citrate and the sodium acetate are uniformly mixed, transferring the mixture into a high-pressure kettle, heating for 10 hours at 200 ℃, washing the product with ethanol, and drying to obtain Big-Fe₃O₄。

8. Big-Fe according to claim 7₃O₄The preparation method is characterized by comprising the following steps: the autoclave is a stainless steel autoclave with a Teflon lining, and the drying temperature is 55-65 ℃.

9. His-Fe according to claim 2₃O₄The preparation method is characterized by comprising the following steps: completely dissolving ferric chloride hexahydrate in ethylene glycol according to the mass ratio of 4: 220: 18: 5, adding sodium acetate and histidine, violently stirring until the mixture is uniformly mixed, then carrying out ultrasonic treatment on the mixture for 10-15 minutes, transferring the mixture into a polymerization reactor, heating the mixture at 200 ℃ for 12 hours, washing the product with ethanol, and drying to obtain His-Fe₃O₄。

10. His-Fe according to claim 9₃O₄The preparation method is characterized by comprising the following steps: the reactor is a polytetrafluoroethylene reactor, and the drying temperature is 55-65 ℃.

Technical Field

The invention belongs to the fields of bioengineering, food microorganisms and industrial microorganisms, and relates to a method for improving fermentation performance of saccharomyces cerevisiae.

Background

Saccharomyces cerevisiae is widely used in industrial fields of alcohol production, food production, fuel alcohol production and the like. However, in the yeast fermentation process, due to the continuous production of ethanol, the cells face high-concentration ethanol at the final stage of fermentation, so that the cells are weakened in multiple toxic functions, the absorption and decomposition of glucose are inhibited, the activity of intracellular glycolytic enzyme is reduced, the integrity of cell membranes is damaged, the production fermentation process is slowed down, and the fermentation efficiency, the sustainability and the ethanol yield of the yeast are affected.

At present, the method for enhancing ethanol tolerance degree and improving ethanol yield of yeast mainly adopts genetic engineering, and the strategy is to utilize a whole genome to screen genes which influence ethanol tolerance and achieve the effect of enhancing cell ethanol tolerance by introducing or knocking out specific mutant genes such as genes influencing cell membrane composition and protein folding. Ethanol tolerance, however, involves multiple physiological processes, each of which relies on many different combinations of alleles and mutations to interact, resulting in complex and time-consuming technical manipulations.

And optimizing a fermentation process, wherein various parameters in the fermentation process are optimized, so that the saccharomycetes are in the optimal fermentation environment for production to further realize the increase of the yield of the ethanol.

The methods limit the application of the method in industrial production, so that it is necessary to develop a convenient and economic new method for improving the fermentation performance of the saccharomyces cerevisiae, enhancing the tolerance of the saccharomyces cerevisiae to ethanol and improving the yield of ethanol.

Disclosure of Invention

The invention aims to solve the technical problem of poor ethanol tolerance of the saccharomyces cerevisiae.

The technical scheme adopted by the invention for solving the technical problems is as follows: fe₃O₄The application of the nano particles in improving the fermentation performance of the saccharomyces cerevisiae.

Fe as described above₃O₄The nano particles are: Small-Fe with grain diameter of 10-50nm₃O₄Big-Fe with particle size of 250-450nm₃O₄Histidine modified His-Fe with particle size of 50-100nm₃O₄Any one of them.

The application of improving the fermentation performance of the saccharomyces cerevisiae is the application of improving the ethanol tolerance of the saccharomyces cerevisiae.

The application of improving the fermentation performance of the saccharomyces cerevisiae is the application of improving the growth rate of the saccharomyces cerevisiae.

When the above-mentioned Fe is present₃O₄The nano particles are His-Fe₃O₄And/or Big-Fe₃O₄The purpose of improving the fermentation performance of the saccharomyces cerevisiae is to improve the electron transfer rate in a system and reduce the oxidation-reduction reaction potential.

When the above-mentioned Fe is present₃O₄The nano particles are His-Fe₃O₄The purpose of improving the fermentation performance of the saccharomyces cerevisiae is to improve the yield of the ethanol acetic acid.

The above Big-Fe₃O₄The preparation method comprises the following steps: completely dissolving ferric chloride and trisodium citrate in ethylene glycol according to the mass ratio of 13: 4: 440: 24, adding sodium acetate, violently stirring until the mixture is uniformly mixed, transferring the mixture into an autoclave, heating for 10 hours at 200 ℃, washing a product with ethanol, and drying to obtain Big-Fe₃O₄。

Further, the above-mentioned vigorous stirring time is 30 to 40 minutes.

Furthermore, the autoclave is a stainless steel autoclave with a Teflon lining, and the drying temperature is 55-65 ℃.

The above His-Fe₃O₄The preparation method comprises the following steps: completely dissolving ferric chloride hexahydrate in ethylene glycol according to the mass ratio of 4: 220: 18: 5, adding sodium acetate and histidine, violently stirring until the mixture is uniformly mixed, then carrying out ultrasonic treatment on the mixture for 10-15 minutes, transferring the mixture into a polymerization reactor, heating the mixture at 200 ℃ for 12 hours, washing the product with ethanol, and drying to obtain His-Fe₃O₄。

Further, the above-mentioned vigorous stirring time is 30 to 40 minutes.

Further, the reactor is a polytetrafluoroethylene reactor, and the drying temperature is 55-65 ℃.

Fe as described above₃O₄The nano particles still have the magnetic recovery capability after the fermentation is finished.

The invention has the beneficial effects that: fe₃O₄Nanoparticles (Fe)₃O₄NPs) as peroxidase-like nanoparticles, participate in the metabolism of peroxides and active oxygen, and can improve the synthesis or degradation of membrane phospholipids by controlling fatty acid composition. Mixing Fe₃O₄The nano particles are used for improving the fermentation performance of the saccharomyces cerevisiae, can effectively improve the tolerance of the saccharomyces cerevisiae to ethanol, and ensure that the saccharomyces cerevisiae keeps stronger growth rate and Fe₃O₄The nano particles still have the magnetic recovery capability after the fermentation is finished.

Meanwhile, when His-Fe specially prepared by the present invention is used₃O₄And Big-Fe₃O₄When the fermentation performance of the saccharomyces cerevisiae is improved, the electron transfer rate in the system can be improved, the oxidation-reduction reaction potential is reduced, the yeast is promoted to perform oxidation reaction at a low potential, the oxidative decomposition of glucose can be accelerated, the cell growth is accelerated, and the saccharomyces cerevisiae can keep a stronger growth rate and higher ethanol production rate.

Wherein, His-Fe₃O₄The introduction of histidine improves the peroxidase-like activity and Fe₃O₄The catalytic efficiency of the nano enzyme reduces the stress of mitochondria, so His-Fe is adopted₃O₄When the fermentation performance of the saccharomyces cerevisiae is improved, the effect of improving the ethanol tolerance of the saccharomyces cerevisiae is the best, the ethanol metabolism is improved, the acetic acid yield can be effectively improved, and the method has great industrial production potential.

Drawings

FIG. 1 is a scanning electron microscope image of Saccharomyces cerevisiae used in the examples of the present invention;

FIG. 2 shows Fe used in examples of the present invention₃O₄TEM images and particle size distributions of NPs;

FIG. 3 is the optical density curve of Saccharomyces cerevisiae during co-cultivation in example 1 of the present invention;

FIG. 4 is a graph of yeast growth after 6 days of incubation in example 2 of the present invention;

FIG. 5 is a CV diagram of example 3 of the present invention;

FIG. 6 is a graph showing the yield of glycolic acid according to example 4 of the present invention;

FIG. 7 is a graph showing the magnetic recovery results in example 5 of the present invention.

Detailed Description

The technical solution of the present invention can be specifically implemented as follows.

Fe₃O₄The application of the nano particles in improving the fermentation performance of the saccharomyces cerevisiae.

Fe as described above₃O₄The nano particles are: Small-Fe with grain diameter of 10-50nm₃O₄Big-Fe with particle size of 250-450nm₃O₄Histidine modified His-Fe with particle size of 50-100nm₃O₄Any one of them.

The application of improving the fermentation performance of the saccharomyces cerevisiae is the application of improving the ethanol tolerance of the saccharomyces cerevisiae and the application of improving the growth rate of the saccharomyces cerevisiae.

When the above-mentioned Fe is present₃O₄The nano particles are His-Fe₃O₄And/or Big-Fe₃O₄The application of improving the fermentation performance of the saccharomyces cerevisiae not only improves the ethanol tolerance of the saccharomyces cerevisiae and improves the growth rate of the saccharomyces cerevisiae; it also has the purpose of improving the electron transfer rate in the system and reducing the oxidation-reduction reaction potential.

When the above-mentioned Fe is present₃O₄The nano particles are His-Fe₃O₄The application of improving the fermentation performance of the saccharomyces cerevisiae comprises the following steps: the application of improving the ethanol tolerance of the saccharomyces cerevisiae, the application of improving the growth rate of the saccharomyces cerevisiae, the application of improving the electron transfer rate in a system, the application of reducing the oxidation-reduction reaction potential and the application of improving the yield of ethanol acetic acid.

The above Big-Fe₃O₄The preparation method comprises the following steps: completely dissolving ferric chloride and trisodium citrate in ethylene glycol according to the mass ratio of 13: 4: 440: 24, adding sodium acetate, violently stirring until the mixture is uniformly mixed, transferring the mixture into an autoclave, heating for 10 hours at 200 ℃, washing a product with ethanol, and drying to obtain Big-Fe₃O₄. Wherein sodium acetate plays a role in electrostatic balance and matching with a reducing agent in the whole system, and solid sodium acetate is added after other raw materials are mixed, so that the concentration of reactants is more uniform.

For better experimental results, it is therefore preferred that the vigorous stirring time is 30-40 minutes, the autoclave is a stainless steel autoclave lined with Teflon and the drying temperature is 55-65 ℃.

The above His-Fe₃O₄The preparation method comprises the following steps: ferric chloride hexahydrate is mixed with glycol, sodium acetate and histidine in the weight ratio of 4 to 220 to 18 to 5After the His-Fe is completely dissolved in ethylene glycol, adding sodium acetate and histidine, violently stirring until the mixture is uniformly mixed, then carrying out ultrasonic treatment on the mixture for 10-15 minutes, transferring the mixture into a polymerization reactor, heating the mixture for 12 hours at 200 ℃, washing the product with ethanol, and drying the product to obtain the His-Fe₃O₄。His-Fe₃O₄Modification of nano Fe for histidine₃O₄Thus first forming Fe₃O₄Then histidine is added to connect the surface of the ferroferric oxide.

For better experimental results, it is therefore preferred that the vigorous stirring time is 30 to 40 minutes, the reactor is a polytetrafluoroethylene reactor, and the drying temperature is 55 to 65 ℃.

Fe as described above₃O₄The nano particles still have the magnetic recovery capability after the fermentation is finished.

The technical solution and effects of the present invention will be further described below by way of practical examples.

Examples

(1) Separation and identification of saccharomyces cerevisiae

Saccharomyces cerevisiae was isolated from commercial yeast powder using 100ml LYPD medium (2g peptone, 2g glucose, 1g yeast extract and 2g agar). The region of the s.cerevisiae 26S rRNAD1/D2 gene was PCR amplified using primers NL1(5'-GCATATCAATAAGCGGAGGAAAAG-3') and NL4 (5'-GGTCCGTGTTTCAAGACGG-3'). The obtained amplified sequences were compared in the GenBank NCBI database and confirmed to be Saccharomyces cerevisiae, and the scanning electron microscopy image of the prepared Saccharomyces cerevisiae is shown in FIG. 1.

(2)Fe₃O₄Synthesis of NPs

①Small-Fe₃O₄: obtained by market purchase.

②Big-Fe₃O₄: 0.65g of ferric chloride and 0.20g of trisodium citrate are completely dissolved in 20mL of ethylene glycol, then 1.20g of sodium acetate are added and stirred vigorously for 30 minutes. The mixture was transferred to a 50ml teflon lined stainless steel autoclave, heated at 200 ℃ for 10h, the product washed three times with ethanol and dried at 60 ℃ for future use.

③His-Fe₃O₄: 0.82g of ferric chloride hexahydrate was completely dissolved in 40mL of ethylene glycol, followed by the addition of 3.6g of sodium acetate and 1g of histidine and vigorous stirring for 30 minutes, followed by sonication for 10 minutes, the mixture was transferred to a 50mL polytetrafluoroethylene reactor, heated at 200 ℃ for 12h, the product washed three times with ethanol and dried at 60 ℃ until use.

Small-Fe obtained in the above₃O₄，Big-Fe₃O₄，His-Fe₃O₄0.16g of each of the solutions was weighed and added to 16mL of distilled water, respectively, to obtain a stock solution having a concentration of 10 mg/mL.

In FIG. 2, ABC is Small-Fe in this example₃O₄，Big-Fe₃O₄，His-Fe₃O₄TEM image of (B), DEF are Small-Fe, respectively₃O₄，Big-Fe₃O₄，His-Fe₃O₄As is clear from FIG. 2, the particle size distribution of Small-Fe used in the examples of the present invention₃O₄An average particle diameter of 18nm, Big-Fe₃O₄Has an average particle diameter of 337nm and His-Fe₃O₄The average particle size was 70 nm.

(3) EXAMPLE 1 cultivation experiment

Small-Fe prepared as above₃O₄，Big-Fe₃O₄，His-Fe₃O₄Adding 400 μ L of the stock solutions into 100ml LYPD culture medium (containing peptone 2g, glucose 2g, yeast extract 1g, and agar 2 g) as three groups of experimental groups, and collecting 100ml LYPD culture medium without Fe₃O₄NPs were used as a control group (NC), and then 8% (v/v) ethanol and 5% (v/v) Saccharomyces cerevisiae were added to the experimental group and the control group, respectively, and cultured for 24 hours (30 ℃, 120 rpm).

And (4) detecting a result: the optical density of Saccharomyces cerevisiae in the culture solution was measured at 600nm with an ultraviolet-visible spectrophotometer during co-culture, and the results are shown in FIG. 3, from FIG. 3, it can be seen that His-Fe was added to the experimental group₃O₄The optical density of the group yeast is obviously higher than that of Big-Fe₃O₄And Small-Fe₃O₄Treated cells, three groups with Fe₃O₄Experimental group of NPsThe density is higher than that of the alloy without adding Fe₃O₄Control group of NPs. Thus, it can be seen that Fe is converted by the method of the present invention₃O₄NPs and saccharomyces cerevisiae are cultured together, so that the yeast can keep stronger growth rate.

(4) EXAMPLE 2 drop plate experiment

A. Small-Fe₃O₄，Big-Fe₃O₄，His-Fe₃O₄Respectively adding 400 μ L of stock solution into 100mLYPD solid culture medium (2% agar) as experimental group, and adding no Fe into 100mLYPD solid culture medium₃O₄NPs are used as a control group (NC), and then 12% (v/v) ethanol is respectively added into the experimental group and the control group for standby;

B. Small-Fe₃O₄，Big-Fe₃O₄，His-Fe₃O₄Respectively adding 400 μ L of stock solution into 100mLYPD solid culture medium (2% agar) as experimental group, and adding no Fe into 100mLYPD solid culture medium₃O₄NPs are used as a control group (NC), and then 16% (v/v) ethanol is respectively added into the experimental group and the control group for standby;

C. drop plate test: the yeast concentration was diluted to 10⁰、10^-1、10^-2、10^-3And 10^-4And respectively sucking 10 mu L of the solution, dropping the solution into the prepared solid culture mediums of the steps A and B, and incubating the solution at 30 ℃ for 6 days.

And (4) detecting a result: FIG. 4 is a graph showing the growth of yeast after 6 days of incubation, and it is apparent from FIG. 4 that Fe was added under the condition of high concentration ethanol₃O₄The yeast of the NPs still maintained good growth, while the control (NC) was significantly weaker. Therefore, the method can effectively improve the ethanol tolerance of the saccharomyces cerevisiae.

(5) EXAMPLE 3 electrochemical experiments

A. Small-Fe₃O₄，Big-Fe₃O₄，His-Fe₃O₄Respectively adding 400 μ L of stock solution into 100mLYPD solid culture medium as group A experimental group, and taking 100mLYPD solid culture medium without Fe₃O₄NPs as group A control group(NC)；

B. Small-Fe₃O₄，Big-Fe₃O₄，His-Fe₃O₄Respectively adding 400 μ L of stock solution into 100mLYPD solid culture medium, respectively inoculating 1% (v/v) Saccharomyces cerevisiae into YPD culture medium, co-culturing for 7 hr to obtain group B experiment group, and taking 100mLYPD solid culture medium without Fe₃O₄NPs were inoculated with 1% (v/v) s.cerevisiae and cultured for 7h as group B control (NC).

And (4) testing results: testing of three Fe's using an electrochemical workstation₃O₄Influence of NPs on the electrochemical activity of Saccharomyces cerevisiae. A three-electrode system with Ag/AgCl (soaked in 3.0M KCl) as a reference electrode, a platinum wire as an auxiliary electrode and a Glassy Carbon Electrode (GCE) as a working electrode is adopted to test a relevant cyclic voltammetry Curve (CV). The scanning potential range was (-0.6) -1.6V, the scanning rate was set at 10mV/s, and the test results are shown in FIG. 5.

As can be seen from fig. 5: no redox reaction was detected in the experimental group A and the control group, but the group A contained His-Fe₃O₄And Big-Fe₃O₄The current of the experimental group (2) is higher than that of the other groups in the range of 1.0-1.6V, which indicates that Fe₃O₄NPs cannot react with the culture medium, but can accelerate the electron transfer rate of the system by reducing the resistance of the culture solution; ② after 7h of culture, the group B experimental group has oxidation peak in the range of 0.8-1.2V and contains His-Fe₃O₄And Big-Fe₃O₄The oxidation peak of the experimental group is shifted to the left relative to the control medium of group B, indicating His-Fe₃O₄And Big-Fe₃O₄Can promote the yeast to have oxidation reaction under low potential, can accelerate the oxidative decomposition of glucose and accelerate the cell growth.

(6) Example 4 fermentation experiments

Small-Fe₃O₄，Big-Fe₃O₄，His-Fe₃O₄Adding 400 μ L of stock solution into 100mLYPD culture medium (40mg/L) to obtain three experimental groups, and adding no Fe into 100mLYPD culture medium₃O₄NPs as control group (NC) were subsequently added to the experimental and control groups, respectively1% (v/v) of Saccharomyces cerevisiae was inoculated and cultured for 48 hours.

And (4) testing results: the cultured culture solution was centrifuged to obtain 900. mu.L of supernatant, 100. mu.L of 2-octanol internal standard solution (10. mu.g/mL) was added to the supernatant, and further filtered to obtain a supernatant, and the finally obtained supernatant was analyzed by Shimadzu TQ8040 gas chromatography-mass spectrometry (GC-MS) using a 30 m.times.0.25 mm.times.0.25 μm Rtx-Wax column. The formula is adopted: the semi-quantitative analysis method is used for quantifying ethanol and acetic acid, and the formula is as follows:wherein C is the compound content (. mu.g/mL); c_I.S2-octanol content in the sample (. mu.g/mL); a. the_CIs the compound peak area; a. the_I.SIs the peak area of 2-octanol. As shown in FIG. 6, A represents ethanol production and B represents acetic acid production, and it can be seen from FIG. 6 that His-Fe₃O₄Can effectively improve the yield of the ethanol acetic acid, and compared with NC ((8.032g/L) of a control group, the ethanol acetic acid is added with His-Fe₃O₄The yield of ethanol in the test group of (1) was improved by about 17.1%, and the yield of acetic acid was the highest.

(7) Example 5 magnetic recovery experiment

Small-Fe₃O₄，Big-Fe₃O₄，His-Fe₃O₄400 mu L of each stock solution is respectively added into 100mLYPD culture medium (40mg/L), and then 1% (v/v) of saccharomyces cerevisiae is respectively inoculated for culture and fermentation.

And (4) testing results: will contain three kinds of Fe respectively₃O₄The culture solution of NPs was put in a 20mL test tube after the fermentation was completed, and was set aside the test tube with a common magnet, and the observation was carried out, and the result is shown in FIG. 7, from which it is apparent that Fe is shown in FIG. 7₃O₄The NPs have magnetic recoverable performance after fermentation.