Extraction method and application of gliocladium neurospora volatile matter with aroma producing capacity

Abstract

The invention provides an extraction method and application of a gliocladium neurospora volatile matter with aroma producing capacity, and relates to the technical field of gliocladium neurospora volatile matter extraction. The extraction method of the gliocladium neurospora volatile matter with the aroma producing capacity specifically comprises the following steps: S1, activating culture; s2, culturing the gliocladium neurospora; s3, screening the extraction heads; s4, volatile matter extraction. The invention provides an extraction method and application of a gliocladium neurospora volatile matter with aroma producing ability, and compared with the prior art, the method for extracting and culturing the gliocladium neurospora volatile matter and the volatile matter component thereof disclosed by the patent has the main advantages that the extraction efficiency and precision of the volatile matter are improved; meanwhile, culture conditions of the gliocladium neurospora are optimized so as to enhance the aroma producing capacity of the gliocladium neurospora, and the research results not only improve the detection sensitivity and the production yield of a target compound, but also reduce the production cost and the environmental influence by reducing the use of chemical reagents, so that a new possibility is provided for development and application of natural spices.

Description

Technical Field

[0001] The invention relates to the technical field of extraction of volatiles from Gliocladium schrenkiana, in particular to a method for extracting volatiles from Gliocladium schrenkiana with fragrance-producing ability and application thereof. Background Art

[0002] Fragrance substances have attracted much attention due to their pleasant and stress-relieving properties. The application of such substances has penetrated into all aspects of life, such as food, light industry, cosmetics and other manufacturing industries. The application of fragrance substances relies on flavors and fragrances. Traditional sources of flavors and fragrances are mostly directly extracted from aromatic plants or animal musk or obtained by chemical synthesis to obtain high-purity fragrance substances, but the above methods are high-cost and low-yield, and cannot meet market demand. Compared with traditional methods, microbial fermentation and biotransformation methods have many advantages such as high efficiency, greenness, and environmental protection, and have now become a research hotspot for the development of flavors and fragrances. Therefore, the discovery of microorganisms with fragrance-producing functions has become an urgent need in the flavor and fragrance market.

[0003] In recent years, fungi have become the focus of attention among microorganisms with aroma-producing functions. The volatile organic compounds (VOCs) produced by fungi include a variety of chemical categories and are used in industrial production. For example, Zhang Peng et al. used the fermentation broth of Aureobasidium melanogenum to improve the aroma of tobacco, and Zhou Sen et al. screened out the aroma-producing Geotrichum candidum with the flavor of liquor. Clonostachys rosea f.catenulata belongs to the genus Clonostachys of the phylum Ascomycota, class Sordariomycetes, order Hypocreales, family Bionectriaceae, and is a variant of Clonostachys rosea. The fungus has a wide distribution area and exists in soils, deserts, and waters in various temperature zones. As a facultative saprophyte, the fungus often coexists with sclerotia, hyphae and cysts of various pathogens in the soil. In addition, the fungus is often isolated from plants, such as barley, onion, strawberry, rose and cocoa, among which roots, leaves and flowers are the main isolated parts, and the most frequent part is the root. At the same time, the fungus can show endogenous ability in different tissues of plants, such as colonization in geranium leaves and cucumber roots. Previous studies on G. chamaemelum have focused on the development of fungicides based on its characteristics as a hyperparasite and on disease control for it as a soil-borne pathogen. Compared with other aroma-producing fungi, G. chamaemelumlum has a rare hyperparasitic ability, which allows it to parasitize other fungi and exchange substances, thereby expanding the source of its metabolites. At present, most of the research on G. chamaemelumlumlum focuses on the non-volatile components in the metabolites, and there are few reports on the research on the volatile components of G. chamaemelumlumlum. Combining the high application value of Gliocladium Alternariae and its ability to expand the source of metabolites, the development of Gliocladium Alternariae with the ability to produce fragrance has great market application prospects. However, currently, there are few studies on the volatiles of Gliocladium Alternariae. Therefore, the development of its application in volatiles has great potential in the field of microbial-derived fragrances. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the shortcomings of the prior art, the present invention provides a method for extracting volatiles from Gliocladium schrenkiana with aroma-producing ability and its application. To make up for it to some extent The problem that there is little research on the volatiles of Gliocladium albicans was addressed.

[0006] (II) Technical solution

[0007] To achieve the above objectives, the present invention is implemented by the following technical scheme: a method for extracting volatiles from Gliocladium schrenkiana with aroma-producing ability, specifically comprising the following steps:

[0008] S1. Activation culture

[0009] The Glechoma nematode was picked out from the cryopreserved tube with an inoculation needle and inoculated in the middle of a 8.5 cm diameter culture dish containing 20 mL of PSA culture medium, and cultured at 28° C. for 7 days for later use;

[0010] S2. Culture of Gliocladium schrenkiana

[0011] Select 500 mL culture flasks as culture containers, and add 120 mL of culture medium to each culture flask;

[0012] S3. Screening of extraction heads

[0013] Use a 50 / 30 μm DVB / CAR / PDMS extraction head and age it at 250°C at the gas chromatography injection port for at least 2 hours until there are no impurity peaks.

[0014] S4. Volatile extraction

[0015] The cultured Glechoma tenuissora culture bottle was placed under the conditions of extraction temperature of 28°C, equilibrium time of 30 minutes, extraction time of 40 minutes, and analysis time of 3 minutes to extract volatile organic compounds (VOCs).

[0016] Preferably, the method steps also include GC-MS analysis.

[0017] Preferably, the GC conditions for the GC-MS analysis are: using an HP-5MS chromatographic column (30m×250μm×0.25μm); the carrier gas is helium, with a flow rate of 0.8mL / min; non-split injection, the injection port temperature is 250°C; the temperature program is set to an initial temperature of 40°C for 5 minutes, heating to 130°C at a rate of 5°C / min and holding for 5 minutes, and then heating to 230°C at a rate of 10°C / min and holding for 2 minutes.

[0018] Preferably, the MS conditions of the GC-MS analysis are: electron ion source, ion source temperature 230° C.; data acquisition method is full scan, mass range m / z: 35-550u.

[0019] Preferably, the culture conditions in the cultivation of Gliocladium albicans in step S2 are: culture medium volume 120 mL, culture temperature 27.5° C., culture days 27 days, carbon source is sucrose, no nitrogen source is added, and pH value is natural.

[0020] The invention discloses an application of a fragrance-producing Gliocladium truncatum volatile in the production of fragrance, flavor and fragrance.

[0021] (III) Beneficial effects

[0022] The present invention provides a method for extracting volatiles from Gliocladium schrenkiana with aroma-producing ability and its application, which has the following beneficial effects:

[0023] The present invention provides a method for extracting volatiles from Gliocladium chainosporum with aroma-producing ability and an application thereof. The present invention uses a carefully selected SPME (solid phase microextraction) extraction head (50 / 30 μm DVB / CAR / PDMS) combined with optimized GC-MS (gas chromatography-mass spectrometry) analysis conditions. The method can efficiently and accurately extract volatile organic compounds (VOCs) from Gliocladium chainosporum. The extraction and analysis methods improve the sensitivity and resolution of detection, making the identification of volatiles more accurate while maintaining the integrity of the components. Through meticulous experimental design and optimization, the optimal culture conditions of Gliocladium chainosporum are found, including culture medium volume, temperature and culture days. The optimization of these conditions not only improves the growth and metabolic activity of the strain, but also significantly enhances its aroma-producing ability. The experimental results fit the model well, verifying the optimized culture conditions. The feasibility and effectiveness of these two technologies are demonstrated. Through the application of these two technologies, not only a standardized method is provided for the extraction and analysis of volatiles from Gliocladium albospora, but also a solid foundation is laid for further research and development of its application in flavors, flavors and fragrances and related fields, especially in the fields of food and cosmetics. This natural source of fragrance has potential market advantages. The use of Gliocladium albospora as a natural fragrance source avoids the environmental and health risks that may be brought about by chemically synthesized fragrances. In addition, the optimized culture conditions reduce the dependence on chemical reagents and the generation of chemical waste during the experiment, which is in line with the principles of green chemistry and sustainable development. By improving the culture efficiency and the accuracy of volatile extraction, the waste of experimental materials and time is reduced, thereby reducing production costs. This provides the possibility for the commercial production of Gliocladium albospora extracts, which is conducive to achieving a win-win situation of economic benefits and environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a graph showing the adsorption capacity of different specifications of extraction heads for VOCs of Gliocladium nematodes in the present invention;

[0025] Figure 2 The effects of carbon source and nitrogen source in the basic culture medium of the present invention on the effective peak of VOCs of Gliocladium nematosporum are shown in (a) carbon source; (b) nitrogen source chart;

[0026] Figure 3 The effects of different culture conditions on the effective peak of VOCs of Glechoma spp. under the single factor test of the present invention are as follows: (a) pH value; (b) culture medium volume; (c) culture days; (d) culture temperature chart;

[0027] Figure 4 This is a table showing the effect of different culture conditions on the number of effective peaks of VOCs of Glechoma nematodes under the response surface experiment of the present invention;

[0028] Figure 5 Schematic diagram of the chemical structure of VOCs of Gliocladium solani of the present invention;

[0029] Figure 6 This is the total ion current chromatogram of VOCs of Gliocladium albicans analyzed by GC-MS of the present invention. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Example 1: Extraction of volatile components from Gliocladium schrenkiana with aroma-producing ability:

[0032] (1) Screening of extraction heads

[0033] The mycelium of Gliocladium albicans with a diameter of 5 mm was inoculated into a 500 mL culture bottle containing 120 mL PSA culture medium and cultured at 28°C for 20 days for use.

[0034] Three types of extraction heads, 50 / 30μm DVB / CAR / PDMS, 75μm CAR / PDMS, and 100μm PDMS, were aged at 250°C at the gas chromatography injection port for at least 2 hours until there were no impurity peaks. A culture bottle of cultured Glechoma spp. was selected and placed under the conditions of extraction temperature of 28°C, equilibrium time of 30 min, extraction time of 40 min, and analysis time of 3 min for VOCs extraction. Then, the VOCs were extracted according to the GC-MS analysis conditions, and the number of effective VOCs peaks was used as an indicator to screen the appropriate extraction head. The adsorption effects of different extraction heads vary due to differences in coating materials. The uncertainty of volatile components will affect the adsorption effect of the extraction head. Therefore, the extraction head screening should be carried out at the beginning of the experiment. Taking the detected effective peaks as indicators, the adsorption capacity of the three extraction heads of different materials was compared. The results are shown in the figure. Figure 1 As shown in the figure, 50 / 30μm DVB / CAR / PDMS has the best adsorption capacity for VOCs of Gliocladium solani, so the 50 / 30μm DVB / CAR / PDMS extraction head is selected as the most suitable extraction head.

[0035] Example 2: GC-MS analysis conditions of volatile substances

[0036] The GC conditions need to be set in combination with the chromatographic peaks of G. cyclosporus volatiles. To obtain the most suitable GC conditions for G. cyclosporus volatiles, the starting temperature is first set to 40°C, and then raised to 230°C at 5°C / min, and the helium flow rate is 1.0mL / min to observe the separation of its chromatographic peaks. The final conditions are obtained by reducing the helium flow rate and changing the heating rate:

[0037] GC conditions: HP-5MS column (30m×250μm×0.25μm); helium as carrier gas, flow rate of 0.8mL / min; splitless injection, injection port temperature of 250℃; heating program: initial temperature of 40℃ for 5min, heating to 130℃ at a rate of 5℃ / min and holding for 5min, then heating to 230℃ at a rate of 10℃ / min and holding for 2min;

[0038] MS conditions: electron ion source; ion source temperature 230°C;

[0039] Data acquisition method: full scan; mass range m / z: 35-550u;

[0040] Example 3: Optimization of the culture medium and culture conditions of Glechoma longituba with aroma production ability

[0041] (1) Activation culture of test fungi

[0042] The test strain was picked out from the preserved frozen tube with an inoculation needle and inoculated in the middle of a 8.5 cm diameter culture dish containing 20 mL of PSA medium, and cultured at 28°C for 7 days for later use.

[0043] (2) Single factor optimization experiment of carbon source and nitrogen source of culture medium

[0044] Agar was added to the potato extract, and the carbon source and nitrogen source of the culture conditions were optimized as the basic culture medium. A 500mL culture bottle was selected as the culture container for G. nematosporum, and 120mL of culture medium was added to each culture bottle. Four different carbon sources (sucrose, glucose, maltose, and soluble starch) were added to the culture medium at 20g / L. After the culture medium was cooled and solidified, a 5mm diameter G. nematosporum cake was inoculated in the center of the culture medium and cultured at 26℃ for 20d. GC-MS analysis was performed with the number of effective peaks of VOCs as an indicator to screen the most suitable carbon source. On the basis of screening out the most suitable carbon source, different types of nitrogen sources (ammonium chloride, ammonium sulfate, peptone, and potassium nitrate) with a mass concentration of 5g / L were added. According to the above culture conditions, the number of effective peaks of VOCs was determined to screen the most suitable nitrogen source. All experiments were repeated 3 times.

[0045] The choice of carbon source will affect the number of effective peaks. Figure 2As shown in the figure, when the carbon source is sucrose, the number of effective peaks of VOCs of Gliocladium nematodes is the largest, followed by maltose, glucose, and soluble starch. On the basis of optimizing the carbon source as sucrose, different nitrogen sources are added, and the number of effective peaks from high to low is potassium nitrate>peptone>ammonium sulfate>ammonium chloride. Compared with the case without adding nitrogen source, the number of effective peaks after adding nitrogen source to the culture medium shows an overall downward trend, so the tested nitrogen source is not suitable for the production of VOCs by Gliocladium nematodes hyphae. The carbon source of the optimized culture medium used in subsequent experiments is sucrose, and no nitrogen source is added.

[0046] (3) Single factor optimization test of culture conditions

[0047] Using the optimized culture medium, the number of effective VOCs peaks was used as an indicator to investigate the effects of pH value (5, 6, 7, 8), culture medium volume (liquid volume) (80 mL, 100 mL, 120 mL, 140 mL), culture temperature (24°C, 26°C, 28°C, 30°C), and culture time (22d, 24d, 26d, 28d) on the volatile components of Gliocladium albicans. All experiments were repeated three times.

[0048] The optimized culture medium was used as the basis for the cultivation of G. serrata, and the effects of different culture conditions on the number of effective peaks of VOCs in G. serrata were studied. Figure 3 As shown. First, the pH value in the culture conditions was changed to explore the effect of different pH values ​​on the effective peaks of VOCs of Glechoma nematodes. The number of effective peaks was the largest at pH 7, but the effect of pH changes in the pH range of pH 5-8 on the number of effective peaks was not obvious, so the pH value of the culture medium was kept at a natural level.

[0049] At the natural pH level, the volume of the culture medium in the culture conditions was changed to study the effect of the volume of the culture medium on the number of effective peaks of VOCs of Glechoma nematodes. The number of effective peaks showed a trend of first decreasing, then increasing, and then decreasing as the volume of the culture medium increased. The number of effective peaks was the largest when the culture medium volume was 120mL, followed by 140mL, 80mL, and 100mL. Therefore, the optimal culture medium volume was selected as 120mL.

[0050] Under the aforementioned culture conditions of natural pH value and 120 mL culture medium volume, the effect of different culture days on the number of effective peaks of VOCs of Glechoma nematodes was studied. With the increase of culture days, the number of effective peaks showed a trend of first increasing and then decreasing. When the number of days was 26 days, the number of effective peaks was the largest, so the optimal culture day was selected as 26 days.

[0051] Based on the optimization of the above factors, the effects of different culture temperatures on the number of effective peaks of VOCs of Glechoma nematodes were studied. As the culture temperature increased, the number of effective peaks first increased and then decreased. The number of effective peaks was the largest when the culture temperature was 28°C, so the optimal culture temperature was selected as 28°C.

[0052] (4) Response surface experiment on the influence of volatile components

[0053] According to the results of the single-factor experiment in step 3 and the Box-Behnken combination design principle, the Design-Expert.V8.0 software was used to design a 3-factor 3-level response surface optimization experiment (Table 1). The culture medium volume (A), culture temperature (B) and culture time (C) were used as the investigation factors to determine the optimal culture conditions for Gliocladium albicans, with 3 replicates for each experimental group.

[0054] Table 1 Response surface experimental factors and levels design

[0055]

[0056]

[0057] The response surface experimental design results obtained based on the response surface experimental factors and level design in Table 1 are shown in Table 2. The results of the quadratic regression equation variance analysis of Table 2 using Design Expert 13.0 are shown in Table 3. The multivariate quadratic regression equation of the effective peak number (Y) of VOCs of Glechoma nematocarpa and the culture medium volume (A), culture temperature (B), and culture days (C) is as follows:

[0058] Table 2 Response surface experimental design results

[0059]

[0060]

[0061] As shown in Table 3, the model F value is 29.79, the P value is less than 0.0001, and the model is extremely significant. The P value of the lack of fit term is 0.4663>0.05, and the difference is not significant, indicating that the regression equation has a good fit and high credibility. The model is suitable for the optimization analysis of the culture conditions and result prediction of VOCs of Glechoma nematodes. From the variance analysis, it can be seen that the first-order culture medium volume A has a significant effect on the results, and the first-order culture temperature B and the second-order A2 have an extremely significant effect on the results. According to the influence of the F value on the number of effective peaks of VOCs, they are ranked from high to low, in the order of culture temperature, culture medium volume, and culture time.

[0062] Table 3 Analysis of variance of quadratic regression equation

[0063]

[0064]

[0065] The model response surface and contour plots drawn using Design Expert 13.0, Figure 4 The figure shows the effect of the interaction between any two variables in the culture medium volume, culture temperature, and culture days on the number of effective peaks of VOCs in Glechoma nematodea. In the response surface analysis diagram, the shape of the response surface curve can reflect the strength of the interaction between the two variables. The steeper the slope of the surface, the closer the contour line graph is to the ellipse, indicating that the interaction between the two variables is more significant; otherwise, it is not significant. It can be seen from the figure that the steepness of each surface is AB>BC>AC, but except for AC, the contour ellipses of AB and BC are not obvious, and there is no maximum value in the AC surface. Therefore, it can be seen that the effect of the pairwise interaction between the three variables A, B, and C on the number of effective peaks of VOCs in Glechoma nematodea is not significant, and the conclusion is consistent with Table 3.

[0066] (5) Prediction and verification of optimal culture conditions

[0067] According to the response surface optimization results, when the culture conditions of G. nematodes were 122.708 mL of culture medium volume, 27.5091 °C of culture temperature, and 26.881 days of culture, the number of effective peaks of G. nematodes VOCs under these conditions was the largest, reaching 6.35087. Combined with the actual experimental conditions, the optimal culture conditions were adjusted to 120 mL of culture medium volume, 27.5 °C of culture temperature, and 27 days of culture. Under these conditions, the verification test was carried out, and the relative error between the results and the predicted values ​​was 0.315%, indicating that the experimental results fit the model well, and the optimized culture conditions obtained by the response surface method are feasible and effective.

[0068] Example 4: Analysis of volatile components of Gliocladium schrenkiana with aroma-producing ability

[0069] The volatile detection conditions obtained in Example 1 were used to detect fungal volatiles under the optimal culture conditions given in Example 2, and VOCs with a matching degree greater than 80 (maximum value 100) were screened. The area normalization method was used for quantification, and the NIST14 standard spectral library was used for qualitative analysis. The results are shown in Table 4. The compound structure is as follows Figure 5 As shown, the VOCs chromatographic peaks are Figure 6 shown.

[0070] Table 4 Composition and relative content of VOCs of Glechoma longituba

[0071]

[0072]

[0073] The VOCs of G. nematodes cultured under optimized culture conditions were analyzed by HS-SPME-GC-MS technology, and their chemical composition and relative content are shown in Table 4. As shown in Table 4, after the response surface culture conditions were optimized, a total of 6 VOCs of G. nematodes were detected, including 3 alcohols, 1 aromatic, and 2 sesquiterpenes, with relative contents of 25.05%, 14.82%, and 59.13%, respectively. Among them, the relative contents of 1,2,3,4,4a,5,6,7-octahydro-4-methyl-7-methylene-1-(1-methylethyl)-,(1S,4R,4aS)-naphthalene (5) and (E)-calamene (6) were relatively high, at 29.96% and 29.17%, respectively, which should be the main components of G. nematodes VOCs.

[0074] Example 5: Analysis of aroma components of Gliocladium schrenkiana with aroma-producing ability

[0075] Alcohols, aromatics and terpenes are the main components of VOCs of G. cholerae, which has the ability to produce fragrance. The unique volatile odor characteristics of the above three types of substances should be the main reason why VOCs have floral and fruity fragrance. This study found that 2,4-dimethyl-1-heptanol can be detected throughout the cultivation process of G. cholerae. Whether it can be used as a characteristic volatile of G. cholerae deserves further study. Among the VOCs of G. cholerae, according to the information of Flavornet (http: / / www.flavornet.org / flavornet.html), octanol is described as the taste of vegetables and is used in large quantities as a flavor and fragrance. In addition, it can also effectively recover low concentrations of N,N-dimethylformamide in wastewater as an extractant. It is particularly worth noting that the most abundant VOCs of G. cholerae are sesquiterpenes. Research on the biosynthesis of such compounds in G. cholerae will provide a reference for the further development and application of G. cholerae as a microbial fragrance.

[0076] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for extracting volatiles from Gliocladium schrenkiana with aroma-producing ability, characterized in that: The specific steps include: S1. Activation culture The Glechoma nematode was picked out from the cryopreserved tube with an inoculation needle and inoculated in the middle of a 8.5 cm diameter culture dish containing 20 mL of PSA culture medium, and cultured at 28° C. for 7 days for later use; S2. Culture of Gliocladium schrenkiana Select 500 mL culture flasks as culture containers, and add 120 mL of culture medium to each culture flask; S3. Screening of extraction heads Use a 50 / 30 μm DVB / CAR / PDMS extraction head and age it at 250°C at the gas chromatography injection port for at least 2 hours until there are no impurity peaks. S4. Volatile extraction The cultured Glechoma tenuissora culture bottle was placed under the conditions of extraction temperature of 28°C, equilibrium time of 30 minutes, extraction time of 40 minutes, and analysis time of 3 minutes to extract volatile organic compounds (VOCs).

2. The method for extracting volatiles from Gliocladium schrenkiana with aroma-producing ability and its application according to claim 1, characterized in that: The method steps also include GC-MS analysis.

3. The method for extracting volatiles from Gliocladium schrenkiana with aroma-producing ability and its application according to claim 2, characterized in that: The GC conditions for the GC-MS analysis are as follows: using an HP-5MS chromatographic column (30m×250μm×0.25μm); the carrier gas is helium with a flow rate of 0.8mL / min; non-split injection, the injection port temperature is 250°C; the temperature program is set to an initial temperature of 40°C for 5 minutes, then increase the temperature to 130°C at a rate of 5°C / min and maintain for 5 minutes, then increase the temperature to 230°C at a rate of 10°C / min and maintain for 2 minutes.

4. The method for extracting volatiles from Gliocladium schrenkiana with aroma-producing ability and its application according to claim 2, characterized in that: The MS conditions of the GC-MS analysis are: electron ion source, ion source temperature 230° C.; data acquisition method is full scan, mass range m / z: 35-550u.

5. The method for extracting volatiles from Gliocladium schrenkiana with aroma-producing ability and its application according to claim 1, characterized in that: The culture conditions in the step S2 of culturing Gliocladium angiosporum are: culture medium volume 120 mL, culture temperature 27.5° C., culture days 27 days, carbon source is sucrose, no nitrogen source is added, and pH value is natural.

6. Use of the volatiles of Gliocladium schrenkiana with aroma-producing ability according to any one of claims 1 to 5 in the production of fragrances, flavors and fragrances.

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