Method for extracting volatile substances of gongronella sp. having aroma-producing capacity and application thereof

Abstract

The application provides an extractive method of volatile substances of Mortierella isabellina with fragrance-producing capacity and application thereof, and relates to the technical field of extractive method of volatile substances of Mortierella isabellina. The extractive method of volatile substances of Mortierella isabellina with fragrance-producing capacity specifically comprises the following steps: S1. activating culture; S2. Mortierella isabellina culture; S3. selection of extraction head; and S4. volatile substance extraction. The application provides the extractive method of volatile substances of Mortierella isabellina with fragrance-producing capacity and application thereof, and the extractive and culture method of Mortierella isabellina and volatile substance components thereof is displayed. Compared with the prior art, the main advantage lies in that the extraction efficiency and precision of volatile substances are improved, and the culture condition of Mortierella isabellina is optimized to enhance the fragrance-producing capacity. These research results not only improve the detection sensitivity and production yield of target compounds, but also reduce the use of chemical reagents, reduce the production cost and environmental impact, and provide a new possibility for the development and application of natural spices.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of Clonostachys rosea volatile substance extraction, in particular to a Clonostachys rosea volatile substance extraction method with fragrance production capacity and application thereof. BACKGROUND

[0002] Fragrance substances are attracting attention due to their pleasant and stress-relieving characteristics, and people's application of such substances has penetrated into various aspects of life, such as food light industry, cosmetic manufacturing, etc. The application of fragrance substances relies on perfume and flavor, and traditional perfume and flavor sources 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 in consumption and low in yield, and cannot meet market demand. Compared with traditional methods, the method of microbial fermentation and biotransformation has many advantages such as high efficiency, green and environmental protection, and has become a research hotspot for the development of perfume and flavor. Therefore, exploring microorganisms with fragrance production function has become an urgent demand of the perfume and flavor market.

[0003] In recent years, fungi have become the focus of attention in the production of flavoring microorganisms. Volatile organic compounds (VOCs) produced by fungi include a variety of chemical classes and are used in industrial production. For example, Zhang Peng et al. used melanin-producing Aureobasidium melanogenum fermentation broth to improve the aroma of tobacco, and Zhou Sen et al. screened out Geotrichum candidum with liquor flavor. Clonostachys rosea f. catenulata belongs to Ascomycota Sordariomycetes Hypocreales Bionectriaceae Clonostachys, which is a variant of Clonostachys rosea. The fungus has a wide distribution area and exists in soil, desert and water in various temperature zones. As a facultative saprophyte, the fungus often coexists with various pathogenic fungi, mycelium and nematode cysts in 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 isolation parts, and the highest frequency part is the root. At the same time, the fungus can show endophytic ability in different tissue parts of plants, such as geranium leaves and cucumber roots. Previous studies on Clonostachys rosea f. catenulata mainly focused on the development of biocontrol agents as hyperparasitic fungi and the control of soil-borne pathogens. Compared with other flavor-producing fungi, Clonostachys rosea f. catenulata has the unique ability to parasitize other fungi and exchange substances, thereby expanding the source of its metabolites. At present, most of the studies on Clonostachys rosea f. catenulata are focused on the non-volatile components in the metabolites, and there are few reports on the volatile components of Clonostachys rosea f. catenulata. Combined with the high application value of Clonostachys rosea f. catenulata and its ability to expand the source of metabolites, the development of Clonostachys rosea f. catenulata with flavor-producing ability has great market application prospect, but at present, there are few studies on the volatile components of Clonostachys rosea f. catenulata, so the development of its application in the field of microorganism-derived flavors has great potential. SUMMARY

[0004] (I) Technical problems solved

[0005] In view of the shortcomings of the prior art, the present application provides a Clonostachys rosea f. catenulata volatile component extraction method and application thereof, To some extent compensate The problem of few studies on Clonostachys rosea f. catenulata in its volatile components is solved.

[0006] (II) Technical solutions

[0007] In order to achieve the above object, the present application is implemented by the following technical scheme: a volatile extraction method of gongronella multiformis with fragrance production capacity, specifically comprising the following steps:

[0008] S1. Activation culture

[0009] The gongronella multiformis is 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 PSA medium, and incubated at 28 DEG C for 7 days for standby;

[0010] S2. Gongronella multiformis culture

[0011] 500 mL culture bottles are selected as culture containers, and 120 mL of culture medium is added to each culture bottle;

[0012] S3. Selection of extraction head

[0013] A 50 / 30 μm DVB / CAR / PDMS extraction head is used, and the gas chromatography inlet is aged at 250 DEG C for at least 2 hours until no impurity peak is obtained for standby;

[0014] S4. Volatile extraction

[0015] The cultured gongronella multiformis culture bottle is placed in the condition of extraction temperature 28 DEG C, equilibrium time 30 minutes, extraction time 40 minutes, and analysis time 3 minutes for extraction of volatile organic compounds (VOCs).

[0016] Preferably, the method step further comprises GC-MS analysis.

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

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

[0019] Preferably, the culture conditions in the step S2 gongronella multiformis culture are: culture medium volume 120 mL, culture temperature 27.5 DEG C, culture time 27 days, carbon source sucrose, no nitrogen source added, and natural pH value.

[0020] The application of a gongronella multiformis volatile with fragrance production capacity in the production of fragrance, essence and spice.

[0021] (III) Beneficial effects

[0022] The present application provides a method for extracting volatile substances from Myceliopthora thermophila with fragrance-producing ability and its application. The method has the following beneficial effects:

[0023] The present application provides a method for extracting volatile substances from Myceliopthora thermophila with fragrance-producing ability and its application. The method can efficiently and accurately extract volatile organic compounds (VOCs) from Myceliopthora thermophila by using a carefully selected SPME (solid phase microextraction) extraction head (50 / 30 μm DVB / CAR / PDMS) and optimized GC-MS (gas chromatography-mass spectrometry) analysis conditions. This extraction and analysis method improves the sensitivity and resolution of detection, making the identification of volatile substances more accurate while maintaining the integrity of the components. Through meticulous experimental design and optimization, the optimal culture conditions for Myceliopthora thermophila are found, including culture medium volume, temperature, and culture time. The optimization of these conditions not only improves the growth and metabolic activity of the strain, but also significantly enhances its fragrance-producing ability. The experimental results are well fitted with the model, verifying the feasibility and effectiveness of the optimized culture conditions. Through the application of these two technologies, not only does it provide a standardized method for the extraction and analysis of Myceliopthora thermophila volatile substances, but also lays a solid foundation for further research and development of its applications in the fields of fragrance, essence, and related fields, especially in the fields of food and cosmetics. This naturally derived fragrance has potential market advantages, and using Myceliopthora thermophila as a natural source of fragrance avoids the environmental and health risks that may be caused by chemical synthesis of fragrances. In addition, the optimized culture conditions reduce the dependence on chemical reagents and the generation of chemical waste during the experiment, in line with the principles of green chemistry and sustainable development. By improving the culture efficiency and the accuracy of volatile substance extraction, the waste of experimental materials and time is reduced, thereby reducing production costs. This makes commercial production of Myceliopthora thermophila extract possible, which is conducive to achieving a win-win situation of economic benefits and environmental protection. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The present application provides a method for extracting volatile substances from Myceliopthora thermophila with fragrance-producing ability and its application. The method has the following beneficial effects:

[0025] Figure 2 The present application provides a method for extracting volatile substances from Myceliopthora thermophila with fragrance-producing ability and its application. The method has the following beneficial effects:

[0026] Figure 3 The present application provides a method for extracting volatile substances from Myceliopthora thermophila with fragrance-producing ability and its application. The method has the following beneficial effects:

[0027] Figure 4 Figure for the influence of the interaction of different culture conditions on the number of VOCs effective peaks of Gliocladium virens under the response surface experiment of the present application;

[0028] Figure 5 Figure for the chemical structure of VOCs of Gliocladium virens of the present application;

[0029] Figure 6 Figure for the total ion current chromatogram of VOCs of Gliocladium virens analyzed by GC-MS. DETAILED DESCRIPTION

[0030] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

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

[0032] (1) Selection of extraction head

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

[0034] Three specifications of extraction heads, 50 / 30 pm DVB / CAR / PDMS, 75 pm CAR / PDMS, and 100 pm PDMS, were aged at a temperature of 250°C for at least 2 hours at the gas chromatography injection port until no impurity peaks were obtained for standby use. The cultured Gliocladium virens culture bottle was selected and placed under the conditions of an extraction temperature of 28°C, an equilibrium time of 30 min, an extraction time of 40 min, and an analysis time of 3 min for VOCs extraction, and then detected according to the GC-MS analysis conditions, with the number of VOCs effective peaks as the index, to select the appropriate extraction head. The adsorption effects of different extraction heads are different due to the differences in coating materials, and the uncertainty of volatile components can affect the adsorption effect of the extraction head, so the extraction head selection should be performed at the beginning of the experiment. The effective peaks detected were used as the index to compare the adsorption capacity of the extraction heads of three different materials. As shown in Table 1, the adsorption capacity of 50 / 30 pm DVB / CAR / PDMS to VOCs of Gliocladium virens was the best, so 50 / 30 pm DVB / CAR / PDMS extraction head was selected as the most suitable extraction head. Figure 1

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

[0036] ​The GC conditions need to be set in combination with the chromatographic peak conditions of Clonostachys rosea f. catenulatum volatile substances. To obtain the optimal GC conditions for Clonostachys rosea f. catenulatum volatile substances, the initial temperature was set to 40°C, and the temperature was increased to 230°C at a rate of 5°C / min, and the helium flow rate was 1.0 mL / min to observe the chromatographic peak separation. The final conditions were obtained by reducing the helium flow rate and changing the temperature increase rate:

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

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

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

[0040] Example 3: Optimization of culture medium and culture conditions of Clonostachys rosea f. catenulatum with aroma-producing ability

[0041] (1) Activation culture of test fungi

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

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

[0044] Agar was added to potato extract to prepare the base medium, and the carbon source and nitrogen source optimization of the culture conditions was carried out. 500 mL culture bottles were used as Clonostachys rosea f. catenulatum culture containers, 120 mL of culture medium was added to each culture bottle, 4 different carbon sources (sucrose, glucose, maltose, soluble starch) were added to the culture medium at a concentration of 20 g / L, and after the culture medium cooled and solidified, a Clonostachys rosea f. catenulatum cake with a diameter of 5 mm was inoculated in the center of the culture medium, and incubated at 26°C for 20 days. The number of effective VOCs peaks was used as an index for GC-MS analysis, and the optimal carbon source was screened. Based on the optimal carbon source, different types of nitrogen sources (ammonium chloride, ammonium sulfate, peptone, potassium nitrate) were added at a concentration of 5 g / L, and the number of effective VOCs peaks was determined under the above culture conditions to screen the optimal nitrogen source. All tests were repeated 3 times.

[0045] The selection of carbon source will affect the number of effective peaks. The results are as follows Figure 2As shown, when the carbon source is sucrose, the number of effective peaks of VOCs of G. avenium is the most, 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 is in the order of KNO3> peptone > (NH4)2SO4> NH4Cl. Compared with no nitrogen source, the number of effective peaks shows a downward trend as a whole after adding nitrogen source to the culture medium, so the tested nitrogen source is not suitable for the production of mycelial VOCs of G. avenium. The optimized culture medium for the subsequent test is sucrose as the carbon source without nitrogen source.

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

[0047] Using the optimized culture medium, the influence of the factors of pH value (5, 6, 7, 8), medium volume (liquid loading amount) (80 mL, 100 mL, 120 mL, 140 mL), culture temperature (24°C, 26°C, 28°C, 30°C) and culture time (22 d, 24 d, 26 d, 28 d) on the volatile components of G. avenium is investigated, with the number of effective peaks of VOCs as the index. All tests are repeated three times.

[0048] Using the optimized culture medium as the basis for the culture of G. avenium, the influence of different culture conditions on the number of effective peaks of VOCs of G. avenium is studied, and the results are shown in Table 3. Figure 3 Firstly, the pH value in the culture conditions is changed to explore the influence of different pH values on the number of effective peaks of VOCs of G. avenium. The number of effective peaks is the most at pH 7, but the change of pH value in the range of pH 5-8 has no obvious influence on the number of effective peaks, so the pH value of the culture medium is kept at the natural level.

[0049] With the pH value at the natural level, the medium volume in the culture conditions is changed to study the influence of the medium volume on the number of effective peaks of VOCs of G. avenium. The number of effective peaks shows a trend of first decreasing, then increasing and then decreasing with the increase of the medium volume, and the number of effective peaks is the most at the medium volume of 120 mL, followed by 140 mL, 80 mL and 100 mL, so the optimal medium volume is selected as 120 mL.

[0050] Under the culture conditions of the aforementioned natural pH value and medium volume of 120 mL, the influence of different culture days on the number of effective peaks of VOCs of G. avenium is studied. With the increase of culture days, the number of effective peaks shows a trend of first increasing and then decreasing, and the number of effective peaks is the most at 26 d, so the optimal culture day is selected as 26 d.

[0051] On the basis of the aforementioned optimization of factors, the influence of different culture temperatures on the number of effective peaks of VOCs of G. avenium is studied. With the increase of culture temperature, the number of effective peaks shows a trend of first increasing and then decreasing, and the number of effective peaks is the most at the culture temperature of 28°C, so the optimal culture temperature is selected as 28°C.

[0052] (4) Volatile components affect response surface test

[0053] According to the results of step 3 single factor test, according to the principle of Box-Behnken combination design, Design-Expert.V8.0 software was used to design 3-factor 3-level response surface optimization scheme test (Table 1), with medium volume (A), culture temperature (B), culture time (C) as the observation factors, to determine the optimal culture conditions of L. theicola, with 3 replicates for each test group.

[0054] Table 1 Response surface experimental factor and level design

[0055]

[0056]

[0057] The response surface experiment design results obtained according to the response surface experiment factor and level design of Table 1 are shown in Table 2. The results of variance analysis of quadratic regression equation of Table 2 by Design Expert 13.0 are shown in Table 3, and the multivariate quadratic regression equation of L. theicola VOCs effective peak number (Y) and medium volume (A), culture temperature (B), culture time (C) is as follows:

[0058] Table 2 Response surface test design results

[0059]

[0060]

[0061] From Table 3, the model F value is 29.79, and the P value is <0.0001, which is extremely significant. The P value of the lack-of-fit term is 0.4663>0.05, which is not significant, indicating that the regression equation has good fitting degree and high reliability, and the model is applicable to the analysis and prediction of the culture conditions of L. theicola VOCs. According to the variance analysis, the effect of medium volume A on the results reaches a significant level, and the effects of culture temperature B and A2 on the results reach an extremely significant level. According to the F value of VOCs effective peak number, the effects of culture temperature, medium volume, and culture time are ranked from high to low.

[0062] Table 3 Variance analysis of quadratic regression equation

[0063]

[0064]

[0065] The model response surface curve and contour plot drawn by Design Expert 13.0, Figure 4 The effects of the interaction between any two variables of culture medium volume, culture temperature and culture time on the number of VOCs effective peaks of G. fimbriatum were shown in the response surface analysis graph. In the response surface curve graph, the shape of the curve can reflect the strength of the interaction between two variables. The steeper the slope of the curve, the closer the contour graph to the elliptical shape, indicating that the interaction between two variables is more significant. Conversely, it is not significant. As can be seen from the figure, the steepness of each curve is AB>BC>AC, but except for AC, the contour ellipse of AB and BC is not obvious, and the AC curve does not present a maximum value. Therefore, it can be known that the interaction between two variables of A, B and C has no significant effect on the number of VOCs effective peaks of G. fimbriatum. The conclusion is consistent with Table 3.

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

[0067] According to the response surface optimization results, when the culture conditions of G. fimbriatum are culture medium volume 122.708 mL, culture temperature 27.5091℃ and culture time 26.881 d, the number of VOCs effective peaks of G. fimbriatum under this condition can reach 6.35087. Combined with the actual experimental conditions, the optimal culture conditions are adjusted to culture medium volume 120 mL, culture temperature 27.5℃ and culture time 27 d. Under this condition, the verification test is carried out, and the relative error between the obtained result and the predicted value is 0.315%, which shows that the experimental result is well fitted with the model. The optimized culture conditions obtained by using the response surface method are feasible and effective.

[0068] Example 4: Analysis of volatile components of G. fimbriatum with aroma-producing ability

[0069] The volatile compounds of the fungus under the optimal culture conditions given in Example 2 were detected using the volatile detection conditions obtained in Example 1. The VOCs with a matching degree greater than 80(maximum value 100) were screened, quantified by area normalization method, and qualitatively analyzed by using NIST14 standard spectral library. The results are shown in Table 4, the compound structures are shown in Figure 5 , and the VOCs chromatographic peaks are shown in Figure 6 .

[0070] Table 4 Composition and relative content of VOCs of G. fimbriatum

[0071]

[0072]

[0073] VOCs of *Nematospora* cultured under optimized conditions were analyzed using HS-SPME-GC-MS. The chemical composition and relative content are shown in Table 4. Table 4 shows that after optimization of the response surface methodology, six VOCs from *Nematospora* were detected, including three alcohols, one aromatic compound, and two sesquiterpenes, with relative contents of 25.05%, 14.82%, and 59.13%, respectively. Among them, 1,2,3,4,4a,5,6,7-octahydro-4-methyl-7-methylene-1-(1-methylethyl)-,(1S,4R,4aS)-naphthalene (5) and (E)-acorene (6) had relatively high contents, at 29.96% and 29.17%, respectively, and should be the main components of *Nematospora* VOCs.

[0074] Example 5: Aroma component analysis of *Nematocephalosporium* with aroma-producing ability

[0075] Alcohols, aromatic compounds, and terpenes constitute the main VOCs of *Nematocystis*, a fungicide, and the unique volatile odor characteristics of these three groups are the primary reason for the floral and fruity aromas of the VOCs. This study found that 2,4-dimethyl-1-heptanol was detectable throughout the entire culture process of *Nematocystis*, and whether it can be considered a characteristic volatile of *Nematocystis* warrants further investigation. Among the VOCs of *Nematocystis*, according to FlavorNet (http: / / www.flavornet.org / flavornet.html), octanol is described as having a vegetable flavor and is widely used as a flavoring agent. Furthermore, it can effectively recover low concentrations of N,N-dimethylformamide from wastewater as an extractant. Of particular note is that sesquiterpenes are the most abundant VOCs in *Nematocystis*. Research on the biosynthesis of these compounds in *Nematocystis* will provide a reference for the further development and application of *Nematocystis* as a microbial fragrance.

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

Claims

1. A method for extracting volatiles from *Nematostella spp.* with aroma-producing capabilities, characterized in that, Specifically comprising the following steps: S1. Activation culture The Clonostachys rosea was picked up from the preserved frozen tube with a inoculation needle and inoculated in the middle of a 8.5 cm diameter culture dish containing 20 mL PSA medium, and incubated at 28℃ for 7 days for standby; S2. Clonostachys rosea culture 500 mL culture bottles were selected as culture containers, and 120 mL medium was added to each culture bottle; S3. Screening of extraction head A 50 / 30 μm DVB / CAR / PDMS extraction head was used, and the gas chromatography inlet was aged at 250℃ for at least 2 hours until no impurity peak was obtained for standby; S4. Volatile extraction The cultured Clonostachys rosea culture bottle was placed in the conditions of extraction temperature 28℃, equilibrium time 30 minutes, extraction time 40 minutes, and analysis time 3 minutes for extraction of volatile organic compounds (VOCs); The method step further comprises GC-MS analysis; The GC conditions of the GC-MS analysis are: using HP-5MS chromatographic column (30 m x 250 μm x 0.25 μm); carrier gas is helium, flow rate is 0.8 mL / min; no split injection, inlet temperature is 250℃; temperature programming is set as initial temperature 40℃ for 5 minutes, temperature rising to 130℃ at a rate of 5℃ / min and keeping for 5 minutes, and then temperature rising to 230℃ at a rate of 10℃ / min and keeping for 2 minutes.

2. The method for extracting volatile substances from Drechslera dictyoides according to claim 1, characterized in that: The MS conditions of the GC-MS analysis are: electron ion source, ion source temperature 230℃; data acquisition method is full scan, mass range m / z: 35-550u.

3. The method of claim 1, wherein the D. hancei volatile compound is extracted by the following steps: (1) inoculating the D. hancei into a culture medium; (2) culturing the D. hancei in the culture medium; (3) collecting the D. hancei; (4) extracting the D. hancei volatile compound. The culture conditions in the step S2 Clonostachys rosea culture are: medium volume 120 mL, culture temperature 27.5℃, culture time 27 days, carbon source is sucrose, no nitrogen source is added, and pH value is natural.

4. Application of a Clonostachys rosea volatile with aroma-producing capacity according to any one of claims 1-3 in the production of fragrance, essence and flavor.

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