A method for producing flavor by co-culturing myceliophthora thermophila and micromyces parcellaris
By co-culturing *Neurospora* and *Cladosporium brevicornum* and using specific combinations and culture conditions, metabolic pathways were activated, resulting in the production of a variety of specific aroma compounds. This solved the problem of insufficient aroma production by a single fungal species and provided abundant natural fragrance resources.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SOUTHWEST FORESTRY UNIVERSITY
- Filing Date
- 2026-01-19
- Publication Date
- 2026-05-12
AI Technical Summary
Existing microbial aroma production technologies mostly rely on single strains, resulting in a limited variety of aroma compounds. Furthermore, research on aroma production through co-culture of filamentous fungi is insufficient, making it difficult to meet market demand for products with rich aroma layers and distinctive characteristics.
A co-culture method using *Neurospora spp.* and *Cladosporium brevicornu* was employed, with the mixture cultured in PSA solid medium using a five-point confrontation method. Headspace solid-phase microextraction and gas chromatography-mass spectrometry were then used to identify co-culture-specific volatile organic compounds.
It activated metabolic pathways that were 'silent' when cultured alone, induced the production of five specific aroma compounds, enriched the microbial aroma resource library, provided a stable fragrance resource, and has clear application potential.
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Figure CN121555582B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, specifically to a method for producing aroma through co-culturing of *Alternaria solani* and *Cladosporium brevicornum*. Background Technology
[0002] Aroma compounds are a class of compounds that include esters, aldehydes, alcohols, ketones, terpenes, and organic acids, and possess characteristic aromas. They are widely used not only in food flavoring, cosmetic fragrance preparation, and other fields, but some also have antioxidant and antibacterial activities, and can be extended to be used as fuels, solvents, or natural insecticides, making them of great value in industrial production.
[0003] Currently, the acquisition of volatile aroma compounds mainly relies on three major approaches: First, direct extraction from natural plants and animals. This method is significantly limited by the seasonality of raw material growth, and the content of target compounds in natural raw materials is generally low, resulting in high extraction costs and limited output, making it difficult to meet the needs of large-scale industrial production. Second, chemical synthesis. Although it dominates the field of artificial fragrances due to the availability and low cost of raw materials, the synthesis process is complex, and toxic and harmful byproducts are easily generated during the reaction. This may not only cause environmental pollution, but the byproduct residues may also affect the safety of aroma compounds. Moreover, chemically synthesized products cannot be labeled as "natural," which contradicts the current consumer demand for healthy and natural products. Third, the biotechnology approach, which utilizes microbial fermentation or biotransformation for production. This method has become a research hotspot in aroma compound development due to its advantages such as mild reaction conditions, environmental friendliness, high product safety, and the ability to be labeled as "natural."
[0004] Current biotechnology research focuses primarily on screening and applying aroma-producing microorganisms related to brewing and sauce fermentation, as well as lactic acid bacteria in dairy fermentation. Exploration of other types of microorganisms, such as filamentous fungi, is limited, resulting in significant limitations in the exploitation of microbial aroma-producing resources. Furthermore, current mainstream microbial aroma-producing technologies mostly employ single-strain fermentation systems. Constrained by the fixed metabolic pathways and limited metabolic capacity of individual strains, the resulting aroma compounds generally exhibit limited variety and low concentration, failing to meet market demands for products with rich and distinctive aromas. Co-culture strategies, by constructing multi-strain interaction culture systems, hold the promise of activating metabolic pathways that are "silent" during individual cultivation, thereby regulating metabolite synthesis and enhancing aroma compound diversity, providing a potential direction for overcoming the technical bottlenecks of single-strain fermentation. However, research reports on aroma production through co-culture of filamentous fungi are extremely scarce, especially regarding *Nematospora* (…). Clonostachys rosea f.catenulata ) and short-haired trichomes ( Lachnum brevipilosumNo research has been published on the co-culture of filamentous fungi to induce the production of specific aroma compounds. There is an urgent need to develop aroma production methods based on the co-culture of these filamentous fungi in order to enrich the microbial aroma production technology pathways and aroma compound resource library. Summary of the Invention
[0005] Technical problems to be solved
[0006] To address the shortcomings of existing technologies, this invention provides a method for aroma production using the co-culture of *Alternaria solani* and *Cladosporium brevicornu*. This method solves the problems of existing microbial aroma production relying on a single species, limited metabolic pathways leading to a limited variety of aroma compounds, insufficient research on aroma production through co-culture of filamentous fungi, and inadequate exploration of natural fragrance resources.
[0007] Technical solution
[0008] To achieve the above objectives, the present invention provides the following technical solution: a method for producing aroma through co-cultivation of *Nepeta spp.* and *Cladosporium brevicornu*, comprising the following steps:
[0009] S1 strain activation: *Alternaria solani* (… Clonostachys rosea f.catenulata ) and short-haired trichomes ( Lachnum brevipilosum The ingredients were inoculated into PSA solid medium for activation culture; each 1L of PSA solid medium consisted of: 200g potato, 20g sucrose, 18g agar, with the remainder being distilled water, and the pH value being natural.
[0010] S2 Co-culture Inoculation and Cultivation: The activated *Neurospora* and *Clerodendrum brevis* from step S1 were inoculated into the same PSA solid medium in a culture container using the five-point confrontation method and incubated in the dark at 27.5℃. The five-point confrontation method is as follows: *Neurospora* mycelium is inoculated in the center of the medium, and *Clerodendrum brevis* mycelium is inoculated at four points 2.5 cm away from the center on a cross line extending from the center point. The diameter of each mycelium is 5 mm.
[0011] S3 Volatile Collection and Analysis: Starting from the second day after inoculation, headspace volatiles of the culture were collected every 48 hours using headspace solid-phase microextraction (HS-SPME). Subsequently, the collected volatiles were analyzed by gas chromatography-mass spectrometry (GC-MS) to identify co-culture-specific volatile organic compounds.
[0012] Preferably, the activation culture conditions described in S1 are: dark culture at 28±1℃ for 14 days.
[0013] Preferably, the culture container in S2 is a 500mL culture flask, and the volume of PSA solid culture medium poured into the culture flask is 120mL.
[0014] Preferably, the headspace solid-phase microextraction method described in S3 uses a 50 / 30μm DVB / CAR / PDMS extraction head, with an adsorption extraction temperature of 27.5℃ and an adsorption extraction time of 40 minutes.
[0015] Preferably, the conditions for the gas chromatography-mass spectrometry (GC-MS) analysis described in S3 are as follows:
[0016] GC conditions: HP-5MS column, 30m×250μm×0.25μm; helium as carrier gas, flow rate 0.8mL / min; injection port temperature 250℃; splitless injection; temperature program: initial temperature 40℃, hold for 5min, increase to 130℃ at 5℃ / min, hold for 5min, then increase to 230℃ at 10℃ / min, hold for 2min.
[0017] MS conditions: Electron impact ion source was used; ion source temperature was 230℃; scan range was 35-550 m / z.
[0018] Beneficial effects
[0019] This invention provides a method for aroma production using the co-culture of *Alternaria solani* and *Cladosporium brevicornu*. It has the following beneficial effects:
[0020] 1. This invention provides a method for aroma production by co-culturing *Neurospora* and *Cladosporium breviculatum*. This method utilizes a specific combination of *Neurospora* and *Cladosporium breviculatum* in an optimized PSA solid medium and a five-point confrontation co-culture mode. By utilizing the hyphal contact or volatile signaling molecules-mediated interspecific interactions between the two fungi, the "silent" secondary metabolic pathways during individual culture are effectively activated, ultimately inducing the production of five specific volatile organic compounds (cyclooctatetraene, n-octyl chloroformate, phenylethanol, cis-muurola-4(15),5-diene, and 1,6-dimethyl-4-isopropyltetrahydronaphthalene). These compounds were not detected in the individual culture systems of the two fungi, greatly enriching the variety of aroma compounds from microorganisms and supplementing the natural fragrance resource library with new functional components.
[0021] 2. This invention provides a method for aroma production using the co-culture of *Neurospora* and *Cladosporium brevis*. This method ensures the stability and controllability of the co-culture system by fixing key process parameters such as the composition of PSA solid culture medium (potato 200 g / L, sucrose 20 g / L, agar 18 g / L, natural pH), a dark static culture temperature of 27.5℃, and a standardized HS-SPME-GC-MS acquisition and analysis process. The appearance time of specific aroma compounds has a clear regularity (e.g., cyclooctatetraene appears on day 15, and n-octyl chloroformate appears on day 25), and can be stably detected in multiple biological replicates. At the same time, among the specific compounds produced, phenylethanol, cis-muurola-4(15),5-diene, and 1,6-dimethyl-4-isopropyltetrahydronaphthalene are all components with clear aroma characteristics reported in the literature, which can be directly applied to food additives, flavoring preparation, and other fields, solving the problems of single product types and unclear application potential in existing microbial aroma production methods. Attached Figure Description
[0022] Figure 1 This is the growth of *Alternaria spp.* and *Cladosporium brevicornum* co-cultured on PDA medium in Example 3 of the present invention;
[0023] Figure 2 This is a schematic diagram of the chemical structure of newly generated VOCs from the co-culture of *Alternaria solani* and *Cladosporium brevicornum* according to the present invention.
[0024] 1: Cyclooctatetraene;
[0025] 2: n-Octyl chloroformate;
[0026] 3: Phenethyl alcohol;
[0027] 4: cis-muurola-4(15),5-diene;
[0028] 5: 1,6-Dimethyl-4-isopropyl-1,2,3,4-tetrahydronaphthalene.
[0029] Figure 3 This is the total ion chromatogram of VOCs from the co-culture of *Alternaria solani* and *Cladosporium brevicornu* as analyzed by GC-MS in this invention. Detailed Implementation
[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0031] The following detailed description of the method for aroma production using the co-culture of *Alternaria solani* and *Cladosporium brevicornum* as described in this invention, based on specific experimental operations and test results, is provided below. Unless otherwise specified, the experimental methods used are all routine operations in the field of microbial culture; the materials and reagents used are all commercially available. This description aims to further clarify the technical solution of this invention, rather than to limit its scope of protection.
[0032] Example 1: Preparation of PSA solid culture medium
[0033] The PSA solid culture medium used in the co-culture system of this invention is prepared according to the following steps for each 1L:
[0034] 1. Raw material preparation: Weigh out 200g of fresh potatoes, 20g of sucrose, and 18g of agar, and set aside;
[0035] 2. Potato processing: Peel the potatoes and cut them into 1cm×1cm pieces. Add 500mL of distilled water and heat on an electric stove until boiling. Maintain a gentle boil for 30 minutes, stirring constantly to prevent burning.
[0036] 3. Filtration and volume adjustment: Filter the above potato decoction through four layers of gauze, collect the filtrate into a 1L volumetric flask, add 300mL of distilled water to the residue and boil again for 10min, filter and combine the two filtrates, add sucrose and agar to the filtrate, stir until completely dissolved, and finally add distilled water to the 1L mark. At this time, the pH of the culture medium is at its natural state (no additional adjustment is required).
[0037] 4. Sterilization and preparation: Dispense the prepared culture medium into Erlenmeyer flasks, stopper them, wrap the mouths with kraft paper, and sterilize them in a 121℃ autoclave for 25 minutes. After sterilization, remove the Erlenmeyer flasks and wait for the culture medium temperature to drop to 50-55℃. Pour the medium onto plates or dispense it into culture containers in a clean bench and allow it to cool and solidify before use.
[0038] Example 2: Activation culture of the test strain
[0039] 1. Culture medium preparation: Take the sterilized and cooled PSA culture medium from Example 1, pour it into sterile glass petri dishes with a diameter of 8.5 cm in a laminar flow hood, pour about 20 mL of culture medium into each dish, and place them horizontally until they are completely solidified to make PSA solid plates.
[0040] 2. Preparation of mycelial cakes: Aseptic techniques were employed, and a 5mm diameter punch was used to extract mycelial cakes from the molds containing *Neurospora* (…). Clonostachys rosea f.catenulata ), *Cladosporium brevicornum* ( Lachnum brevipilosum On the slant culture medium, obtain the mycelial cakes with vigorous edge growth to ensure that the mycelial cakes are of uniform thickness and free from contamination by other microorganisms;
[0041] 3. Inoculation and culture: The mycelial cakes of the two fungi mentioned above were inoculated into the center of PSA solid plates. After inoculation, the plates were inverted and placed in a constant temperature incubator. They were cultured in the dark at 28±1℃ for 14 days. During this period, the growth status of the strains was observed regularly to ensure that there was no contamination by other microorganisms. After the culture was completed, the activated pure strains were obtained and ready for use.
[0042] Example 3: Solid co-culture of *Alternaria solani* and *Cladosporium brevicornum*
[0043] 1. Preparation of culture container: Take a 500mL sterile Erlenmeyer flask, pour 120mL of PSA solid culture medium prepared in Example 1 into the ultra-clean workbench, and use it as a co-culture carrier after cooling and solidification.
[0044] 2. Inoculation procedure: The five-point confrontation method was used for inoculation. Specifically, the activated *Streptomyces spp.* from Example 2 was picked up with sterile forceps and inoculated into the center of the culture medium in the Erlenmeyer flask. Then, four activated *Clerodendrum spp.* mycelium ...
[0045] 3. Control setup: Three control experiments were set up simultaneously: "Alternaria spp. culture group" (Alternaria spp. mycelium was inoculated only in the center of the culture medium), "Alternaria spp. culture group" (Alternaria spp. mycelium was inoculated only at the four points of the cross on the culture medium), and "blank culture group" (no mycelium was inoculated). Each group had 3 biological replicates.
[0046] 4. Cultivation process: The Erlenmeyer flasks of the co-culture group and the control group were inverted and placed in a constant temperature incubator. They were then incubated statically at 27.5℃ in the dark. During the cultivation period, the mycelial growth was observed daily, and the time of mycelial convergence between the two mycelia and the changes in the morphology of the culture medium surface were recorded.
[0047] Example 4: Headspace sampling of volatiles from a co-culture system
[0048] 1. Sampling preparation: Starting from the day of inoculation, volatile matter was collected from the co-culture group and the control group every 48 hours from the second day onwards. Before collection, the Erlenmeyer flasks were removed from the incubator and sealed with sterile sealing film in a laminar flow hood, and equilibrated to room temperature (27.5℃).
[0049] 2. Extraction procedure: Headspace solid-phase microextraction (HS-SPME) was used. A 50 / 30μm DVB / CAR / PDMS coated extraction head was selected and inserted into the headspace area of the Erlenmeyer flask (avoiding contact with the culture medium and mycelium). Adsorption extraction was performed at 27.5℃ for 40 minutes. During the extraction process, the Erlenmeyer flask was kept still to avoid shaking, which would affect the extraction efficiency.
[0050] 3. Post-extraction processing: After extraction, immediately remove the extraction head and quickly insert it into the injection port of the gas chromatography-mass spectrometry (GC-MS) instrument for desorption analysis. After each sample is collected, age the extraction head in the injection port at 250℃ for 5 minutes to remove residual impurities and avoid cross-contamination.
[0051] Example 5: GC-MS Analysis of Volatile Compounds
[0052] 1. GC conditions: An HP-5MS capillary column (30m × 250μm × 0.25μm) was used; high-purity helium (purity ≥ 99.999%) was used as the carrier gas, and the flow rate was controlled at 0.8mL / min; the injection port temperature was set to 250℃, and splitless injection mode was used; the temperature program was as follows: initial temperature 40℃, hold for 5 minutes; then increase the temperature to 130℃ at a rate of 5℃ / min, hold for 5 minutes; then increase the temperature to 230℃ at a rate of 10℃ / min, hold for 2 minutes.
[0053] 2. MS settings: Electron impact ionization (EI) source was used, with an ion source temperature of 230℃, an electron energy of 70 eV, a scan range of m / z 35-550, and a solvent delay time of 3 minutes to avoid interference from solvent peaks on the detection of the target compound.
[0054] 3. Data Acquisition and Processing: Start the GC-MS system. After the instrument stabilizes, insert the extraction head containing the volatiles from Example 4 into the injection port for desorption for 5 minutes, and start data acquisition simultaneously. After acquisition, use the instrument's data analysis software and the NIST14 standard spectral library for qualitative analysis of the compounds, retaining only compounds with a library matching degree greater than 80 (maximum value is 100). The relative content of each compound is calculated using the area normalization method for semi-quantitative analysis.
[0055] Example 6: Identification and Result Analysis of Co-cultured Specific Volatile Compounds
[0056] 1. Screening for specific compounds: Comparison of GC-MS total ion chromatograms of the co-culture group, the *Alternaria* monoculture group, the *Cladosporium brevis* monoculture group, and the blank culture group (see...) Figure 3 Compounds that appear only in the co-culture group and are not detected in the single culture group and blank group are identified as co-culture specific volatile organic compounds.
[0057] 2. Identification Results: Analysis revealed that five volatile organic compounds were specifically produced in the co-culture system. Specific details are as follows:
[0058] Compound 1: Cyclooctatetraene, with a retention time (RT) of 8.916 min, was first detected on day 15 of co-culture;
[0059] Compound 2: n-Octyl chloroformate, RT 13.312 min, first detected on day 25 of co-culture;
[0060] Compound 3: Phenethyl alcohol, RT 15.347 min, was stably detected on co-culture days 31 and 33;
[0061] Compound 4: cis-muurola-4(15),5-diene, RT 29.496 min, was stably detected on co-culture days 25 and 41;
[0062] Compound 5: 1,6-Dimethyl-4-isopropyl-1,2,3,4-tetrahydronaphthalene, with a reaction time of 30.628 min, was detected for the first time on day 35 of co-culture;
[0063] 3. Application Value Analysis: Based on existing literature reports, among the above five specific compounds, phenylethanol has a sweet floral aroma, while cis-muurola-4(15),5-diene and 1,6-dimethyl-4-isopropyltetrahydronaphthalene both have terpene-like aromas. All three meet the application attributes of fragrances and food additives, indicating that the volatiles generated by the co-cultivation method of this invention have clear industrial application potential. At the same time, in three biological replication experiments, the detection time and relative content of the above compounds remained stable, proving that this method has good repeatability and controllability.
[0064] 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 producing aroma using *Nematostella spp.* and *Cladosporium brevicornum* co-culture, characterized in that... Includes the following steps: S1 strain activation: *Alternaria solani* (… Clonostachys rosea f.catenulata ) and short-haired trichomes ( Lachnum brevipilosum They were inoculated separately into PSA solid medium for activation culture; The composition of each 1L of the PSA solid culture medium includes: 200g potato, 20g sucrose, 18g agar, and the remainder is distilled water with a natural pH value. S2 Co-culture Inoculation and Cultivation: The activated *Neurospora* and *Clerodendrum brevis* from step S1 were inoculated into the same PSA solid medium in a culture container using the five-point confrontation method and incubated in the dark at 27.5℃. The five-point confrontation method is as follows: *Neurospora* mycelium is inoculated in the center of the medium, and *Clerodendrum brevis* mycelium is inoculated at four points 2.5 cm away from the center on a cross line extending from the center point. The diameter of each mycelium is 5 mm. S3 Volatile Collection and Analysis: Starting from the second day after inoculation, headspace volatiles of the culture were collected every 48 hours using headspace solid-phase microextraction (HS-SPME). Subsequently, the collected volatiles were analyzed by gas chromatography-mass spectrometry (GC-MS) to identify co-culture-specific volatile organic compounds.
2. The method for producing aroma using *Nematostella spp.* and *Cladosporium brevicornum* co-culture according to claim 1, characterized in that: The activation culture conditions described in S1 are: 28±1℃ dark culture for 14 days.
3. The method for producing aroma using *Nematostella spp.* and *Cladosporium brevicornum* co-culture according to claim 1, characterized in that: The culture container mentioned in S2 is a 500mL culture flask, and the volume of PSA solid culture medium poured into the culture flask is 120mL.
4. The method for producing aroma by co-culturing *Alternaria solani* and *Cladosporium brevicornu* according to claim 1, characterized in that: The headspace solid-phase microextraction method described in S3 uses a 50 / 30μm DVB / CAR / PDMS extraction head, with an adsorption extraction temperature of 27.5℃ and an adsorption extraction time of 40 minutes.
5. The method for producing aroma by co-culturing *Alternaria solani* and *Cladosporium brevicornu* according to claim 1, characterized in that: The conditions for gas chromatography-mass spectrometry (GC-MS) analysis described in S3 are as follows: GC conditions: HP-5MS column, 30m×250μm×0.25μm; helium as carrier gas, flow rate 0.8mL / min; injection port temperature 250℃; splitless injection; temperature program: initial temperature 40℃, hold for 5min, increase to 130℃ at 5℃ / min, hold for 5min, then increase to 230℃ at 10℃ / min, hold for 2min. MS conditions: Electron impact ion source was used; ion source temperature was 230℃; scan range was 35-550 m / z.