CN-121555582-B - Method for producing aroma by co-culturing gliocladium catenulatum and Mao Limao dish fungus

CN121555582BCN 121555582 BCN121555582 BCN 121555582BCN-121555582-B

Abstract

The invention provides a method for producing fragrance by co-culturing gliocladium catenulatum and Mao Limao dish fungus, and relates to the technical field of microorganisms. The method for producing the perfume by co-culturing the gliocladium catenulatum and the short Mao Limao tray fungus comprises the following steps of S1 strain activation, S2 co-culturing inoculation and culture, wherein the gliocladium catenulatum and the short Mao Limao tray fungus are respectively inoculated into a PSA solid culture medium for activation culture, the activated gliocladium catenulatum and the short Mao Limao tray fungus in the step S1 are inoculated into the PSA solid culture medium of the same culture container by adopting a five-point counter method, and S3 volatile matter collection and analysis are carried out, namely, from the 2 nd day after inoculation, the headspace volatile matter of the culture is collected by adopting a headspace solid-phase microextraction method every 48 hours, and then the co-culturing specific volatile organic matters are identified. The method is stable and repeatable, the product contains definite aroma components, and a high-efficiency green new strategy is provided for natural spice development.

Inventors

ZOU LIHUA
WU BOYU
ZHAO PING
ZHU GUOLEI
YANG XIAOQIN
JIANG QIAN

Assignees

西南林业大学

Dates

Publication Date: 20260512
Application Date: 20260119

Claims (5)

1. The method for producing the incense by co-culturing the gliocladium catenulatum and the Mao Limao dish fungus is characterized by comprising the following steps of: s1, strain activation, namely respectively inoculating gliocladium catenulatum (Clonostachys rosea f. Catenulata) and Pantoea brachiocephalum (Lachnum brevipilosum) into a PSA solid culture medium for activation culture, wherein each 1L of the PSA solid culture medium comprises 200g of potato, 20g of sucrose, 18g of agar, the balance of distilled water and natural pH value; S2, co-culturing, namely inoculating the activated gliocladium catenulatum and the activated short Mao Limao tray fungus in the step S1 into a PSA solid culture medium of the same culture container by adopting a five-point counter method, and performing dark static culture at the temperature of 27.5 ℃, wherein the five-point counter method comprises inoculating a gliocladium catenulatum cake in the center of the culture medium, and respectively inoculating the gliocladium catenulatum cake into four points 2.5cm away from the center of the culture medium on a cross line expanding from the center point, wherein the diameter of each bacterial cake is 5mm; S3, collecting and analyzing the volatile matters, namely collecting the headspace volatile matters of the culture by adopting a headspace solid-phase microextraction method (HS-SPME) every 48 hours from the 2 nd day after inoculation, and then analyzing the collected volatile matters by using a gas chromatography-mass spectrometry (GC-MS) technology to identify the co-culture specific volatile organic matters.
2. The method for producing aroma by co-cultivation of Gliocladium catenulatum and Mao Limao dish fungus according to claim 1, wherein the condition of the activation cultivation in S1 is 28+ -1deg.C dark cultivation for 14 days.
3. The method for producing aroma by co-cultivation of Gliocladium catenulatum and Brevibacterium brevis Mao Limao according to claim 1, wherein the cultivation container in S2 is a 500mL cultivation bottle, and the volume of the PSA solid medium poured into the cultivation bottle is 120mL.
4. The method for producing aroma by co-culture of Gliocladium catenulatum and Brevibacterium brevis Mao Limao as claimed in claim 1, wherein the head of the headspace solid-phase microextraction method in S3 is 50/30 mu mDVB/CAR/PDMS head, the adsorption extraction temperature is 27.5 ℃, and the adsorption extraction time is 40 minutes.
5. The method for producing aroma by co-cultivation of Gliocladium catenulatum and Mao Limao b.breve according to claim 1, wherein the conditions of gas chromatography-mass spectrometry (GC-MS) analysis in S3 are as follows: GC conditions including adopting an HP-5MS chromatographic column, 30m multiplied by 250 mu m multiplied by 0.25 mu m, helium as carrier gas, a flow rate of 0.8mL/min, a sample inlet temperature of 250 ℃, adopting non-split sample injection, and a temperature-raising program, wherein the initial temperature is kept at 40 ℃ for 5min, the temperature is raised to 130 ℃ at 5 ℃ for 5min, and the temperature is raised to 230 ℃ at 10 ℃ for 2min; MS conditions are that electron bombardment is adopted to the ion source, the temperature of the ion source is 230 ℃, and the scanning range is 35-550 m/z.

Description

Method for producing aroma by co-culturing gliocladium catenulatum and Mao Limao dish fungus Technical Field The invention relates to the technical field of microorganisms, in particular to a method for producing aroma by co-culturing gliocladium catenulatum and Mao Limao frisbania breve. Background The aroma compounds are compounds which cover esters, aldehydes, alcohols, ketones, terpenes, organic acids and the like and have characteristic aroma, are generally called, have wide application in the fields of food aroma enhancement, cosmetic aroma enhancement, spice preparation and the like, have antioxidant and antibacterial activities partially, can be used as fuels, solvents or natural pesticides, and have important values in industrial production. At present, the acquisition of volatile aroma compounds mainly depends on three major ways, namely, firstly, direct extraction from natural animals and plants, the way is obviously limited by the growing season of raw materials, the content of target compounds in natural raw materials is generally low, the extraction cost is high, the yield is limited, and the large-scale industrial production requirements are difficult to meet, secondly, the chemical synthesis method is characterized in that the raw materials are easily available, the cost is low, the field of artificial spices is dominant, but the synthesis process is complex, toxic and harmful byproducts are easily generated in the reaction process, the environmental pollution is possibly caused, the safety of the aroma compounds is influenced by byproduct residues, the chemical synthesis products cannot be marked as natural, and the current consumer demand trend for healthy and natural products is contrary, and thirdly, the way of biotechnology, namely, the microbial fermentation or biotransformation production is utilized, and the way has the advantages of mild reaction conditions, environmental friendliness, high product safety, natural product marking and the like, so that the way becomes a research on the development of aroma compounds. In the prior art research, screening and application of aroma-producing microorganisms are concentrated on saccharomycetes related to brewing and sauce product fermentation and lactic acid bacteria in dairy product fermentation, so that exploration of other types of microorganisms such as filamentous fungi is less, and obvious limitation exists in the excavation of aroma-producing resources of microorganisms. Meanwhile, the current mainstream microbial aroma producing technology mostly adopts a single strain fermentation system, is limited by the fixed metabolic pathway and limited metabolic capacity of a single strain, and the produced aroma compound has the problems of few types and low concentration, so that the market demand for products with rich aroma levels and distinct characteristics is difficult to meet. The co-culture strategy is expected to activate the silent metabolic pathway of the microorganism during the independent culture by constructing a multi-strain interaction culture system, thereby regulating and controlling the synthesis of metabolites, improving the diversity of aroma compounds and providing a potential direction for solving the technical bottleneck of single strain fermentation. However, few reports on the co-culture of filamentous fungi for aroma production are reported at present, and particularly, the related researches on the co-culture induction of gliocladium catenulatum (Clonostachys rosea f. Catenulata) and Paniculate brevis (Lachnum brevipilosum) for producing specific aroma compounds are not disclosed yet, and development of an aroma production method based on the co-culture of the filamentous fungi is needed to enrich microbial aroma production technology paths and aroma compound resource libraries. Disclosure of Invention Technical problem to be solved Aiming at the defects of the prior art, the invention provides a method for producing aroma by co-culturing gliocladium catenulatum and short Mao Limao dish fungus, which solves the problems of single strain dependence on aroma production by the existing microorganism, few aroma compound types caused by limited metabolic pathways, few research on aroma production by co-culturing filamentous fungi and insufficient excavation of natural spice resources. Technical proposal The invention aims at realizing the purposes by adopting the following technical scheme that the method for producing fragrance by co-culturing the gliocladium catenulatum and the Mao Limao dish fungus comprises the following steps: s1, strain activation, namely respectively inoculating gliocladium catenulatum (Clonostachys rosea f. Catenulata) and Pantoea brachiocephalum (Lachnum brevipilosum) into a PSA solid culture medium for activation culture, wherein each 1L of the PSA solid culture medium comprises 200g of potato, 20g of sucrose, 18g of agar, the balance of distilled water and natural pH value; S2, co-culturin