Yeast composition for accelerating aging of folium artemisiae argyi and folium artemisiae argyi aging accelerating method

Abstract

The invention provides a yeast composition for accelerating aging of folium artemisiae argyi. The yeast composition at least contains aspergillus oryzae, saccharomyces cerevisiae or bouquet yeast. The invention further provides a folium artemisiae argyi aging accelerating method. Different from an existing single-bacterium fermentation method, the method has the advantages that the combination of the mould and the two yeasts is adopted, the advantages of the aspergillus oryzae in the aspects of extracellular enzymolysis and substrate release and the effects of the yeasts in glycometabolism and terpenoid synthesis are fully played, a mould-yeast synergistic system is formed, the content of key active ingredients such as borneol is effectively increased, and the yield of the borneol is increased. And irritant components such as camphor and eucalyptol are reduced. The process is simple, conditions are mild, exogenous chemical treatment is not needed, and the toxic residue risk is avoided; and large-scale application can be realized under conventional facilities of traditional Chinese medicinal material processing enterprises, and the method has a good popularization prospect.

Description

Technical Field

[0001] This invention relates to a yeast composition for aging mugwort leaves and a method for aging mugwort leaves. Background Technology

[0002] Artemisia argyi (Levl. et Vant.), a perennial herbaceous plant belonging to the Asteraceae family, is widely distributed throughout most parts of my country. my country has abundant Artemisia argyi resources, high yields, and a long history of application, making it an important traditional Chinese medicine with both medicinal and economic value. In traditional Chinese medicine theory, Artemisia argyi has the effects of warming the meridians and stopping bleeding, dispelling cold and relieving pain, regulating menstruation and calming the fetus. It is widely used in various forms such as acupuncture, medicinal baths, internal and external applications, with moxibustion being the most common. Based on differences in storage time and changes in medicinal properties, Artemisia argyi is usually divided into "new Artemisia" and "aged Artemisia." New mugwort refers to mugwort leaves harvested fresh in the current year or dried and stored for no more than one year. It is hot and irritating in nature and is suitable for external use such as foot baths, mosquito repellents, and herbal packs, but it is not suitable for direct moxibustion. Aged mugwort, on the other hand, is the first choice for moxibustion. During long-term storage, the volatile oily irritating components (such as eucalyptol, monoterpenes, sesquiterpenes, etc.) are significantly reduced, the moxibustion smoke is mild, and the medicinal properties are stable, making it more suitable for long-term moxibustion and health care.

[0003] However, due to the long natural aging process required for aged mugwort, the supply-demand imbalance is becoming increasingly prominent. Traditional aging methods generally suffer from problems such as long aging cycles, large land resource requirements, and difficulty in controlling product quality, leading to a worsening supply-demand imbalance. Therefore, there is an urgent need to develop a new method for accelerating the aging of mugwort leaves that features a scientific combination of fungal strains, a controllable process, and stable results, in order to meet the urgent demand of the moxibustion industry for high-quality aged mugwort.

[0004] In the existing technology, CN115518094A discloses a systematic method for aging Artemisia argyi and its supporting tools. Although it achieves standardized management of the aging process, it does not significantly improve the aging efficiency and still suffers from problems such as long aging cycle, long site occupation time, and high quality control costs. To shorten the aging time, some studies have proposed accelerating aging through microbial assistance. For example, CN114099560B and CN118542887A disclose the anaerobic fermentation method of Lactic Acid Bacteria and the fermentation method of Fusarium solani, respectively, to promote the rapid aging of Artemisia argyi. Although the above methods shorten the aging time to a certain extent, they have the following shortcomings: (1) the microbial strains used are relatively simple, and the aging effect is limited; (2) there is a lack of quantitative objective indicators to evaluate the degree of aging, which makes it difficult to stably control the aging process; (3) it is difficult to achieve large-scale and standardized application in production practice. Therefore, it is still urgent to develop a new method for accelerating the aging of Artemisia argyi with scientific microbial combinations, controllable process, and stable results to meet the urgent needs of the moxibustion industry for high-quality aged Artemisia argyi. Summary of the Invention

[0005] This invention provides a yeast composition for aging Artemisia argyi and a method for aging Artemisia argyi.

[0006] The present invention provides a yeast composition for aging Artemisia argyi, which contains at least Aspergillus oryzae, Saccharomyces cerevisiae or Saccharomyces cerevisiae.

[0007] The mass ratio of the raw materials is as follows:

[0008] 1-3 parts Aspergillus oryzae, 1-3 parts Saccharomyces cerevisiae, 1-3 parts Saccharomyces cerevisiae.

[0009] The mass ratio of the raw materials is as follows:

[0010] 1 part Aspergillus oryzae, 1 part brewing yeast, 1 part wine aroma yeast.

[0011] This invention provides a method for accelerating the aging of Artemisia argyi, which includes the following steps:

[0012] a. Take unaged mugwort leaves;

[0013] b. Preparation of inoculum: Weigh the yeast composition, wherein *Saccharomyces cerevisiae* and *Saccharomyces cerevisiae* are inoculated into YM liquid medium, and *Aspergillus oryzae* is inoculated into PDA liquid medium, and the mixture is placed in a shaker at 28℃ and 100rpm for activation culture; after activation, the bacterial concentration is adjusted to 1×10⁻⁶. 8 -1×10 9 CFU / mL, Aspergillus oryzae spore suspension concentration is 1×10 7 –1×10 8 The bacterial culture was diluted with sterile water at a volume ratio to prepare a single-cell inoculum solution;

[0014] The mixed inoculum is prepared by mixing Aspergillus oryzae, Saccharomyces cerevisiae and Saccharomyces cerevisiae in the specified proportions to form a mixed bacterial solution with an equivalent concentration to the single bacterial solution.

[0015] c. Spray the inoculation solution from step b onto the surface of the unaged Artemisia argyi from step a, and let it stand after spraying;

[0016] d. Aging:

[0017] First, pre-aging is carried out by culturing at a relative humidity of 65% and a temperature of 30℃, and then the temperature is raised to 50℃ for accelerated aging, which takes 2-30 days.

[0018] Preferably, the shaking incubation time in step b is 3 days; the dilution ratio of the bacterial solution to sterile water is 1:8. The pre-aging time in step d is 3 days; the accelerated aging time is 30 days.

[0019] Compared with existing methods, this invention has the following advantages: Unlike existing single-strain fermentation methods, this invention uses a combination of mold and two types of yeast, fully leveraging the advantages of Aspergillus oryzae in extracellular enzymatic hydrolysis and substrate release, and the role of yeast in sugar metabolism and terpene synthesis, forming a "mold-yeast" synergistic system. This effectively increases the content of key active ingredients such as borneol and reduces irritating components such as camphor and eucalyptol. By setting the inoculation concentration, culture conditions, and process flow, and combining physicochemical indicators (color, active ingredient content, antioxidant capacity) to evaluate the aging progress, the aging process can be quantitatively monitored and stably controlled, avoiding the drawbacks of traditional methods that rely on experience and sensory judgment. The method of this invention is simple, mild, requires no exogenous chemical treatment, and avoids the risk of toxic residues; it can be applied on a large scale in the conventional facilities of Chinese medicinal material processing enterprises and has good prospects for promotion. Attached Figure Description

[0020] Figure 1 Graph showing the change in borneol content under different aging times;

[0021] Figure 2 Comparison of the ABTS+ free radical scavenging activity of Artemisia argyi under different aging conditions;

[0022] Figure 3 Burning rate of mugwort floss under different aging conditions. Detailed Implementation

[0023] Example 1: Method for accelerating the aging of Artemisia argyi in this invention

[0024] 1. Strains preparation

[0025] The strains were sourced as follows: *Saccharomyces cerevisiae* (batch number: HZB660070, *Gyrophyte*), *Saccharomyces cerevisiae* (batch number: HZB126346, *Gyrophyte*), and *Aspergillus oryzae* (batch number: HZB307840, *Gyrophyte*). Culture media included YM liquid medium (batch number: HZB875006, *Gyrophyte*) and PDA liquid medium (batch number: HB0233-8, *Gyrophyte*), both autoclaved at 121℃ for 20 min. *Saccharomyces cerevisiae* and *Saccharomyces cerevisiae* were inoculated into YM liquid medium, and *Aspergillus oryzae* was inoculated into PDA liquid medium, and both were activated and cultured in a shaker at 28℃ and 100 rpm for 3–5 days. The inoculum was prepared with sterile water at a volume ratio of 8:1; the mixed bacterial solution was prepared by mixing the three bacterial solutions in a mass ratio of 1:1:1 to obtain an equal volume of the other bacterial solutions.

[0026] 2. Methods to accelerate aging

[0027] This experiment set up five groups of accelerated aging treatments: *Saccharomyces cerevisiae* group (code J), ​​*Saccharomyces cerevisiae* group (code N), *Aspergillus oryzae* group (code M), mixed culture group (code H, *Saccharomyces cerevisiae*: *Aspergillus oryzae* (mass ratio) = 1:1:1), and a temperature control group (code W, sprayed with sterile inoculum as a blank control). Samples for each treatment were collected at three time points and numbered using "group code + number," where the numbers 2, 6, 10, 15, 20, and 30 represent the number of days of accelerated aging (i.e., days 2, 6, 10, 15, 20, and 30) starting from when the incubator temperature was raised to 50℃. For example, J2 represents the sample from the *Saccharomyces cerevisiae* group on day 2 of accelerated aging, and H10 represents the sample from the mixed culture group on day 10 of accelerated aging. The specific operating procedure is as follows: Place the inoculum of each treatment group in a sterile sprayer and spray it evenly onto the surface of the unaged Artemisia argyi leaves at a dosage of 20 mL; after spraying, let it stand for about 1 hour, and after the surface is free of obvious droplets, compact the Artemisia argyi leaves and place them in a drug stability test chamber. First, pre-age them by incubating them at a relative humidity of 65% and a temperature of 30℃ for 3 days, and then raise the temperature to 50℃ for accelerated aging; take samples on the 2nd, 6th, 10th, 15th, 20th and 30th days from the date of temperature increase and preserve them according to the above numbering for subsequent analysis.

[0028] 3. Analytical Methods

[0029] 3.1 Color Measurement

[0030] The color parameters of Artemisia argyi samples after aging were measured using a colorimeter. The instrument was also used to measure the color parameters of fresh Artemisia argyi. Record the sample at time t And calculate the difference: Where, ΔL * <0 indicates brighter, ΔL * >0 indicates darker; Δa * >0 indicates a reddish tint, Δa * <0 indicates a greenish tint; Δb * >0 indicates a yellowish tint, Δb * <0 indicates a bluish tint. The total color difference is calculated using the following formula: ΔE * This represents the total color difference; the larger the value, the more significant the color difference compared to fresh mugwort leaves. The formula is:

[0031]

[0032] 3.2 Determination of Borneol Content

[0033] 3.2.1 Preparation of Artemisia argyi samples

[0034] Take an appropriate amount of mugwort leaves, grind them in a grinder, and pass them through a No. 3 sieve for later use.

[0035] 3.2.2 HS-HPME-GC-MS Method

[0036] HS-HPME conditions: Accurately pipette 0.1g of Artemisia argyi powder aged for 2, 6, 10, 15, 20, and 30 days into separate 20mL inert headspace vials, seal them, and set aside. After equilibration at 50℃ for 5 min, transfer the headspace vials containing the samples to the extraction apparatus. Insert the SPME arrow solid-phase extraction head into the headspace vial through the PTFE headspace septum, without contacting the sample. Extract and adsorb at 50℃ for 15 min. After the autosampler removes the extraction head, quickly insert it into the GC-MS / MS inlet and desorb at 250℃ for 2 min. The solid-phase extraction head is a composite of DVB / CWR / PDMS materials, with a film thickness of 120μm and a length of 20mm. The solid-phase extraction head is aged at 250℃ for 3 min before and after sample injection.

[0037] Chromatographic conditions: The capillary column was an SH-Polar Wax (60 mm × 0.25 mm × 0.25 μm), the injection port temperature was 250 °C, the injection mode was split injection with a split ratio of 5:1, the carrier gas control mode was constant linear velocity mode with a linear velocity of 25.5 cm·s⁻¹, the carrier gas was helium with a purity of 99.999%, the purge flow rate was set to 3.0 mL·min⁻¹, the column temperature was programmed, the initial temperature was 40 °C, held for 5 min, then increased to 250 °C at 3 °C·min⁻¹, held for 15 min; the column equilibration time was 3 min.

[0038] Mass spectrometry conditions: Electron impact ionization (EI) was used for ionization, with an ionization energy of 70 eV, an ion source temperature of 200℃, an interface temperature of 250℃, a full scan mode for mass acquisition, a scan mass number range of 35-500 amu, a detector voltage of +0.1 kV relative to the tuning result, and a solvent delay time of 1 min.

[0039] 3.3 Antioxidant Activity Experiment

[0040] Determination of ABTS+ free radical scavenging ability. Accurately weigh 384.076 mg of ABTS and dilute to 100 mL with deionized water in a volumetric flask to prepare an ABTS solution with a concentration of 7 mmol / L. Weigh 66.284 mg of K2S2O8 and dilute to 100 mL with deionized water in a volumetric flask to prepare a K2S2O8 aqueous solution with a concentration of 2.45 mmol / L. Mix 10 mL of each of the two solutions thoroughly and let stand at room temperature in the dark for 12–16 h to obtain the ABTS+ working solution stock solution. Dilute the stock solution with an appropriate amount of anhydrous ethanol to a concentration of 0.7 ± 0.02 absorbance at 734 nm to prepare the ABTS+ working solution. The formula for ABTS+ free radical scavenging rate is as follows: ABTS+ free radical scavenging rate = (1 - A sample / A blank) × 100%

[0041] 3.4 Calorific Value

[0042] 3.4.1 Preparation of Artemisia floss with different aging times

[0043] Artemisia leaves aged for 2, 6, 10, 15, 20 and 30 days under 5 different aging conditions were selected and made into 3:1 Artemisia floss for combustion parameter measurement.

[0044] 3.4.2 Thermogravimetric Study

[0045] Oxygen was selected as the carrier gas, with an oxygen flow rate of 100 mL / min. The temperature was programmed to rise from 28 to 600 °C at a rate of 10 °C / min. An empty Al2O3 crucible was used as a control. The sample amount was 6.0 mg, and ≥3 samples of each type were measured.

[0046] 4. Results and Conclusions

[0047] 4.1 Color changes of Artemisia argyi under different aging conditions

[0048] In the preliminary experiment, different gradient ratios of bacterial solutions were used to accelerate aging for 6 days based on the degree of color change in Artemisia argyi leaves. The results are shown in Table 1. The results showed that under different mixed bacterial solution conditions, the total color difference value (△E*) of Artemisia argyi leaves gradually decreased with the extension of aging time, a trend consistent with the color change during natural aging. Among them, the aging-accelerating effect was most significant when Aspergillus oryzae, Saccharomyces cerevisiae, and Saccharomyces cerevisiae were mixed in a mass ratio of 1:1:1. Therefore, this ratio was determined as the formula for subsequent mixed bacterial solutions.

[0049] Table 1. Color determination results of Artemisia argyi under different bacterial suspension ratios and aging time.

[0050]

[0051] The color changes of Artemisia argyi during the aging process under different bacterial solutions were analyzed. The results measured by a colorimeter are shown in Table 2. As can be seen from the table, under the five different aging conditions, the color of Artemisia argyi increased with the extension of aging time, ΔL * Value decreases, Δa * Value increases, Δb * The decrease in the value indicates that the color brightness of the mugwort gradually darkens during the aging process, and the overall color gradually changes towards red and yellow. Comparing the total color difference ΔE* values ​​of mugwort under five aging conditions, the total color difference values ​​of mugwort under all five aging conditions decrease with increasing aging time, which is consistent with the color change trend of mugwort during natural aging. In addition, under the aging effect of mixed yeast (1:1:1), ΔL * The value increased from 7.91 on the second day to 22.66 on the 30th day, ΔL * The value increased from 5.16 to 17.40, Δa * The value increased from 0.37 to 2.04, Δb *The value increased from 5.98 to 14.37, showing a significant change in color parameters compared to other groups. This indicates that the mixed bacterial group had a more pronounced effect on color change.

[0052] Table 2. Color determination results of Artemisia argyi under different aging conditions and times.

[0053]

[0054] Note: △E*=(a* 2 +b* 2 +*L 2 ) 1 / 2 The difference between the △E* value and the previous time period is divided into three levels: a, b, and c. If there is no significant difference, the level is not labeled. Different letters indicate significant differences (a: P < 0.005, b: P < 0.01, c: P < 0.05).

[0055] 4.2 Changes in borneol content under different aging conditions

[0056] Field investigations revealed that the unique aroma of mugwort gradually intensifies during natural aging, with borneol becoming one of the main contributors to this fragrance. Therefore, this invention uses changes in borneol content as a key indicator for evaluating the aging effect.

[0057] according to Figure 1 The data show that under the conditions of brewing yeast and mixed bacteria aging, the borneol content in Artemisia argyi showed a continuous upward trend with the extension of aging time, and the increase was significant; while under the conditions of brewing yeast and Aspergillus oryzae aging, the borneol content first increased and then decreased slightly, with a relatively small change; the control group (temperature aging) showed a trend of gradual decrease in borneol content.

[0058] This result indicates that the combination of wine yeast and mixed bacteria can effectively promote the enrichment process of borneol in Artemisia argyi, mimicking the aroma enhancement phenomenon during natural aging. Therefore, borneol content can serve as a quantifiable and objective indicator reflecting the degree of aging of Artemisia argyi, and has important reference value in the evaluation of actual aging processes.

[0059] 4.3 Antioxidant activity under different aging conditions

[0060] The ability of Artemisia argyi to scavenge ABTS+ free radicals under different aging times is as follows: Figure 2As shown in the figure. After 30 days of accelerated aging, the scavenging rates of ABTS+ free radicals for each sample were 52.87% for *Saccharomyces cerevisiae*, 51.09% for *Saccharomyces cerevisiae*, 50.25% for *Aspergillus oryzae*, 59.24% for mixed cultures, and 44.03% for temperature. There was a significant difference between mixed cultures and single cultures. The scavenging ability of ABTS+ free radicals was in the order of mixed cultures (1:1:1) > *Saccharomyces cerevisiae* > *Aspergillus oryzae* > *Saccharomyces cerevisiae* > temperature, indicating that the *Artemisia argyi* treated with mixed cultures had the best ABTS+ free radical scavenging ability, followed by *Saccharomyces cerevisiae*. Overall, the antioxidant capacity of *Artemisia argyi* leaves treated in each fermentation group was better than that in the control group. In particular, the mixed culture treatment group showed better ABTS+ free radical scavenging ability. + The strain exhibits the strongest free radical scavenging ability and the most significant antioxidant effect, indicating that the synergistic effect of the microorganisms can enhance the expression of functional components in Artemisia argyi and promote its application in health care and moxibustion.

[0061] 4.4 Combustion rate of Artemisia argyi under different aging conditions

[0062] Aging is a natural drying and slow oxidation process. During this process, the moisture in the mugwort leaves continuously evaporates. Low moisture content means that a large amount of heat is not needed to vaporize the moisture during combustion, allowing the heat to be more concentrated on maintaining the combustion reaction of the mugwort itself. Therefore, the combustion is more stable and longer, and the heat released per unit time (i.e., the combustion rate) appears more "gentle" (relatively slow). The combustion rate of mugwort leaves under different aging times is as follows: Figure 3 As shown. After 30 days of aging, the burning rates of each sample were 54.09% for *Saccharomyces cerevisiae*, 50.55% for *Saccharomyces cerevisiae*, 49.11% for *Aspergillus oryzae*, 60.12% for mixed bacteria (1:1:1), and 43.22% for temperature. The burning rate was in the order of mixed bacteria (1:1:1) < *Saccharomyces cerevisiae* < *Aspergillus oryzae* < *Saccharomyces cerevisiae* < temperature, indicating that the *Artemisia argyi* under the aging effect of mixed bacteria had better combustibility.

[0063] 4.5 Results Summary and Technical Effects

[0064] This invention establishes a fermentation and aging process for Artemisia argyi based on three microorganisms: wine yeast, brewing yeast, and Aspergillus oryzae. The aging effect is evaluated by multiple parameters such as color change, borneol content, antioxidant activity, and combustion characteristics.

[0065] The main conclusions are as follows:

[0066] Under the aging conditions induced by wine yeast, the color change trend of Artemisia argyi was consistent with that of natural aging, and the color difference value was significantly reduced, indicating that it can effectively simulate the natural aging process. Under the aging conditions induced by wine yeast and mixed bacteria, the borneol content in Artemisia argyi was significantly increased, consistent with the phenomenon of enhanced Artemisia argyi aroma after natural aging, exhibiting good sensory characteristics. Under the action of mixed bacteria, the Artemisia argyi sample showed a lower ABTS content. +The highest free radical scavenging ability indicates enhanced antioxidant capacity; the mixed bacteria treatment group has the lowest moxa wool burning rate, with gentle and stable heat, which better meets the standards for the use of moxibustion materials.

[0067] In summary, microorganisms such as wine yeast, brewer's yeast, and Aspergillus oryzae can all play a role in promoting aging during the aging process, with the synergistic effect of mixed strains being the best. The microbial aging method for Artemisia argyi provided by this invention can significantly shorten the aging cycle, improve the quality of moxibustion materials, and has good practicality, controllability, and industrial promotion value. It is of great significance for promoting the efficient utilization of Artemisia argyi resources and the high-quality development of the moxibustion industry.

Claims

1. A yeast composition for aging Artemisia argyi, characterized in that: It is made from at least Aspergillus oryzae, Saccharomyces cerevisiae, or Saccharomyces cerevisiae.

2. The yeast composition for aging Artemisia argyi according to claim 1, characterized in that: The mass ratio of the raw materials is as follows: 1-3 parts Aspergillus oryzae, 1-3 parts Saccharomyces cerevisiae, 1-3 parts Saccharomyces cerevisiae.

3. The yeast composition for aging Artemisia argyi according to claim 2, characterized in that: The mass ratio of the raw materials is as follows: 1 part Aspergillus oryzae, 1 part brewing yeast, 1 part wine aroma yeast.

4. A method for accelerating the aging of Artemisia argyi, characterized in that: It includes the following steps: a. Take unaged mugwort leaves; b. Preparation of inoculum: Weigh the yeast composition according to any one of claims 1-3, wherein *Saccharomyces cerevisiae* and *Saccharomyces cerevisiae* are inoculated into YM liquid medium, and *Aspergillus oryzae* is inoculated into PDA liquid medium, and the mixture is placed in a shaker at 28°C and 100 rpm for activation culture; after activation, the bacterial concentration is adjusted to 1×10⁻⁶. 8 -1×10 9 CFU / mL, Aspergillus oryzae spore suspension concentration is 1×10 7 –1×10 8 The bacterial culture was diluted with sterile water at a volume ratio to prepare a single-cell inoculum solution; The mixed inoculation solution is prepared by mixing Aspergillus oryzae, Saccharomyces cerevisiae and Saccharomyces cerevisiae in the proportions described in claims 2-3, and preparing a mixed bacterial solution with an equivalent concentration to the single bacterial solution; c. Spray the inoculation solution from step b onto the surface of the unaged Artemisia argyi from step a, and let it stand after spraying; d. Aging: First, pre-aging is carried out by culturing at a relative humidity of 65% and a temperature of 30℃, and then the temperature is raised to 50℃ for accelerated aging, which takes 2-30 days.

5. The method for accelerating the aging of Artemisia argyi according to claim 4, characterized in that: The shaking incubation time in step b is 3 days; the dilution ratio of the bacterial solution to sterile water is 1:

8.

6. The method for accelerating the aging of Artemisia argyi according to claim 4, characterized in that: Step d: Pre-aging time is 3 days; Accelerated aging time is 30 days.

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