A process for the preparation of a hypnea musciformis extract enriched in mono-arylated compounds

Abstract

The application discloses a preparation method of tree moss extract rich in mono-aryl compounds. The application utilizes aged fermentation of fresh tree moss by Aspergillus niger to prepare the tree moss extract rich in mono-aryl compounds. The method provided by the application can degrade macromolecular substances in the tree moss into small-molecule aroma substances, i.e., mono-aryl compounds, so as to improve the content of aroma components and active components, i.e., mono-aryl compounds, in the tree moss extract. The prepared tree moss extract rich in mono-aryl compounds can be used in the field of cigarettes and the like to highlight the style characteristics of tree moss. The tree moss extract prepared by the method has strong antioxidant activity, and can further meet the demand of daily chemical products for natural antioxidant active components in the field of daily chemicals.

Description

Technical Field

[0001] This invention belongs to the field of plant extraction technology, specifically relating to a method for preparing a tree moss extract rich in monoaryl compounds. Background Technology

[0002] Tree moss is a type of lichen belonging to the genus Usnea in the family Usneaceae. It grows epiphytically on the bark of coniferous trees such as fir and pine. Tree moss extract, made from tree moss, is an important mossy fragrance internationally due to its unique natural fresh aroma of moss and rich resinous scent. It is widely used in the daily chemical and cigarette flavoring industries.

[0003] Tree moss contains a large amount of condensed phenolic acids, which are high molecular weight, non-volatile, odorless, and poorly soluble. These compounds negatively impact the solubility of tree moss extract, making the solution cloudy and difficult to clarify. After standing, a viscous solid settles at the bottom, affecting the state and effectiveness of the extract. Currently, in the application of tree moss extract, these high molecular weight condensed phenolic acids are often discarded as residues during the oil extraction process, wasting resources. Patent CN113180286A invented a method for separating and purifying high molecular weight condensed phenolic acids from tree moss extract, obtaining solid condensed phenolic acids that can be applied to the preparation of tobacco sheets. Although condensed phenolic acids are odorless, after aging, alcoholysis, or decarboxylation, they can be degraded to yield some low molecular weight, aromatic monoaryl compounds. These strongly aromatic monoaryl compounds constitute the characteristic aroma of tree moss. In addition, some monoaryl compounds, such as teurophenol and ethyl teuronate, have also shown significant antibacterial and antioxidant biological activities.

[0004] Aspergillus niger is an important microbial strain in the fermentation and food processing industries. It is a strain used to produce enzyme preparations (protease, amylase, pectinase) and has a certain ability to decompose proteins, tannic acid, starch, cellulose, pectin, etc. Summary of the Invention

[0005] To address the aforementioned deficiencies and shortcomings of existing technologies, the present invention aims to provide a method for preparing a tree moss extract rich in monoaryl compounds. Compared with existing methods, adding Aspergillus niger to the tree moss raw material helps promote the degradation of phenolic acids in the tree moss, generating monoaryl compounds. This can increase the content and antioxidant activity of small-molecule monoaryl compounds in the extract, improve solubility and aroma intensity, and broaden the application of the extract in the fields of daily chemicals and cigarette flavoring.

[0006] The first objective of this invention is to provide a method for preparing a tree moss extract rich in monoaryl compounds, comprising the following steps:

[0007] S1. Inoculate fresh tree moss with Aspergillus niger, age and ferment for 4-8 months, turning it over as needed to obtain aged and fermented tree moss; the preservation number of Aspergillus niger is CCTCC NO:M 2016767.

[0008] S2. The aged and fermented tree moss is dried and pulverized to obtain crude powder of aged and fermented tree moss, and then extracted with alcohol to obtain tree moss extract rich in monoaryl compounds.

[0009] Preferably, the *Aspergillus niger* is inoculated in the form of a *Aspergillus niger* spore suspension at an inoculation amount of 2% of the mass of the fresh moss, and the concentration of the *Aspergillus niger* spore suspension is 1.0 × 10⁻⁶. 7 cfu / mL.

[0010] Preferably, the aging and fermentation conditions are: temperature 30℃ and relative humidity 70%. The aging and fermentation time is 6 months, and the timely turning is done once every 15 days.

[0011] Preferably, the drying is carried out at 55°C until the moisture content is 8%; the pulverization is carried out using a pulverizer and passing through a 40-mesh sieve.

[0012] Preferably, the method of extracting tree moss extract rich in monoaryl compounds by alcohol extraction involves adding aged fermented tree moss powder to a 95% (v / v) aqueous ethanol solution, heating and refluxing for 2 hours, filtering, concentrating the filtrate under reduced pressure, adding a 95% (v / v) aqueous ethanol solution, stirring for 2 hours, letting stand for 1 hour, filtering, and concentrating the filtrate under reduced pressure to obtain the tree moss extract rich in monoaryl compounds.

[0013] Preferably, the method of extracting tree moss extract rich in monoaryl compounds by alcohol extraction involves adding aged fermented tree moss powder to a 95% (v / v) aqueous ethanol solution and heating under reflux for 2 hours, wherein the mass ratio of aged fermented tree moss powder to the 95% (v / v) aqueous ethanol solution is 1:5; filtering, concentrating the filtrate under reduced pressure at 60°C to 40% of the mass of the aged fermented tree moss powder to obtain a concentrated filtrate, then adding three times the mass of the concentrated filtrate to a 95% (v / v) aqueous ethanol solution, stirring at 200 rpm for 2 hours, allowing to stand for 1 hour, filtering, and concentrating the filtrate under reduced pressure at 60°C to obtain the tree moss extract rich in monoaryl compounds.

[0014] A second object of the present invention is to provide a tree moss extract rich in monoaryl compounds prepared according to the method.

[0015] A third objective of this invention is to provide the application of the aforementioned tree moss extract rich in monoaryl compounds in the preparation of products with characteristic tree moss aromas and natural antioxidant functions.

[0016] Preferably, the product is cigarettes and / or daily chemical products.

[0017] The fourth objective of this invention is to provide the application of Aspergillus niger with accession number CCTCC NO:M 2016767 in the biotransformation of tree moss into monoaryl compounds, wherein the monoaryl compounds are moss phenol, moss ethyl ester and 5-pentylresorcinol.

[0018] The beneficial effects of this invention are:

[0019] This invention provides a method for preparing tree moss extract rich in monoaryl compounds. Compared with traditional methods, this method degrades macromolecular phenolic acids in tree moss into small-molecule aroma compounds, increasing the content of aroma and active monoaryl compounds in the tree moss extract, thus obtaining a tree moss extract rich in monoaryl compounds. The tree moss extract prepared by this method has a rich, characteristic tree moss aroma, good solubility and condition, and can be used in cigarette manufacturing and other fields to highlight the tree moss style and enrich the tobacco aroma. Furthermore, the tree moss extract prepared by this method has strong antioxidant activity, which can further meet the demand for natural antioxidant active ingredients in daily chemical products. The preparation of tree moss extract through this method achieves efficient utilization of tree moss resources. Attached Figure Description

[0020] Figure 1 The image shows the total ion chromatogram of the aged fermented tree moss extract by gas chromatography-mass spectrometry. The numbers in the image represent the following compounds: 1. moss black phenol; 2. sugar; 3. synthetic oak moss; 4. ethyl moss ester; 5. ethyl erythropoietin; 6. 5-pentylresorcinol; 7. ethyl erythropoietin; the same applies below.

[0021] Figure 2 The image shows the total ion chromatogram of a fresh tree moss extract obtained by gas chromatography-mass spectrometry.

[0022] Figure 3 The image shows the total ion chromatogram of the aged tree moss extract by gas chromatography-mass spectrometry.

[0023] Figure 4 The figure shows the comparison results of the antioxidant activity of tree moss extracts. Detailed Implementation

[0024] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0025] The Aspergillus niger strains used in the following examples were provided by Boton (Shanghai) Biotechnology Co., Ltd., with accession number CCTCCNO:M 2016767.

[0026] Example 1: Preparation of aged fermented tree moss extract

[0027] The aged and fermented tree moss extract was prepared by the following method.

[0028] Step 1: Prepare a suspension of Aspergillus niger spores (1.0 × 10⁻⁶). 7 The strain (cfu / mL) was added to freshly harvested tree moss at a ratio of 2% (by mass). The tree moss with the strain was then aged and fermented for 6 months at a temperature of 30°C and a relative humidity of 70%, with the fermentation pile being turned over every 15 days.

[0029] Step 2: Dry the tree moss aged and fermented in Step 1 at 55℃ until the moisture content is 8%, then pulverize it in a grinder and pass it through a 40-mesh sieve to obtain coarse aged and fermented tree moss powder. Take 500g of the coarse aged and fermented tree moss powder and place it in a three-necked flask. Add 2.5kg of 95% ethanol aqueous solution and heat under reflux for 2 hours. After extraction, filter the solution and concentrate the filtrate under reduced pressure at 60℃ to 200g. Add 600g of 95% ethanol aqueous solution and stir with a magnetic stirrer at 200rpm for 2 hours. After stirring, let it stand for 1 hour, filter the solution, and concentrate the filtrate under reduced pressure at 60℃ to 125g to obtain the aged and fermented tree moss extract.

[0030] The aged fermented tree moss extract prepared in this embodiment is a brownish-black, flowable paste with a rich aroma characteristic of tree moss, and it is well soluble in a 95% (v / v) aqueous ethanol solution.

[0031] The components in the aged and fermented tree moss extract were determined by gas chromatography-mass spectrometry (GC-MS), and the results are as follows: Figure 1 As shown.

[0032] Comparative Example 1: Preparation of Fresh Tree Moss Extract

[0033] Fresh tree moss was dried at 55℃ to a moisture content of 8%, then pulverized in a grinder and passed through a 40-mesh sieve to obtain coarse fresh tree moss powder. 500g of the coarse fresh tree moss powder was placed in a three-necked flask, and 2.5kg of 95% ethanol aqueous solution was added. The mixture was heated under reflux for 2 hours. After extraction, the mixture was filtered, and the filtrate was concentrated under reduced pressure at 60℃ to 200g. 600g of 95% ethanol aqueous solution was added, and the mixture was stirred with a magnetic stirrer at 200rpm for 2 hours. After stirring, the mixture was allowed to stand for 1 hour, filtered, and the filtrate was concentrated under reduced pressure at 60℃ to 125g to obtain the fresh tree moss extract.

[0034] The fresh tree moss extract obtained in this comparative example was a reddish-brown flowing liquid with a weak characteristic aroma of tree moss.

[0035] The components in fresh tree moss extract were determined by gas chromatography-mass spectrometry (GC-MS), and the results are as follows: Figure 2 As shown.

[0036] Comparative Example 2: Preparation of Extracts from Aged Tree Moss

[0037] Step 1: Place the freshly picked tree moss in an environment with a temperature of 30℃ and a relative humidity of 70% for 12 months, turning it over every 15 days.

[0038] Step 2: Dry the aged tree moss at 55℃ until the moisture content is 8%, then pulverize it in a grinder and pass it through a 40-mesh sieve to obtain coarse aged tree moss powder. Take 500g of the coarse aged tree moss powder and place it in a three-necked flask. Add 2.5kg of 95% ethanol aqueous solution and heat under reflux for 2 hours. After extraction, filter the solution and concentrate the filtrate under reduced pressure at 60℃ to 200g. Add 600g of 95% ethanol aqueous solution and stir with a magnetic stirrer at 200rpm for 2 hours. After stirring, let it stand for 1 hour, filter the solution, and concentrate the filtrate under reduced pressure at 60℃ to 125g to obtain the aged tree moss extract.

[0039] The aged tree moss extract prepared in this comparative example is a brownish-black, flowable paste with the characteristic aroma of tree moss.

[0040] The components in the extract of aged tree moss were determined by gas chromatography-mass spectrometry (GC-MS), and the results are as follows: Figure 3 As shown.

[0041] The GC MS spectra obtained from Examples 1, Comparative Example 1, and Comparative Example 2 were searched using the NIST spectral library and analyzed manually to identify the substances using GC MS. The relative content (peak area percentage) of the main components was calculated using the peak area normalization method. The results are shown in Table 1. The results show that the relative content of monoaryl compounds (teosinol, synthetic oak moss, ethyl erythritol, ethyl teosinol, and ethyl teosinol) in the fresh tree moss extract of Comparative Example 1 was 37.531%, the relative content of monoaryl compounds in the aged tree moss extract of Comparative Example 2 was 49.306%, and the relative content of monoaryl compounds in the aged fermented tree moss extract of Example 1 was 63.800%. The fresh tree moss extract of Comparative Example 1 had the highest content of sugar compounds, followed by the aged tree moss extract of Comparative Example 2, while the aged fermented tree moss extract of Example 1 had the lowest content of sugar compounds. This indicates that Aspergillus niger fermentation accelerates the degradation of macromolecules such as sugars in tree moss and promotes the formation of monoaryl compounds. Monoaryl compounds such as ethyl styrosine, styrosine, ethyl erythropoietin, and synthetic oakmoss are the main components that constitute the strong mossy aroma. As a result, the aged fermented tree moss extract obtained by the method in Example 1 has a more concentrated characteristic aroma of tree moss compared to extracts obtained by other methods, and also has better solubility.

[0042] Table 1. Analysis results of major components in fresh and aged fermented tree moss extracts.

[0043]

[0044]

[0045] Note: - indicates not detected.

[0046] Example 2: In vitro antioxidant effect (DPPH scavenging experiment)

[0047] 1. Experimental Materials

[0048] DPPH was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; L-ascorbic acid (Vc) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; and anhydrous ethanol (analytical grade) was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0049] 2. Experimental Methods

[0050] 3.9 mg of DPPH powder was weighed and dissolved in 50 mL of anhydrous ethanol. The solution was shaken well and fully dissolved to prepare the DPPH working solution. Using anhydrous ethanol as the solvent, solutions of aged fermented tree moss extract (prepared in Example 1), fresh tree moss extract (prepared in Comparative Example 1), and aged tree moss extract (prepared in Comparative Example 2) were prepared as test samples. A 0.01 mg / mL L-ascorbic acid sample was prepared using deionized water as a positive control. 100 μL of the test sample was mixed with 100 μL of DPPH working solution to form the sample group. 100 μL of the test sample was mixed with 100 μL of anhydrous ethanol to form the control group. 100 μL of anhydrous ethanol was mixed with 100 μL of DPPH working solution to form the blank group. Each group was shaken at room temperature for 2 min, allowed to stand for 30 min, and then the absorbance at 517 nm was measured using a microplate reader. The experiment was repeated 4 times and the average value was taken. The DPPH free radical scavenging rate was calculated according to formula (1):

[0051]

[0052] In formula (1): As is the absorbance of the sample group, Ac is the absorbance of the control group, and Ab is the absorbance of the blank group.

[0053] 3. Experimental Results

[0054] The results of in vitro antioxidant (DPPH scavenging assay) are shown in Table 2 and Figure 4 The results showed that fresh tree moss extract had the worst DPPH free radical scavenging effect, while aged and fermented tree moss extract had the best DPPH free radical scavenging effect, which was slightly weaker than the positive control sample.

[0055] Example 3: In vitro antioxidant effect (ABTS scavenging experiment)

[0056] 1. Experimental Materials

[0057] ABTS(2,2-adiazon-bis(3-ethyl- benzothiazolium-6- sulfonic acid Diammonium salt was purchased from Merck Sigma Chemical Reagent Co., Ltd.; K2S2O8 was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd.; L-ascorbic acid (Vc) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; and anhydrous ethanol (analytical grade) was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0058] 2. Experimental Methods

[0059] Weigh 200 mg ABTS and 34.4 mg K2S2O8, dissolve in 50 mL deionized water, shake well, and use as the ABTS stock solution. Let stand at room temperature for 24 hours before use. Dilute the ABTS stock solution with anhydrous ethanol to an absorbance within 0.7 ± 0.02 to prepare the ABTS working solution. Using anhydrous ethanol as the diluent, prepare 0.01 mg / mL solutions of aged fermented tree moss extract (prepared in Example 1), fresh tree moss extract (prepared in Comparative Example 1), and aged tree moss extract (prepared in Comparative Example 2) as test samples. Prepare a 0.01 mg / mL L-ascorbic acid sample using deionized water as the solvent as a positive control. 100 μL of the test sample and 100 μL of ABTS working solution were mixed to form the sample group. 100 μL of the test sample and 100 μL of anhydrous ethanol were mixed to form the control group. 100 μL of anhydrous ethanol and 100 μL of ABTS working solution were mixed to form the blank group. After shaking at room temperature for 2 min, each group was allowed to stand for 6 min, and the absorbance value at 734 nm was measured using an ELISA reader.

[0060] The experiment was repeated 4 times and the average value was taken. The ABTS free radical scavenging rate was calculated according to formula (2):

[0061]

[0062] In formula (2): As is the absorbance of the sample group, Ac is the absorbance of the control group, and Ab is the absorbance of the blank group.

[0063] 3. Experimental Results

[0064] The results of in vitro antioxidant (ABTS scavenging assay) are shown in Table 2 and Figure 4 The results showed that fresh tree moss extract had the worst scavenging effect on ABTS free radicals, while aged and fermented tree moss extract had the best scavenging effect on ABTS free radicals, which was stronger than the positive control sample.

[0065] Table 2. Results of antioxidant tests on fresh and aged fermented tree moss extracts.

[0066] Sample Name DPPH% ABTS% Fresh tree moss extract (Comparative Example 1) 35.60％ 65.60％ Aged tree moss extract (Comparative Example 2) 78.22％ 91.71％ Aged and fermented tree moss extract (Example 1) 95.11％ 98.77％ L-Ascorbic acid (positive control) 96.18％ 94.25％

Claims

1. A method for preparing a tree moss extract rich in monoaryl compounds, characterized in that, Includes the following steps: S1. Inoculate fresh tree moss with Aspergillus niger, age and ferment for 4-8 months, turning it over as needed to obtain aged and fermented tree moss; the preservation number of Aspergillus niger is CCTCC NO:M 2016767. S2. The aged and fermented tree moss is dried and pulverized to obtain crude powder of aged and fermented tree moss, and then extracted with alcohol to obtain tree moss extract rich in monoaryl compounds.

2. The method according to claim 1, characterized in that, The *Aspergillus niger* was inoculated in the form of a *Aspergillus niger* spore suspension at a rate of 2% of the mass of fresh moss, and the concentration of the *Aspergillus niger* spore suspension was 1.0 × 10⁻⁶. 7 cfu / mL.

3. The method according to claim 1, characterized in that, The aging and fermentation conditions are: temperature 30℃ and relative humidity 70%; the aging and fermentation time is 6 months; and the timely turning is once every 15 days.

4. The method according to claim 1, characterized in that, The drying process involves drying at 55°C until the moisture content reaches 8%; the pulverization process involves pulverizing using a pulverizer and passing the pulverized material through a 40-mesh sieve.

5. The method according to claim 1, characterized in that, The method of extracting tree moss extract rich in monoaryl compounds by alcohol extraction involves adding aged fermented tree moss powder to a 95% (v / v) aqueous ethanol solution, heating and refluxing for 2 hours, filtering, concentrating the filtrate under reduced pressure, adding another 95% (v / v) aqueous ethanol solution, stirring for 2 hours, letting stand for 1 hour, filtering, and concentrating the filtrate under reduced pressure to obtain the tree moss extract rich in monoaryl compounds.

6. The method according to claim 1, characterized in that, The method for extracting tree moss extract rich in monoaryl compounds by alcohol extraction involves adding aged fermented tree moss powder to a 95% (v / v) aqueous ethanol solution and heating under reflux for 2 hours, wherein the mass ratio of aged fermented tree moss powder to the 95% (v / v) aqueous ethanol solution is 1:

5. After filtration, the filtrate is concentrated under reduced pressure at 60°C to 40% of the mass of the aged fermented tree moss powder to obtain a concentrated filtrate. Then, three times the mass of the concentrated filtrate is added to a 95% (v / v) aqueous ethanol solution, and the mixture is stirred at 200 rpm for 2 hours. After standing for 1 hour, the mixture is filtered, and the filtrate is concentrated under reduced pressure at 60°C to obtain the tree moss extract rich in monoaryl compounds.

7. A tree moss extract rich in monoaryl compounds prepared by the method according to any one of claims 1-6.

8. The use of the tree moss extract rich in monoaryl compounds as described in claim 7 in the preparation of products with tree moss characteristic aroma and natural antioxidant function.

9. The application according to claim 8, characterized in that, The products mentioned are cigarettes and / or daily chemical products.

10. Application of Aspergillus niger with accession number CCTCC NO:M 2016767 in the biotransformation of tree moss into monoaryl compounds, wherein the monoaryl compounds are moss phenol, moss ethyl ester and 5-pentylresorcinol.

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