WO2011134355A1 - Preparation method of dihydrocanadensolide and use thereof - Google Patents

Abstract

The present invention discloses a preparation method and use of dihydrocanadensolide. The preparation method of the invention comprises the steps of solid fermentation, hydrolysis, separation and purification to obtain the natural dihydrocanadensolide compound by using Aspergillus oryzae as a strain and grain as a medium. The dihydrocanadensolide compound prepared by the method of the invention is a bicyclolactone compound that has pure soy sauce flavor, and it can be used as an essence or spice for enhancing the flavor of soybean oil and sauce.

Description

 Preparation method of dihydrobicyclolactone and application thereof

Technical field

 The invention relates to the field of food biotechnology, Specifically, the invention relates to a method for preparing dihydrobicyclolactone by using Aspergillus oryzae, Aspergillus niger, and the use of dihydrobicyclolactone in preparing a flavor and fragrance and a flavor regulator.

Background technique

Dihydrobicyclic lactone was originally isolated from the metabolite of Penicillium. This compound has anti-ulcer activity and is currently mainly used as a drug (Aldridge, David C. et al. Pharmaceutical Compositions. US patent 4103023). However, the production of the compound by Penicillium may result in non-food ingredients in the fermentation broth, which has a certain food safety risk. AJ Birch et al. reported that other Aspergillus can produce dihydrobicyclic lactone (AJ) Birch, Metabolites of Aspergillus indicus: The structure and some aspects of The biosynthesis of dihydrocanadensolide, Australian Journal of Chemistry ,1968, 21(11) :2775 - 2784), but due to the use of semi-synthetic media, some ingredients that are not allowed to be added by the food industry have been introduced, which is difficult to purify and is not suitable for use in the food industry. In addition, none of the above documents reported the flavor characteristics and flavor use of the compound.

Summary of the invention

The invention provides a method for preparing a dihydrobicyclic lactone having a sauce flavor by fermentation of Aspergillus oryzae, which adopts a natural medium and a food strain, and has good safety.

The dihydrobicyclic lactone and Aspergillus oryzae fermentation preparation method of the invention comprises the following steps:

(1) Using grain as raw material, cooking, inoculation with 0.03% (w/w) Aspergillus oryzae or Aspergillus niger to obtain Daqu;

(2) mixing Daqu with 15-20% (w/w) saline solution at 1:2.5-5 (w/w), and hydrolyzing at 20-30 °C for 30-60 days to obtain a hydrolyzate;

(3) centrifuging the hydrolyzate to remove the precipitate to obtain a hydrolyzed supernatant;

(4) The hydrolyzed supernatant was extracted three times with an equal volume of ethyl acetate, and the ethyl acetate phase was combined.

(5) The crude extract is dissolved in ethyl acetate, and then mixed with 230-400 mesh silica gel G powder, and the column is packed and eluted with a n-hexane-acetone mixture to obtain an eluent. The weight of the crude extract is 1-3 times, and the weight of the silica gel G powder is 1-3 times of the mixed solution of the crude extract and ethyl acetate;

(6) The eluate is concentrated and distilled to obtain a colorless transparent granular crystal, which is dihydrobicyclic lactone.

In the step (1), the grain is one or more of soybean, soybean meal, wheat, flour, and bran. The cooking temperature is from 100 ° C to 125 ° C, and the cooking time is from 6 min to 20 min. The culture temperature is 28 ° C to 36 ° C, the humidity is 70% to 95%, and the culture time is 40 h to 72 h.

In the step (3), the centrifugal speed is 1500 rpm to 4800 rpm, and the centrifugation time is 10 to 20 minutes.

In the step (4), the temperature under reduced pressure is 30 to 55 ° C, and the degree of vacuum is 0.08 MPa to 0.1 MPa.

In the step (5), elution is carried out in n-hexane:acetone = 99%: 1% to 50%: 50%.

The use of dihydrobicyclic lactones as flavors, flavors, flavor regulators and the preparation of complex flavors and fragrances can enhance and improve the flavor of soy sauces and sauces.

Compared with the prior art, the invention has the following advantages: the method uses natural food ingredients and microorganisms allowed in the food industry to produce dihydrobicyclic lactone, no fine chemical products, no secondary pollution, and mild reaction conditions, Low cost and good security.

 DRAWINGS

 Figure 1 is a mass spectrum of dihydrobicyclolactone prepared in a specific embodiment.

 2 is a single crystal structure analysis diagram of dihydrobicyclic lactone obtained in a specific embodiment.

Figure 3 is a ¹³ C nuclear magnetic resonance spectrum of dihydrobicyclic lactone prepared in a specific embodiment.

Figure 4 is a ¹ H NMR spectrum of dihydrobicyclic lactone prepared in a specific embodiment.

 detailed description

The specific embodiments of the present invention are further described below in conjunction with the embodiments, but the scope of the present invention is not limited thereto.

Example 1:

1. Soybean was soaked in water for 6h, heated at 121 °C for 10min, cooled, mixed with flour at 4:1 (w / w), and evenly mixed with 0.03% (w / w) of Aspergillus oryzae 3.042 spores The culture was carried out for 48 hours in an incubator at 30 ° C and a humidity of 90% to obtain Daqu.

2. The Daqu and the concentration of 20% (w/w) saline were mixed at a weight of 1:2.5 and hydrolyzed at 30 ° C for 30 d to obtain a hydrolyzate.

3. The hydrolyzate was centrifuged at 4,800 rpm for 10 min to remove the precipitate to obtain a hydrolyzed supernatant.

4. Extract the hydrolyzed supernatant three times with an equal volume of ethyl acetate. The crude extract was obtained by concentrating to dryness at 30 ° C under a vacuum of 0.08 MPa.

5. The crude extract was dissolved in ethyl acetate, and mixed with 300 mesh silica gel G powder, and the column was packed with a gradient of n-hexane-acetone mixture to obtain an eluent, the crude extract and ethyl acetate. The weight ratio of the mixture is 1:1, the weight ratio of the crude extract and ethyl acetate mixed solution to the silica gel G powder is 1:1, and the weight ratio of n-hexane to acetone in the n-hexane-acetone mixture is 50%: 50%; after the eluent is allowed to stand in a volatile solvent, a colorless transparent granular crystal is obtained, mp 90~92°C, confirmed by gas chromatography-mass spectrometry, nuclear magnetic resonance, single crystal X-ray diffraction as 6β-n-butyl-3,3aα,6,6aα-tetrahydro-3a-methylfur[3,4 -b]-furan-2,4-dione, as shown in Figures 1-4, respectively, the mass spectrum of dihydrobicyclic lactone, single crystal structure analysis, 13C nuclear magnetic resonance spectrum and 1H nuclear magnetic resonance spectrum, the structural formula is as follows:

6. After the sensory evaluation, dihydrobicyclic lactone has a natural sauce and aroma. Adding to the condiment enhances the aroma of the sauce and improves the flavor.

Example 2

1. Solid culture: The soybean meal is slightly moistened with water and heated at 110 ° C for 20 min. After cooling, the ratio of flour to flour is 4:1 (w/w), mixed, and evenly mixed into the production of 0.05% (w/w) of soybean meal and flour. The spores of Aspergillus niger were cultured in an incubator at 36 ° C and a humidity of 90% for 40 h, and Daqu was obtained after 45 h.

2. Mix Daqu with a concentration of 18% (w/w) saline in 1:3 and hydrolyze at 20 °C for 60 d to obtain a hydrolyzate.

3. The hydrolyzate was centrifuged at 2000 rpm for 20 min to remove the precipitate to obtain a hydrolyzed supernatant.

4. The hydrolyzed supernatant was extracted three times with an equal volume of ethyl acetate, and concentrated to dryness at 45 ° C under a vacuum of 0.1 MPa to give a crude extract.

5. The crude extract was dissolved in ethyl acetate, and mixed with 250 mesh silica gel G powder, and the column was packed with a gradient of n-hexane-acetone mixture to obtain an eluent, the crude extract and ethyl acetate. The weight ratio of 1:1.5, the weight ratio of the crude extract and ethyl acetate mixed solution to the silica gel G powder is 1:2, and the weight ratio of n-hexane to acetone in the n-hexane-acetone mixture is 80%: 20%; The eluate was concentrated to give a colorless transparent granular crystal, which was dihydrobicyclic lactone.

6. After the sensory evaluation, the isolated compound has a natural sauce and aroma. Adding to the condiments improves the flavor.

Example 3

1. Solid culture: Soybean meal moistened with water, heated at 125 ° C for 6 min, cooled, and mixed with fried and ground wheat flour in a ratio of 1:1, and then evenly mixed into the soybean meal and wheat flour quality of 0.03% (w / w) production The Aspergillus oryzae spores were cultured in an incubator at 28 ° C and a humidity of 90% for 50 h, and Daqu was obtained after 50 h.

2. Mix Daqu with 19% (w/w) saline to 1:5 and hydrolyze at 25 °C for 50 d to obtain a hydrolyzate.

3. Centrifuge the hydrolysate at 3500 rpm for 15 min to remove the precipitate to obtain a hydrolyzed supernatant.

4. The hydrolyzed supernatant was extracted three times with an equal volume of ethyl acetate, and concentrated to dryness under reduced pressure at a vacuum of 0.08 MPa to give a crude extract.

5. The crude extract was dissolved in ethyl acetate, and mixed with 400 mesh silica gel G powder, and the column was packed, and the gradient was eluted with a n-hexane-acetone mixture to obtain an eluate, the crude extract and ethyl acetate. The weight ratio of 1:3, the weight ratio of the crude extract and ethyl acetate mixed solution to the silica gel G powder is 1:3, and the weight ratio of n-hexane to acetone in the n-hexane-acetone mixture is 98%: 2%; the eluted component was allowed to stand for the volatile solvent to obtain a colorless transparent granular crystal, which was identified as dihydrobicyclolactone.

6. It can be obtained by sensory evaluation that the dihydrobicyclolactone obtained by the separation is mellow and thick, and added to the food can improve the flavor of the sauce.

Claims (1)

1. A method for preparing dihydrobicyclic lactone, which comprises the steps of:

   (1) Using grain as raw material, by cooking, inoculating Aspergillus oryzae or Aspergillus niger with a weight of 0.03%-0.05% of the grain to obtain Daqu;

   (2) Mixing Daqu with a brine having a mass concentration of 15 to 20% at a weight ratio of 1:2.5-5, and hydrolyzing at 20 to 30 ° C for 30-60 days to obtain a hydrolyzate;

   (3) centrifuging the hydrolyzate to remove the precipitate to obtain a hydrolyzed supernatant;

   (4) The hydrolyzed supernatant is extracted with an equal volume of ethyl acetate, and the ethyl acetate phase is combined, and concentrated to dryness under reduced pressure to give a crude extract;

   (5) The crude extract was dissolved in ethyl acetate, and mixed with 230-400 mesh silica gel G powder, and the column was packed and eluted with a n-hexane-acetone mixture to obtain an eluent. The weight of the ethyl acetate was 1-3 times of the crude extract, the weight of the silica gel G powder is 1-3 times of the mixed solution of the crude extract and ethyl acetate;

   (6) The eluate is concentrated and distilled to obtain a colorless transparent granular crystal, which is dihydrobicyclic lactone.

   2. The method according to claim 1, wherein in the step (1), the grain is one or more of soybean, soybean meal, wheat, flour, and bran.

   3. The method according to claim 1, wherein in the step (1), the cooking temperature is from 100 ° C to 125 ° C, and the cooking time is from 6 min to 20 min.

   4. The method according to claim 1, wherein in the step (1), the culture temperature is from 28 ° C to 36 ° C, the humidity is from 70% to 95%, and the culture time is from 40 h to 72 h.

   The method according to claim 1, wherein in the step (3), the centrifugal rotation speed is 1500 rpm to 4800 rpm, and the time is 10 to 20 minutes.

   6. The method according to claim 1, wherein in the step (4), the temperature under reduced pressure is 30 to 55 ° C, and the degree of vacuum is 0.08 MPa to 0.1 MPa.

   7. The method according to claim 1, wherein in the step (5), the weight ratio of n-hexane to acetone in the mixture of n-hexane and acetone is 99%: 1% to 50%: 50%. .

   8. A method according to any one of claims 1 to 7, wherein the number of extractions in step (4) is three.

   9. Use of the dihydrobicyclic lactone of claim 1 as a fragrance, fragrance or flavor modulator.

   10. The use of the dihydrobicyclic lactone of claim 1 for the preparation of a perfume flavor.

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