Method for rapidly characterizing content variations of triterpenoids in liquid fermentation process of Antrodia camphorata

Abstract

The present invention discloses a method for rapidly characterizing content variations of triterpenoids in a liquid fermentation process of Antrodia camphorata, belonging to the field of microbial fermentation. According to the method of the present invention, in the liquid fermentation of Antrodia camphorata, an analysis method of rapidly judging the content variations of triterpenoids by rapid on-line or off-line detection and analysis of the content of a volatile aromatic substance -terpineol is utilized to implement automatic control of the fermentation process. Predictive analysis of triterpenoids in the fermentation process based on variations in on-line real-time parameters increases the controllability and production predictability of the fermentation process. This is of great significance for the development and utilization of Antrodia camphorata products having various bioactivities and application thereof in industrial production.

Claims

1. A method of rapid characterizing of production of triterpenoids in a liquid fermentation process of Antrodia camphorate, comprising: providing Antrodia camphorate in a liquid fermentation apparatus under fermentation conditions, quantitating the content of a volatile substance in the liquid fermentation process, wherein the volatile substance is -terpineol, measuring a variation trend of the content of the volatile substance over time, and correlating triterpenoids production in the liquid fermentation process with the variation trend of the volatile substance, thereby determining the production of triterpenoids in the liquid fermentation process.

2. The method of claim 1, wherein the variation trend of content of the -terpineol is consistent with a variation trend of content of triterpenoids.

3. The method of claims 1 wherein the content of -terpineol is determined by an on-line detection of -terpineol or an off-line detection of -terpineol.

4. The method of claim 3, wherein the fermentation apparatus comprises a process mass spectrometer, an electronic nose, and other on-line detection instruments, and a parameter acquisition system.

5. The method of claim 4, wherein the method further comprises: detecting -terpineol in an exhaust gas of the liquid fermentation apparatus, connecting an electronic nose through a hose to form a circulation loop to allow passage of the exhaust gas generated in the liquid fermentation process, monitoring the concentration of -terpineol in the exhaust gas by the electronic nose, and calculating the amount of -terpineol in a fermentation broth of the liquid fermentation.

6. The method of claim 3, wherein the off-line detection method further comprises: sampling a fermentation broth of the Antrodia camphorate fermentation liquid by a headspace solid phase microextraction-gas chromatography-mass spectrometer, and quantitating the -terpineol in the sampled fermentation broth.

7. A method of quantifying triterpenoids produced in an Antrodia camphorate liquid fermentation process, which comprises: incubating Antrodia camphorate in a first fermentation broth under fermentation conditions, quantifying over time an amount of -terpineol in the first fermentation broth, quantifying over time an amount of triterpenoids in the first fermentation broth, calculating a correlation between the amount of -terpineol in the first fermentation broth and the amount of triterpenoids in the first fermentation broth, such that for any given amount of -terpineol in the fermentation broth at any time in fermentation the amount of triterpenoids is known under the fermentation conditions without directly quantifying the triterpenoids in the fermentation process, fermenting Antrodia camphorate in a second fermentation broth under the fermentation conditions, and quantifying the amount of -terpineol in the second fermentation broth, determining the amount of triterpenoids in the second fermentation broth based on the correlation and based on the amount of -terpineol detected in the second fermentation broth, and wherein the first fermentation broth is the same as the second fermentation broth.

8. The method of claim 7, wherein determining the amount of triterpenoids in the second fermentation broth does not require directly assaying for triterpenoids and does not require the steps of: (1) solid-liquid separation, (2) room-temperature leaching, decoction, or heat reflux extraction on mycelium with organic solvents, and/or (3) direct assay determination of triterpenoids in the extraction.

9. The method of claim 7, wherein quantifying the amount of -terpineol comprises obtaining a sample of exhaust gas from the second fermentation broth, and quantifying -terpineol in the exhaust gas.

10. The method of claim 7, wherein quantifying the amount of -terpineol occurs in real time.

11. The method of claim 7, wherein the first fermentation broth comprises bran, glucose, MgSO.sub.4. 7H.sub.2O, and KH.sub.2PO.sub.4, at pH 5.5.

12. The method of claim 7, wherein the correlation between the amount of -terpineol in the first fermentation broth and the amount of triterpenoids in the first fermentation broth is a Pearson correlation coefficient of at least 0.9.

13. The method of claim 12, wherein the amount of -terpineol in the first or second fermentation broth is determined by solid phase microextraction-gas chromatography-mass spectrometry of a centrifuged sample of the first or second fermentation broth.

Description

BRIEF DESCRIPTION OF FIGURES

(1) FIG. 1 shows the yields of -terpineol and triterpenoids as a function of time in the fermentation process of Example 1.

(2) FIG. 2 shows the yields of -terpineol and triterpenoids as a function of time in the fermentation process of Example 2.

(3) FIG. 3 shows the yields of -terpineol and triterpenoids as a function of time in the fermentation process of Example 3.

(4) FIG. 4 shows the yields of -terpineol and triterpenoids as a function of time in the fermentation process of Example 4.

(5) FIG. 5A-5J show the yields of other volatile substances and triterpenoids as a function of time in the fermentation process of Example 1; FIG. 5A shows 1,2-decanediol, FIG. 5B shows linalool, FIG. 5C shows 1-octen-3-ol, FIG. 5D shows methyl furan-3-carboxylate, FIG. 5E shows 3-octanone, FIG. 5F shows phenethyl alcohol, FIG. 5G shows cubenol, FIG. 5H shows n-caprylic Octanal, FIG. 5I shows Trans-(E)-Nerolidol, FIG. 5J shows trans-2-octen-1-ol.

(6) FIG. 6A-6J show the yields of other volatile substances and triterpenoids as a function of time in the fermentation process of Example 2; FIG. 6A shows 1,2-decanediol, FIG. 6B shows linalool, FIG. 6C shows 1-octen-3-ol, FIG. 6D shows methyl furan-3-carboxylate, FIG. 6E shows 3-octanone, FIG. 6F shows phenethyl alcohol, FIG. 6G shows cubenol, FIG. 6H shows n-caprylic Octanal, FIG. 6I shows Trans-(E)-Nerolidol, FIG. 6J shows trans-2-octen-1-ol.

DETAILED DESCRIPTION

(7) Determination Method of Triterpenoids:

(8) 1) The mycelium obtained by centrifugation of a fermentation broth was rinsed with deionized water (washing off medium components), and freeze-dried to obtain a dried mycelium. The dried mycelium was accurately weighed 500 mg after liquid nitrogen milling, 15 mL of anhydrous ethanol was added, and the mixture was leached with 90 C. hot water for 1 h, the extraction being repeated three times. The final extraction solution was centrifuged for 10 min under a centrifugal force of 7104g, and the volume was adjusted to 25 mL. The solution was diluted to an appropriate concentration, and 1 mL was sampled and determined according to a standard curve determination method. By using a reagent blank as a control, the absorbance was measured at 550 nm, and the content of the triterpenoids was calculated according to the standard curve.

(9) 2) Determination of oleanolic acid standard curve: 5.00 mg of oleanolic acid standard sample was accurately weighed, and dissolved in anhydrous ethanol to a 0.1 g.Math.L.sup.1 solution. 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8 and 2.0 mL were respectively put in a 25 mL colorimetric tube, evaporated to dryness in boiling water, 0.3 mL of freshly prepared 5% (mass to volume) vanillin-glacial acetic acid and 1.00 mL of perchloric acid were added, and treatment is performed in a 60 C. water bath for 20 min. After cooling, 4 mL of glacial acetic acid was added, shaken well to make it fully react. By using a reagent blank as a control, the absorbance was measured at 550 nm.

(10) Off-line detection method of -terpineol (headspace solid phase microextraction-gas chromatography-mass spectrometer):

(11) 1) The -terpineol was subjected to content detection, the added internal standard being chromatographically pure n-butanol.

(12) 2) The content of the -terpineol was calculated by an internal standard method: a certain weight of n-butanol was added as an internal standard to a certain amount of the Antrodia camphorata liquid fermentation broth mixture, then the sample containing n-butanol was subjected to chromatographic analysis to respectively determine the peak areas and relative correction factors of the n-butanol and the measured component, and the percentage of the measured component in the sample could be calculated according to the equation and method.

(13) 3) The headspace solid phase microextraction conditions: The extraction head was aged at the GC injection port until no impurity peak was found, and then extraction was performed at 55 C. for 30 min. The adsorbed volatile compounds were desorbed for 5 min at 250 C. at the GC injection port and flushed into the GC chromatographic column. At the same time, the instrument was started to collect data. Extraction head: DVB/CAR/PMDS.

(14) 4) Gas chromatography conditions: Chromatographic column: DB-WAX; carrier gas: helium, 1.2 mL.Math.min.sup.1; temperature programmed condition: holding at 40 C. for 1 min, heating at 4 C..Math.min.sup.1 to 120 C., heating at 10 C..Math.min.sup.1 to 240 C., and holding for 6 min.

(15) 5) Mass spectrometry conditions: Interface temperature 250 C.; ionization source temperature: 250 C.; quadrupole temperature 150 C.; ionization potential: 70 eV; ionization mode: EI.sup.+; mass scanning range m/z 30-450 amu.

EXAMPLE 1

(16) (1) Culture Conditions 1

(17) Antrodia camphorata strain: ATCC200183

(18) PDA slant medium (g.Math.L.sup.1): Potato 200, glucose 20, agar 20.

(19) Seed liquid medium (g.Math.L.sup.1): Bran 10, corn flour 10, glucose 20, MgSO.sub.4.7H.sub.2O 2, VB.sub.1 0.1, KH.sub.2PO.sub.4 3.

(20) Fermentation liquid medium (g.Math.L.sup.1): Bran 10, corn flour 10, glucose 20, MgSO.sub.4.7H.sub.2O 2, VB.sub.1 0.1, KH.sub.2PO.sub.4 3, pH 5.5.

(21) Seed slant: The Antrodia camphorata strain stored at 4 C. was activated, the PDA slant medium was inoculated with the activated Antrodia camphorata strain, and the activated Antrodia camphorata strain was cultured at 28 C. for 20 days.

(22) Primary seed liquid fermentation: The Antrodia camphorata strain stored at 4 C. was activated, and the liquid seed medium was inoculated with a small square of 1 cm.sup.2. Each bottle was filled with 80 mL of seed medium (250 mL), and after the inoculation, culture was carried out under the conditions of 28 C. and 110 r.Math.min.sup.1 for 10 days.

(23) Secondary shake flask liquid fermentation: The well-grown primary seed liquid was mixed, 500 mL shake flasks were uniformly inoculated with the mixed well-grown primary seed liquid, the amount of each bottle being 150 mL, and the inoculum volume being 10 mL, and after the inoculation, culture was carried out under the conditions of 28 C. and 110 r.Math.min.sup.1 for 15 days.

(24) (2) Off-Line Determination of Contents of -Terpineol and Triterpenoids

(25) Under the culture conditions of Example 1, the variation trend of contents of the -terpineol and triterpenoids in the fermentation process are shown in FIG. 1. The variation trends of the two are consistent. Correlation analysis and unary linear regression analysis of -terpineol and triterpenoids with SPSS21.0 software show that the -terpineol is positively correlated with triterpenoids in terms of content and the Pearson correlation coefficient is 0.958. Further, unary linear regression analysis was performed on the two to obtain the quantitative regression equation between the two as Y=0.460X+8.976.

(26) In the present Example, after the Antrodia camphorata fermentation broth was sampled, at least 10 mL of the fermentation broth was centrifuged for 10 minutes under the centrifugal force of greater than or equal to 7104g, then 5 mL of the supernatant was put into the headspace bottle, and the -terpineol was determined by the headspace solid phase microextraction-gas chromatography-mass spectrometry according to the method as described in the specification.

EXAMPLE 2

(27) (1) Culture Conditions 2

(28) Antrodia camphorata strain: ATCC200183

(29) PDA slant medium (g.Math.L.sup.1): Potato 200, glucose 20, agar 20.

(30) Seed liquid medium (g.Math.L.sup.1): Bran 10, corn flour 10, glucose 20, MgSO.sub.4.7H.sub.2O 2, VB.sub.1 0.1, KH.sub.2PO.sub.4 3.

(31) Fermentation liquid medium (g.Math.L.sup.1): Bran 10, corn flour 10, glucose 20, MgSO.sub.4.7H.sub.2O 2, VB.sub.1 0.1, KH.sub.2PO.sub.4 3, pH 4.5.

(32) Seed slant: The Antrodia camphorata strain stored at 4 C. was activated, the PDA slant medium was inoculated with the activated Antrodia camphorata strain, and the activated Antrodia camphorata strain was cultured at 28 C. for 20 days.

(33) Primary seed liquid fermentation: The Antrodia camphorata strain stored at 4 C. was activated, and the liquid seed medium was inoculated with a small square of 1 cm.sup.2. Each bottle was filled with 80 mL of seed medium (250 mL), and after the inoculation, culture was carried out under the conditions of 28 C. and 110 r.Math.min.sup.1 for 10 days.

(34) Secondary shake flask liquid fermentation: The well-grown primary seed liquid was mixed, 500 mL shake flasks were uniformly inoculated with the mixed well-grown primary seed liquid, the amount of each bottle being 150 mL, and the inoculum volume being 10 mL, and after the inoculation, culture was carried out under the conditions of 28 C. and 110 r.Math.min.sup.1 for 15 days.

(35) (2) Off-Line Determination of Contents of -Terpineol and Triterpenoids

(36) Under the culture conditions of Example 2, the variation trend of contents of the -terpineol and triterpenoids in the fermentation process are shown in FIG. 2. The variation trends of the two are consistent. Correlation analysis and unary linear regression analysis of -terpineol and triterpenoids under such culture conditions show that the -terpineol is positively correlated with triterpenoids in terms of content and the Pearson correlation coefficient is 0.942. Further, unary linear regression analysis was performed on the two to obtain the quantitative regression equation between the two as Y=2.721X+12.057.

(37) The determination of the -terpineol is the same as in Example 1.

EXAMPLE 3

(38) (1) Culture Conditions 3

(39) Antrodia camphorata strain: JMA 01, preserved by KYORI IND (SHENZHEN) Co. Ltd., Shenzhen.

(40) PDA slant medium (g.Math.L.sup.1): Potato 200, glucose 20, agar 20.

(41) Seed liquid medium (g.Math.L.sup.1): Bran 2.0, glucose 20, peptone 10, MgSO.sub.4 1.5, VB.sub.1 0.1, KH.sub.2PO.sub.4 3.

(42) Fermentation liquid medium (g.Math.L.sup.1): Bran 2.0, glucose 20, peptone 10, MgSO.sub.4 1.5, VB.sub.1 0.1, KH.sub.2PO.sub.4 3.

(43) Seed slant: The Antrodia camphorata strain stored at 4 C. was activated, the PDA slant medium was inoculated with the activated Antrodia camphorata strain, and the activated Antrodia camphorata strain was cultured at 28 C. for 20 days.

(44) Primary seed liquid fermentation: The Antrodia camphorata strain stored at 4 C. was activated, and the liquid seed medium was inoculated with a small square of 1 cm.sup.2. Each bottle was filled with 80 mL of seed medium (250 mL), and after the inoculation, culture was carried out under the conditions of 28 C. and 110 r.Math.min.sup.1 for 10 days.

(45) Secondary shake flask liquid fermentation: The well-grown primary seed liquid was mixed, 500 mL shake flasks were uniformly inoculated with the mixed well-grown primary seed liquid, the amount of each bottle being 150 mL, and the inoculum volume being 10 mL, and after the inoculation, culture was carried out under the conditions of 28 C. and 110 r.Math.min.sup.1 for 15 days.

(46) (2) Off-Line Determination of Contents of -Terpineol and Triterpenoids

(47) Under the culture conditions of Example 3, the variation trend of contents of the -terpineol and triterpenoids in the fermentation process are shown in FIG. 3. The variation trends of the two are consistent. Correlation analysis and unary linear regression analysis of -terpineol and triterpenoids under such culture conditions show that the -terpineol is positively correlated with triterpenoids in terms of content and the Pearson correlation coefficient is 0.961. Further, unary linear regression analysis was performed on the two to obtain the quantitative regression equation between the two as Y=2.360X+2.927.

(48) The determination of the -terpineol is the same as in Example 1.

EXAMPLE 4

(49) The conditions of implementation were the same as in Example 1. Compared to FIG. 1, the -terpineol in FIG. 4 did not rise but fall after the 8.sup.th to 9.sup.th days, suggesting that the fermentation was abnormal. Microbiological contamination was found after sampling and microscopy, and the microbes causing contamination were bacteria. This indicates that the present invention can not only characterize the content variation of triterpenoids in the normal fermentation process, but also emit a prompt signal when the fermentation is abnormal.

COMPARATIVE EXAMPLE 1

(50) Under the culture conditions of Example 1, the correlation analysis of other volatile compounds detected under the same conditions and triterpenoids was carried out, and the correlation coefficients were low, and were all lower than the correlation coefficient between -terpineol and triterpenoids. The statistical analysis results are shown in Table 1.

(51) Among them, the 1, 2-decanediol is negatively correlated with triterpenoids in terms of content, and the Pearson correlation coefficient is 0.630. The linalool is negatively correlated with triterpenoids in terms of content, and the correlation coefficient is 0.301. The 1-octen-3-ol is negatively correlated with triterpenoids in terms of content, and the Pearson correlation coefficient is 0.436. The methyl furan-3-carboxylate is positively correlated with triterpenoids in terms of content, and the Pearson correlation coefficient is 0.899. The 3-octanone is positively correlated with triterpenoids in terms of content, and the Pearson correlation coefficient is 0.233. The phenethyl alcohol is positively correlated with triterpenoids in terms of content, and the Pearson correlation coefficient is 0.869. The cubenol is positively correlated with triterpenoids in terms of content, and the Pearson correlation coefficient is 0.875. The n-caprylic octanal is negatively correlated with triterpenoids in terms of content, and the Pearson correlation coefficient is 0.202. The Trans-(E)-Nerolidol is positively correlated with triterpenoids in terms of content, and the Pearson correlation coefficient is 0.512. The trans-2-octen-1-ol is positively correlated with triterpenoids in terms of content, and the Pearson correlation coefficient is 0.326.

(52) TABLE-US-00001 TABLE 1 Correlation analysis of other volatile substances and triterpenoids in the fermentation process of Example 1 Pearson Pearson Correlation Volatile Correlation Volatile Substance Coefficient Substance Coefficient 1,2-decanediol 0.630 phenethyl alcohol 0.869 linalool 0.301 cubenol 0.875 1-octen-3-ol 0.436 n-caprylic octanal 0.202 methyl furan-3- 0.899 Trans-(E)-Nerolidol 0.512 carboxylate 3-octanone 0.233 trans-2-octen-1-ol 0.326

COMPARATIVE EXAMPLE 2

(53) Under the culture conditions of Example 2, the correlation analysis of other volatile compounds detected under the same conditions and triterpenoids was carried out, and the Pearson correlation coefficients were low, and were all lower than the correlation coefficient between -terpineol and triterpenoids. The statistical analysis results are shown in Table 2.

(54) Among them, the 1, 2-decanediol is negatively correlated with triterpenoids in terms of content, and the Pearson correlation coefficient is 0.601. The linalool is positively correlated with triterpenoids in terms of content, and the correlation coefficient is 0.405. The 1-octen-3-ol is negatively correlated with triterpenoids in terms of content, and the Pearson correlation coefficient is 0.171. The methyl furan-3-carboxylate is positively correlated with triterpenoids in terms of content, and the Pearson correlation coefficient is 0.585. The 3-octanone is positively correlated with triterpenoids in terms of content, and the Pearson correlation coefficient is 0.306. The phenethyl alcohol is positively correlated with triterpenoids in terms of content, and the Pearson correlation coefficient is 0.809. The cubenol is positively correlated with triterpenoids in terms of content, and the Pearson correlation coefficient is 0.501. The n-caprylic Octanal is positively correlated with triterpenoids in terms of content, and the Pearson correlation coefficient is 0.530. The Trans-(E)-Nerolidol is positively correlated with triterpenoids in terms of content, and the Pearson correlation coefficient is 0.853. The trans-2-octen-1-ol is positively correlated with triterpenoids in terms of content, and the Pearson correlation coefficient is 0.120.

(55) TABLE-US-00002 TABLE 2 Correlation analysis of other volatile substances and triterpenoids in the fermentation process of Example 2 Pearson Pearson Correlation Volatile Correlation Volatile Substance Coefficient Substance Coefficient 1,2-decanediol 0.601 phenethyl alcohol 0.809 linalool 0.405 cubenol 0.501 1-octen-3-ol 0.171 n-caprylic octanal 0.530 methyl furan-3- 0.585 Trans-(E)-Nerolidol 0.853 carboxylate 3-octanone 0.306 trans-2-octen-1-ol 0.120