Optimization Technology of the LHS-1 Strain for Degrading Gallnut Water

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Article Optimization Technology of the LHS-1 Strain for Degrading Gallnut Water Extract and Appraisal of Benzene Ring Derivatives from Fermented Gallnut Water Extract Pyrolysis by Py-GC/MS

Chengzhang Wang 1,2,3,4,5,* and Wenjun Li 1,2,3,4,5

1 Institute of Chemical Industry of Forest Products, Chinese Academy Forestry, Nanjing 210042, China; [email protected] 2 National Engineering Laboratory for Biomass Chemical Utilization, Nanjing 210042, China 3 Key and Open Laboratory on Forest Chemical Engineering, State Forestry Administration, Nanjing 210042, China 4 Key Laboratory of Biomass Energy and Material, Nanjing 210042, China 5 Institute of New Technology of Forestry, Chinese Academy Forestry, Beijing 100091, China * Correspondence: [email protected]; Tel./Fax: +86-025-8548-2471

Received: 31 October 2017; Accepted: 11 December 2017; Published: 20 December 2017

Abstract: Gallnut water extract (GWE) enriches 80~90% of gallnut tannic acid (TA). In order to study the biodegradation of GWE into gallic acid (GA), the LHS-1 strain, a variant of Aspergillus niger, was chosen to determine the optimal degradation parameters for maximum production of GA by the response surface method. Pyrolysis–gas chromatography–mass spectrometry (Py-GC/MS) was ﬁrst applied to appraise benzene ring derivatives of fermented GWE (FGWE) pyrolysis by comparison with the pyrolytic products of a tannic acid standard sample (TAS) and GWE. The results showed that optimum conditions were at 31 ◦C and pH of 5, with a 50-h incubation period and 0.1 g·L−1 of TA as substrate. The maximum yields of GA and tannase were 63~65 mg·mL−1 and 1.17 U·mL−1, respectively. Over 20 kinds of compounds were identiﬁed as linear hydrocarbons and benzene ring derivatives based on GA and glucose. The key benzene ring derivatives were 3,4,5-trimethoxybenzoic acid methyl ester, 3-methoxy-1,2-benzenediol, and 4-hydroxy-3,5-dimethoxy-benzoic acid hydrazide.

Keywords: gallnut water extract; tannic acid; benzene ring derivatives; biodegradation; Py-GC/MS

1. Introduction Gallnut is an abnormal gall in China produced by parasitic aphids on the host trees of Rhus chinensis, Rhus potaninii, or Rhus punjabensis, etc. Chinese gallnut can be divided into the three categories of Du-ensiform gall, horned gall, and gall ﬂowers, according to aphid species and host differences. Du-ensiform gall enriches 55~65% of tannin (Figure1), and is the most traditional and important raw material for producing gallnut water extract (GWE) which contains 80~90% tannin. GWE has been used to produce gallic acid through the hydrolysis of alkali, and acid catalysis. However, chemical degradation of GWE not only provides very low yields of gallic acid, but causes serious environmental pollution and equipment corrosion. Therefore, it is urgent to probe more environmentally friendly degradation technology in order to produce gallic acid and its derivatives [1,2].

Molecules 2017, 22, 2253; doi:10.3390/molecules22122253 www.mdpi.com/journal/molecules Molecules 2017, 22, 2253 2 of 11 Molecules 2017, 22, 2253 2 of 11

(a) (b)

FigureFigure 1. 1. ChineseChinese Du-ensiform Du-ensiform gallnut, gallnut, (a ()a )fresh fresh gallnut; gallnut; (b (b) )dry dry gallnut. gallnut.

The enzymatic hydrolysis of GWE using the microorganisms of Aspergillus and Penicillium, The enzymatic hydrolysis of GWE using the microorganisms of Aspergillus and Penicillium, particularly Aspergillus niger, has great potential for the industrialized production of gallic acid [3]. particularly Aspergillus niger, has great potential for the industrialized production of gallic acid [3]. Wang [4] separated one strain of A. niger from Chinese Gall “Beihua” and fermented gallnut directly Wang [4] separated one strain of A. niger from Chinese Gall “Beihua” and fermented gallnut directly to produce GA with a yield of 78.5~89.5%. Yang [5] used the same strain to ferment gallotannins and to produce GA with a yield of 78.5~89.5%. Yang [5] used the same strain to ferment gallotannins purified macroporous resin to obtain a high yield of 85.6% gallic acid. Banerjee modified a tannin- and purified macroporous resin to obtain a high yield of 85.6% gallic acid. Banerjee modified a rich substrate of solid-state fermentation (MSSF) to produce tannase and gallic acid through co- tannin-rich substrate of solid-state fermentation (MSSF) to produce tannase and gallic acid through culture method to obtain maximum yield of 94.8% gallic acid (GA) [6]. However, no literature has co-culture method to obtain maximum yield of 94.8% gallic acid (GA) [6]. However, no literature has reported on fermented GWE chemical constitutes and structures so far, particularly with respect to reported on fermented GWE chemical constitutes and structures so far, particularly with respect to the the biodegradation mechanism of GWE biotransformation. biodegradation mechanism of GWE biotransformation. Since the microbial transformation of tannins was discovered by Tieghem [7], only a few Since the microbial transformation of tannins was discovered by Tieghem [7], only a few analytical analytical techniques, such as high-performance liquid chromatography (HPLC), thin-layer techniques, such as high-performance liquid chromatography (HPLC), thin-layer chromatography chromatography (TLC), or pyrolysis–gas chromatography–mass spectrometry (Py-GC/MS) have (TLC), or pyrolysis–gas chromatography–mass spectrometry (Py-GC/MS) have been applied to been applied to the identification of natural products and their metabolites by both conventional the identification of natural products and their metabolites by both conventional destructive or destructive or non-destructive methods in the process of chemical synthesis and biotransformation non-destructive methods in the process of chemical synthesis and biotransformation [8–11]. In order [8–11]. In order to develop gallic acid by microbial degradation of GWE, Min previously reported the to develop gallic acid by microbial degradation of GWE, Min previously reported the extraction and extraction and screening of tannic acid degradation by the LHS-1 strain according to the indexes of screening of tannic acid degradation by the LHS-1 strain according to the indexes of tannic acid tannic acid degradation rate, gallic acid accumulation concentration, and tannin enzyme activity [12]. degradation rate, gallic acid accumulation concentration, and tannin enzyme activity [12]. This study This study focused on the optimization technology of LHS-1 strain degradation of GWE, and Py- focused on the optimization technology of LHS-1 strain degradation of GWE, and Py-GC/MS was GC/MS was applied to probe the chemical constituents and structural characterization of the linear applied to probe the chemical constituents and structural characterization of the linear and benzene and benzene ring derivatives, based on phenolic and glucose structure from fermented GWE (FGWE) ring derivatives, based on phenolic and glucose structure from fermented GWE (FGWE) pyrolysis. pyrolysis. It will be very helpful for us to research the biodegradation mechanism and the It will be very helpful for us to research the biodegradation mechanism and the corresponding corresponding metabolites of GWE for the future. metabolites of GWE for the future. 2. Results 2. Results

2.1.2.1. Determination Determination of of Tannic Tannic Acid Acid and and Gallic Gallic Acid Acid in in FGWE FGWE Folin–CiocalteauFolin–Ciocalteau and and HPLC HPLC were were both both used used to to moni monitortor the the changes changes in in gallic gallic acid acid and and tannic tannic acid acid inin GWE GWE biodegradation biodegradation with with the the LHS-1 LHS-1 strain. strain. The The standard standard curves curves of tannic of tannic acid and acid gallic and gallic acid had acid 2 higherhad higher linearity. linearity. Tannic Tannic acid was acid dete wascted detected at 630 atnm, 630 and nm, its and equation its equation was y was= 69.848y =x 69.848 + 0.0011x + 0.0011(R = 2 0.9993),(R2 = 0.9993) while, the while equation the equation of gallic of acid gallic was acid y = was0.0029y =x 0.0029+ 0.0039x + (R 0.0039 = 0.9998). (R2 = The 0.9998). analysis The showed analysis GWEshowed contained GWE contained81.6% of tannin 81.6% acid of tannin and 5.6% acid of and gallic 5.6% acid. of gallicWhen acid.GWE When was incubated GWE was at incubated 28 °C with at a28 pH◦C of with 5.0 for a pH 50 ofh, 5.0HPLC for 50showed h, HPLC that showed the levels that of thetannic levels acid of decreased tannic acid and decreased those of and gallic those acid of increasedgallic acid significantly increased signiﬁcantly in FGWE (Figure in FGWE 2). (Figure2).

Molecules 2017, 22, 2253 3 of 11 Molecules 2017, 22, 2253 3 of 11 Molecules 2017, 22, 2253 3 of 11 mAU mA U mAU mA U 270nm,4nm (1.00) 270nm,4nm (1.00) 270nm,4nm (1.00) 270nm,4nm (1.00) 100 100 2000 2000

50 50 1000 1000

0 0 0 0 0.0 5.0 10.0 15.0 20.0 min 0.0 5.0 10.0 15.0 20.0 25.0 30.0 min 0.0 5.0 10.0 15.0 20.0 min 0.0 5.0 10.0 15.0 20.0 25.0 30.0 min (a) (b) (a) (b) Figure 2. HPLC of a standard gallic acid sample and fermented gallnut water extract (FGWE), (a) FigureFigure 2. 2. HPLCHPLC of ofa standard a standard gallic gallic acid acid sample sample and and fermented fermented gallnut gallnut water water extract extract (FGWE), (FGWE), (a) standard sample of gallic acid; (b) the fermentation broth of GWE. standard(a) standard sample sample of gallic of gallic acid; acid; (b) the (b) fermentation the fermentation broth broth of GWE. of GWE. The degradation rate of GWE tannic acid exceeded 85.6%. The concentration of gallic acid TheThe degradation degradation rate rate of of GWE GWE tannic tannic acid acid exceeded exceeded 85.6%. 85.6%. The The concentration concentration of of gallic gallic acid acid approached 63~65 mg·mL−1, and tannin enzyme activity was 1.17 U·mL−1. This indicated that the LHS- approachedapproached 63~65 63~65 mg·mL mg·mL−1,− and1, and tannin tannin enzyme enzyme activity activity was was1.17 1.17U·mL U−·1mL. This−1 .indicated This indicated that the that LHS- the 1 strain could produce tannase to transform tannic acid into gallic acid (Figure 3). 1LHS-1 strain straincould couldproduce produce tannase tannase to transform to transform tannic tannic acid into acid gallic into gallicacid (Figure acid (Figure 3). 3).

Gallic acid Glucose Gallic acid Glucose Tannic acid Tannic acid Figure 3. The degradation of GWE tannic acid into gallic acid. FigureFigure 3. 3. TheThe degradation degradation of of GWE GWE tannic tannic acid acid into into gallic gallic acid. acid. 2.2. Box–Behnken Design and Variance Analysis of the LHS-1 Strain for Degrading Tannic Acid 2.2.2.2. Box–Behnken Box–Behnken Design Design and and Variance Variance Analysis Analysis of of the the LHS-1 LHS-1 Strain Strain for for Degrading Degrading Tannic Tannic Acid Acid Since the incubation temperature, the initial pH, and the incubation period obviously affected Since the incubation temperature, the initial pH, and the incubation period obviously affected tanninSince enzyme the incubationactivities in temperature, the culture media, the initial and pH, the andability the of incubation the LHS-1 period strain obviouslyto degrade affected tannic tannin enzyme activities in the culture media, and the ability of the LHS-1 strain to degrade tannic acidtannin was enzyme measured activities through in the the culture degradation media, rate and of the tannic ability acid, of the the LHS-1 concentration strain to degrade of gallic tannic acid, and acid acid was measured through the degradation rate of tannic acid, the concentration of gallic acid, and thewas activity measured of tannase, through the the Box– degradationBehnken experiment rate of tannic was acid, designed the concentration and the results of are gallic shown acid, in and Table the the activity of tannase, the Box–Behnken experiment was designed and the results are shown in Table 1.activity The regression of tannase, equation the Box–Behnken was determined experiment as follows: was designed and the results are shown in Table1. 1.The The regression regression equation equation was was determined determined as as follows: follows: Y = 1.18 + 0.039X + 0.002X + 0.35X − 0.003X X − 0.007X X + Y = 1.18 + 0.039X 1+ 0.002X 2+ 0.35X 3− 0.003X 1X 2− 0.007X 1X 3+ 1 2 3 1 2 1 3 Y = 1.18 + 0.039X + 0.002X + 0.35X − 0.003X X − 0.007X X + 0.045X X − 0.101X 2 − 0.13X2 2 − 0.153X 2 1 2 1 3 0.045XX X2X −3− 0.10XX 212−− 0.13XX 22−− 0.15XX 232 0.045 22 3 3 0.10 11 0.13 22 0.15 33 Variance analysis and significant tests of this regression model are shown in Table 2. The VarianceVariance analysis andand significant significant tests tests of thisof this regression regression model model are shown are shown in Table in2. TheTable quadratic 2. The quadratic model was very significant and meaningful in statistics (F = 23.62, p = 0.0002 < 0.001). The quadraticmodel was model very was significant very significant and meaningful and meaningful in statistics in (statisticsF = 23.62, (Fp = 23.62, 0.0002 p < = 0.001).0.0002 The< 0.001). lack-of-fit The lack-of-fit item was good (p = 0.3680 > 0.05), and no loss factors existed. Therefore, the regression lack-of-fititem was item good was (p = good 0.3680 (p >= 0.05),0.3680 and > 0.05), no loss and factors no loss existed. factors Therefore,existed. Therefore, the regression the regression equation equation could substitute the real experiment to analyze and direct experiments. For the monomial equationcould substitute could substitute the real the experiment real experiment to analyze to analyze and direct and experiments.direct experiments. For the For monomial the monomial items items (X1, X2, and X3), the effects of culture temperature and incubation time on tannase activities items(X1, X(X2,1, and X2, Xand3), theX3), effectsthe effects of culture of culture temperature temperature and incubationand incubation time time on tannase on tannase activities activities were were significantly different (p <0.05). The effects of the three monomial factors on tannase activities weresignificantly significantly different different (p <0.05). (p <0.05). The effects The effects of the of three the monomialthree mono factorsmial factors on tannase on tannase activities activities ranging ranging from strong to weak were in the following order: incubation temperature (X1) > incubation rangingfrom strong from tostrong weak to were weak in were the followingin the following order: order: incubation incubation temperature temperature (X1) >(X incubation1) > incubation time time (X2) > incubation initial pH (X3). For interactive items of X2X3 (p = 0.0352 < 0.05), initial incubation time(X2) ( >X2 incubation) > incubation initial initial pH pH (X 3().X3 For). For interactive interactive items items of ofX X2X2X33( (pp= = 0.0352 < 0.05), 0.05), initial initial incubation incubation pH and the incubation time showed a great interaction, while X1X2 and X1X3 had no interaction pHpH and and the the incubation incubation time time showed showed a a great interaction, while X1XX22 andand XX11XX3 3hadhad no no interaction interaction (p > 0.05). For the binomial items of the equation, the effects of the incubation temperature, initial pH, (p(p >> 0.05). 0.05). ForFor the the binomial binomial items items of of the the equation, equation, th thee effects effects of of the the incubation incubation temperature, temperature, initial initial pH, pH, and incubation time on tannin enzyme activities reached significant and extreme levels (p < 0.0001). and incubation time on tannin enzyme activities reached significant and extreme levels (p < 0.0001).

Molecules 2017, 22, 2253 4 of 11 and incubation time on tannin enzyme activities reached signiﬁcant and extreme levels (p < 0.0001). Through comparing the p-value, the primary and secondary factors could be judged for the LHS-1 strain with respect to the degradation of tannic acid [13].

Table 1. The Box–Behnken experimental design and corresponding tannase activity for the LHS-1 strain.

◦ −1 No. X1 Temperature ( C) X2 Initial pH X3 Incubation Time (h) Y Tannase Activity (U·mL ) 1 −1 −1 0 0.892 2 1 −1 0 0.968 3 −1 1 0 0.944 4 1 1 0 1.016 5 −1 0 −1 0.852 6 1 0 −1 0.948 7 −1 0 1 0.936 8 1 0 1 1.004 9 0 −1 −1 0.944 10 0 1 −1 0.812 11 0 −1 1 0.924 12 0 1 1 0.972 13 0 0 0 1.164 14 0 0 0 1.14 15 0 0 0 1.192 16 0 0 0 1.22 17 0 0 0 1.204

Table 2. Variance analysis of items in the regression equation.

Sources of Variation Sum of Squares df Mean Square F-Value p-Value model 0.016 9 0.028 23.62 0.0002 −5 X1 7.605 × 10 1 0.012 10.19 0.0152 −6 −5 X2 2.000 × 10 1 3.200 × 10 0.027 0.8746 −4 −3 X3 6.125 × 10 1 9.800 × 10 8.21 0.0242 −7 −6 X1X2 2.500 × 10 1 4.00 × 10 0.0033 0.9555 −5 −4 X1X3 1.225 × 10 1 1.960 × 10 0.16 0.6975 −4 −3 X2X3 5.063 × 10 1 8.100 × 10 6.78 0.0352 2 −3 X1 2.819 × 10 1 0.045 37.77 0.0005 2 −3 X2 4.145 × 10 1 0.066 55.53 0.0001 2 −3 X3 5.571 × 10 1 0.089 74.64 <0.0001 residual 5.225 × 10−4 7 1.194 × 10−3 lack-of-ﬁt item 2.665 × 10−4 3 1.421 × 10−3 1.39 0.3680 pure error 2.560 × 10−4 4 1.024 × 10−3 total 0.016 16

2.3. The Optimization Technology for the LHS-1 Strain to Degrade Tannin According to the regression analysis and Box–Behnken experimental optimal model, the contour plots and 3D response surface plots were obtained by the Design Expert software, as shown in Figure4, which are confined in the smallest ellipse in the contour diagram. The tannin enzyme activities were obtained with two continuous variables, while the other variables were fixed constantly. Elliptical contours were obtained when there was a perfect interaction between the independent variables. For X2X3 there existed a significant interaction (p < 0.05) and the contour map was shown to be elliptical. However, X1X2 and X1X3 showed round contours with no significant interaction (p > 0.05), indicating that X1X2 and X1X3 had less of an effect on tannin enzyme activities. Hence, initial pH and incubation time had more obvious effects. The maximum tannin enzyme activities were obtained when the temperature was around 29~33 ◦C, with pH of 4.5~5.5, and an incubation time of 42~54 h. MoleculesMolecules2017 2017, 22, 22, 2253, 2253 5 5of of 11 11

In the Box–Behnken design, the optimal conditions were determined as follows: cultivating In the Box–Behnken design, the optimal conditions were determined as follows: cultivating temperature 30.92◦ °C, initial pH 5.03, and incubation time of 49.44 h, with the corresponding tannin temperatureenzyme activity 30.92 of C,1.192 initial U·mL pH−1. 5.03,Considering and incubation the practical time experime of 49.44 h,nts, with the optimal the corresponding parameters tanninwere · −1 enzymeadjusted activity to a temperature of 1.192 U mL of 31 .°C, Considering initial pH theof 5, practical and incubation experiments, time of the 50 optimal h, with parameterstannin enzyme were ◦ adjustedactivity toof a1.17 temperature U·mL−1. The of relative 31 C, initialerror of pH the of model 5, and was incubation 1.68% (Rtime < 5%). of Thus, 50 h, withthe response tannin enzymemodel −1 activitycould reflect of 1.17 the U· mLexpected. The optimization. relative error Compar of the modeled with was an enzyme 1.68% (R activity< 5%). of Thus, 0.973 the U·mL response−1, the LHS- model −1 could1 strain reﬂect could the expectedincrease optimization.the tannin enzyme Compared activi withty by an enzymemore 20.2% activity in the of 0.973 optimal U·mL fermentation, the LHS-1 straincondition. could increase the tannin enzyme activity by more 20.2% in the optimal fermentation condition.

(a)

(b)

(c)

FigureFigure 4. 4. ContourContour plots plots and and 3D-response 3D-response surface surface plots showing plots showing (a) the interactive (a) the interactive effects of incubation effects of temperature and initial pH (X1X2); (b) incubation temperature and incubation time (X1X3); and (c) incubation temperature and initial pH (X1X2); (b) incubation temperature and incubation time (X1X3); initial pH and incubation time (X2X3). and (c) initial pH and incubation time (X2X3).

Molecules 2017, 22, 2253 6 of 11 Molecules 2017, 22, 2253 6 of 11 2.4. The Appraisal of Linear and Benzene Ring Derivatives Based on Phenolic and Glucose Structure from FGWE2.4. The Pyrolysis Appraisal by Py-GC/MS of Linear and Benzene Ring Derivatives Based on Phenolic and Glucose Structure from FGWEPy-GC/MS Pyrolysis was by Py-GC/MSused to appraise the pyrolytic products of FGWE pyrolysis through comparison with thePy-GC/MS tannic acid was standard used to appraisesample (TAS) the pyrolytic and GWE products data, ofcombined FGWE pyrolysis with GC-MS through to determine comparison gallicwith acid the and tannic tannic acid acid standard levels samplein samples. (TAS) Their and total GWE ion data, chromatography combined with peaks GC-MS all consisted to determine of lineargallic and acid aromatic and tannic products, acid levels with inretention samples. times Their ranging total ion from chromatography 9 to 13 min and peaks 13 allto 16 consisted min, respectively,of linear and as aromaticwell as gallic products, acid structural with retention products times with ranging retention from times 9 to ranging 13 min from and 1316 toto 1621.15 min, min.respectively, Through ascomparison well as gallic of acid the structural mass spectra products of withGC-MS retention retention times times ranging with from corresponding 16 to 21.15 min. standards,Through over comparison 20 kinds of of the compounds mass spectra were of identified GC-MS retention as linear timesand benzene with corresponding ring matrix structures standards, (Figureover 20 5). kinds The linear of compounds compounds, were marked identiﬁed with asnumb linearers and1~9, benzenewere identified ring matrix because structures of the pyrolysis (Figure5 ). ofThe glucose, linear and compounds, benzene ring marked compounds with numbers were 1~9,marked were with identiﬁed numbers because 10~20, of which the pyrolysis were appraised of glucose, asand the benzeneresults of ring pyrolysis compounds of ester were bonds marked between with gallic numbers acid 10~20, and glucose which and were epside appraised bonds as between the results gallicof pyrolysis acids. of ester bonds between gallic acid and glucose and epside bonds between gallic acids.

Linear pyrolytic 16 10 12 Aromatic pyrolytic products of tannic acid 14 products of tannic acid 2 4 6

5 9 1 7 (a) 3 8 19

1415 12 19 10 16 Pyrolytic products (b) 2 of gallic acid 17 18 3 6 1 19 14 20 46 15 2 18 (c) 10 16

6 8 10 12 14 16 18 20 22 24 26 28 Minutes

FigureFigure 5. 5.TheThe total total ion ion chromatography chromatography of ofthree three samp samplesles with with pyrolysis–gas pyrolysis–gas chromatography–mass chromatography–mass spectrometryspectrometry (Py-GC/MS). (Py-GC/MS). (a) (Tannica) Tannic acid acid standard standard sample sample (TAS); (TAS); (b ()b GWE;) GWE; (c) ( cFGWE.) FGWE.

AfterAfter pyrolysis, pyrolysis, TAS TAS produced produced 9 9linear linear compounds compounds and and 5 benzene 5 benzene ring ring derivatives, derivatives, while while GWE GWE pyrolyzedpyrolyzed into into 6 linear 6 linear compounds compounds and and 10 10 aromatic aromatic ring ring products, products, and and FGWE FGWE was was found found to toproduce produce onlyonly 3 linear 3 linear compounds compounds and and 7 benzene 7 benzene ring ring derivatives. derivatives. The The differences differences originated originated from from the the contents contents ofof tannic tannic acid acid and and gallic gallic acid. acid. FGWE FGWE contained contained more more gallic gallic acid and acid little and tannic little tannic acid, while acid, whileTAS and TAS GWEand GWEcontained contained over 90% over of 90% tannin. of tannin. Therefore, Therefore, the results the results indicated indicated that that the the LHS-1 LHS-1 strain strain could could produceproduce tannase tannase to to transform transform the the tannic tannic acid acid of of GWE GWE into into gallic gallic acid. acid.

2.5.2.5. The The Pyrolytic Pyrolytic Products Products of ofTAS, TAS, GWE, GWE, and and FGWE FGWE TheThe pyrolytic pyrolytic products products of TAS, of TAS, GWE, GWE, and andFGWE FGWE werewere composed composed of linear of linearand benzene and benzene ring compounds.ring compounds. The Py-GC/MS The Py-GC/MS signal signalpeaks peaksintegrated integrated the therelated related abundances abundances of ofindividual individual compoundscompounds and and determined determined the pyrolytic the pyrolytic products products of TAS, GWE, of TAS, and GWE,FGTA, andas shown FGTA, in Table as shown 3. It canin be Table seen3 that. Itthe can pyrolytic be seen products that of the TAS pyrolytic had 14 compounds, products of in TASwhich had 3-methoxy-benzene-1,2- 14 compounds, in diol was 20.77% of the highest content, and the content of 2,6-dimethoxyphenol, 1,2,3-

Molecules 2017, 22, 2253 7 of 11 which 3-methoxy-benzene-1,2-diol was 20.77% of the highest content, and the content of Molecules 2017, 22, 2253 7 of 11 2,6-dimethoxyphenol, 1,2,3-trimethoxybenzene and 3,4-dimethoxyphenol were between 11.18% andtrimethoxybenzene 11.84%. GWE contained and 3,4-dimethoxyphenol analogous linear were and between benzene 11.18% ring pyrolytic and 11.84%. products GWE contained such as TAS. Sixteenanalogous pyrolytic linear products and benzene were ring appraised pyrolytic in products GWE pyrolysis. such as TAS. Among Sixteen them, pyrolytic 3,4,5-trimethoxybenzoic products were acidappraised methyl esterin GWE and pyrolysis. 3-methoxy-1,2-benzenediol Among them, 3,4,5-trimethoxybenzoic represented 13.77% acid and methyl 12.70%, ester respectively. and 3- Onlymethoxy-1,2-benzenediol four compounds represented represented between 13.77% 5.32% and 12.70%, and 8.22%. respectively. However, Only FGWE four onlycompounds produced pyrolyticrepresented products between of 105.32% compounds. and 8.22%. However, For FGWE, FGWE 3,4,5-trimethoxy-benzoic only produced pyrolytic acidproducts methyl of 10 ester representedcompounds. 41.55% For FGWE, (the highest 3,4,5-trimethoxy-benzoic percentage), and 4-hydroxy-3,5-dimethoxy-benzoicacid methyl ester represented 41.55% acid(the highest hydrazide representedpercentage), 7.42%. and 4-hydroxy-3,5-dimethoxy-benzoic The pyrolysis products of benzene acid hydrazide ring derivatives represented from 7.42%. FGWE The werepyrolysis further identiﬁedproducts as of 3,4-dimethoxy-phenol, benzene ring derivatives 1,2,3-trimethoxybenzene, from FGWE were further 3-methoxy-benzoicidentified as 3,4-dimethoxy-phenol, acid methyl ester, 2,6-dimethoxy-phenol,1,2,3-trimethoxybenzene, 3,4-dimethoxy-benzoic 3-methoxy-benzoic acid acid methyl methyl ester, ester, 3,4,5-trimethoxybenzoic 2,6-dimethoxy-phenol, acid 3,4- methyl dimethoxy-benzoic acid methyl ester, 3,4,5-trimethoxybenzoic acid methyl ester, and 4-hydroxy-3,5- ester, and 4-hydroxy-3,5-dimethoxy-benzoic acid hydrazide. The contents of main pyrolytic products dimethoxy-benzoic acid hydrazide. The contents of main pyrolytic products in TAS, GWE, and in TAS, GWE, and FGWE are shown in Figure6. FGWE are shown in Figure 6.

O O O O

O O O O O O

O O 5 2 4 O

O OH O O

O O O

HO O O O O OH OH 10 12 14 15 16

O O

O HO

O O O O

O HN 19 20 NH2

FigureFigure 6. The6. The chemical chemical structures structures ofof thethe main py pyrolyticrolytic products products in inTAS, TAS, GWE, GWE, and and FGWE. FGWE.

Table 3. The relative intensities of pyrolytic products of TAS, GWE, and FGWE. Table 3. The relative intensities of pyrolytic products of TAS, GWE, and FGWE. Retention Sample Total Peak Area, % Peak MW Formula Compound Time (min) Retention TASSample TotalGWE Peak FGWE Area, % Peak1 9.946 MW146 FormulaC6H10O4 Succinic acid,Compound dimethyl ester 1.68 0.62 - Time (min) TAS GWE FGWE 2 10.656 160 C7H12O4 Succinic acid, methyl-, dimethyl ester 8.11 1.8 2.57 13 9.94611.182 146124 CC6H7H108OO24 SuccinicPhenol, acid, 2-methoxy- dimethyl ester 2.17 1.68 1.37 0.62 - - 24 10.65611.348 160174 CC78HH1214O44 SuccinicDimethyl acid, 2,3-dimethylsuccinate methyl-, dimethyl ester 6.33 8.11 0.74 1.8 2.69 2.57 35 11.18211.520 124158 CC77H108O24 DimethylPhenol, ethylidene 2-methoxy- malonate 1.89 2.17 0.67 1.37 - - 4 11.348 174 C H O Dimethyl 2,3-dimethylsuccinate 6.33 0.74 2.69 6 11.749 174 C88H1414O44 Pentane dioic acid, 2-methyl-, dimethyl ester 5.35 0.71 2.62 5 11.520 158 C7H10O4 Dimethyl ethylidene malonate 1.89 0.67 - 7 12.184 174 C8H14O4 Butane dioic acid, ethyl-, dimethyl ester 1.30 - - 6 11.749 174 C8H14O4 Pentane dioic acid, 2-methyl-, dimethyl ester 5.35 0.71 2.62 8 12.584 188 C9H16O4 Dimethyl 2-methyladipate 0.73 - - 7 12.184 174 C8H14O4 Butane dioic acid, ethyl-, dimethyl ester 1.30 - - 9 12.784 176 C9H20O3 1,2,6-Trimethoxy-hexane 1.86 - - 8 12.584 188 C9H16O4 Dimethyl 2-methyladipate 0.73 - - 10 13.963 154 C8H10O3 Phenol, 3,4-dimethoxy- 11.84 6.22 0.92 9 12.784 176 C9H20O3 1,2,6-Trimethoxy-hexane 1.86 - - 11 14.009 152 C9H12O2 3,5-Dimethoxytoluene - 0.81 - 10 13.963 154 C8H10O3 Phenol, 3,4-dimethoxy- 11.84 6.22 0.92 12 14.512 140 C7H8O3 1,2-Benzenediol, 3-methoxy- 20.77 12.70 - 11 14.009 152 C9H12O2 3,5-Dimethoxytoluene - 0.81 - 13 14.844 140 C7H8O3 2-Methoxyresorcinol - 1.62 - 12 14.512 140 C7H8O3 1,2-Benzenediol, 3-methoxy- 20.77 12.70 - 9 12 3 1314 14.84414.964 140168 CC7H8O3 1,2,3-Trimethoxybenzene2-Methoxyresorcinol 11.18 - 7.63 1.62 4.39 - 1415 14.96415.439 168166 CC99HH1210O33 3-Methoxybenzoic1,2,3-Trimethoxybenzene acid methyl ester - 11.18 5.32 7.63 1.81 4.39 1516 15.43915.640 166154 CC98HH1010O33 3-MethoxybenzoicPhenol, 2,6-dimethoxy- acid methyl ester 11.24 - 8.22 5.32 0.71 1.81 1617 15.64015.937 154166 CC89HH1010O33 MethylPhenol, 4-methoxybenzoate 2,6-dimethoxy- - 11.24 0.91 8.22 -0.71 1718 15.93718.793 166196 CC910HH1012O34 Benzoic acid,Methyl 3,4-dimethoxy-, 4-methoxybenzoate methyl ester - - 1.02 0.91 1.12- 1819 18.79320.383 196226 CC1011HH1214O54 BenzoicBenzoic acid, acid, 3,4,5-trimethoxy-, 3,4-dimethoxy-, methyl methyl ester ester 3.57 - 13.77 1.02 41.55 1.12 19 20.383 226 C11H14O5 BenzoicBenzoic acid, 3,4,5-trimethoxy-,4-hydroxy-3,5-dimethoxy-, methyl ester 3.57 13.77 41.55 20 21.150 212 C9H12N2O4 - - 7.42 20 21.150 212 C9H12N2O4 Benzoic acid, 4-hydroxy-3,5-dimethoxy-,hydrazide hydrazide - - 7.42 Note:Note: “-” “-” indicates indicates a compounda compound peak peak area area of <0.6%of <0.6% in thein the table. table.

Molecules 2017, 22, 2253 8 of 11

3. Discussion Garnier et al. [8] reported temperatures effected on the pyrolytic process of hydrolysis–methylation of between 250 ◦C and 500 ◦C. When the temperature was above 500 ◦C, sufficient thermal energy probably led the formation of free radicals in the gas phase and caused thermal fragmentation reactions to occur. Therefore, we carried out pyrolytic processes with gallnut tannic acid at 380 ◦C for 5 s in order to control the radical fragmentations. All experiments were conducted in triplicate to study the reliability and stability, and the results provided enough evidence for good reproducibility of the method [10]. In general, Chinese gallnut contained 55~75% tannic acid, and 2~5% gallic acid and ellagic acid. GWE contained more than 90% of tannin [14]. Gallnut tannic acid is a β-glycoside bond polymer with one glucose core and five gallic acid esters. It is very easy for the LHS-1 strain or pyrolysis to break the ester bonds between the glucose core and gallic acid, like the benzoic acid and phenolic hydroxyl group. Those pyrolytic products with a benzene ring might be derived from splitting between the ester bonds in gallic acid and glucose, and the depside bonds among gallic acid, while liner pyrolytic products probably result because of the splitting among glucose. Compared to TAS, GWE gave rise to linear and aromatic pyrolytic products of tannic acid as major products. It was obvious that both the varieties and quantities of linear products were reduced. Pyrolysis products like the ethyl-, dimethyl ester of butanedioic acid, dimethyl 2-methyladipate, and 1,2,6-trimethoxyhexane disappeared. While the aromatic products became more complex, new pyrolytic products, such as 3-methoxybenzoic acid methyl ester and 4-methoxy benzoic acid methyl ester, appeared. Benzoic acid 3,4,5-trimethoxybenzoic acid methyl ester had a greater presence, of about 13.77%. All these results might be caused due to the effects of water extraction, which decreased the degrees of polymerization of tannic acid, resulting in an increase in free gallic acid and polyphenols so the structures of methylate with the benzene ring were more complex. Besides, the other water-soluble impurities mixed in the Chinese gall medicinal materials could influence the pyrolytic products. However, the pyrolysis products of FGWE were further simplified as compared with TAS and GWE. The total abundances of 3,4,5-trimethoxybenzoic acid methyl ester and 4-hydroxy-3,5-dimethoxy- benzoic acid hydrazide (pyrolysis and methylated products of gallic acid) were about 48.97%, which was more than 3.6 times greater than in GWE. The results might be because there was no tannic acid with a high polymerization degree in FGWE. The LHS-1 strain degraded most of tannic acid in GWE into gallic acid and small amounts of low molecular weight phenolic substances and glucose in FGWE. Then, glucose continued to be degraded as the carbon source and formed the linear pyrolysis products. Meanwhile, FGWE contained few other sources of mycelium except the metabolite of the LHS-1 strain. These results showed that the LHS-1 strain gave a high degradation rate of tannic acid, resulting in the formation of gallic acid. From the above, the biodegradation mechanism of gallnut tannic acid was different from that of the Chinese gallnut aqueous extract. Obviously, the differences in the pyrolytic products that existed in the three samples originated from the compositions of tannic acid and gallic acid. The benzene ring or aromatic compounds came from the pyrolysis of the epside bonds of gallic acid. The main pyrolytic products of FGWE were 3,4,5-trimethoxybenzoic acid methyl ester, 3-methoxy-1,2-benzenediol, and 4-hydroxy-3,5-dimethoxy- benzoic acid hydrazide.

4. Materials and Methods

4.1. Raw Materials and Chemicals Chinese gallnut powder (containing 56.8% total tannic acid) was provided as a raw material by the Nanjing Longyuan Natural Polyphenol Synthesis Factory (Nanjing, China) in China, and was kept at 4 ◦C before used. The other chemicals were purchased from commercial suppliers (Sigma-Aldrich, St. Louis, MO, USA; Merck, Darmstadt, Germany; laddin, Beijing, China). GWE, FGWE, and the LHS-1 strain were prepared by our lab. Molecules 2017, 22, 2253 9 of 11

4.2. Basal Culture Mediums Plate sieve culture medium (tannic acid medium): The basal medium contained sucrose 20 g, NaNO3 2.0 g, K2HPO4 1.0 g, MgSO4·7H2O 0.5 g, KCl 0.5 g, FeSO4 0.01 g, 1% brom ophenol blue, gallnut tannin 10 g, and agar 20 g in 1000 mL tap water. Liquid fermentation medium: The basal medium contained sucrose 20 g, NaNO3 2.0 g, K2HPO4 1.0 g, MgSO4·7H2O 0.5 g, KCl 0.5 g, FeSO4 0.01 g, and gallnut tannins 100 g in 1000 mL tap water. The above mediums were autoclaved at 121 ◦C for 20 min.

4.3. Microorganism and Culture Conditions The LHS-1 strain was isolated from GWE in our laboratory previously, and was identiﬁed as a new variant of A. niger through traditional morphological identiﬁcation and the phylogenetic tree of 18 s rDNA sequencing. It was then used for further study. The LHS-1 strain was kept for the culture of tannic acid agar slants stored at 4 ◦C and sub-cultured for regular intervals of four weeks. The cells were grown at 30 ◦C for 4 days shaking at 150 rpm.

4.4. The Preparation of GWE and FGWE Samples Took 5 g of Chinese gallnut powder to mix with 50 mL distilled water in a 150-mL extractor at 50 ◦C for 1 h by vacuum cavitation extraction, then filtered and centrifuged. The filtrate was freeze-dried at −50 ◦C for 72 h to obtain GWE, and stored at 4 ◦C before use. 250 mL of GWE solution was then sterilized at 121 ◦C for 20 min. After being cooled to room temperature, the inoculated strain LHS-1 was added to the sterilized GWE solution, and the mixture was shaken at 150 rpm at 30 ◦C for 4 days. After the GWE was fermented by the LHS-1 strain, the fermented broth was filtered in a vacuum, and the filtrate was freeze-dried at −50 ◦C until the LHS-1 strain fermentation sample of GWE (FGWE) was obtained. The sample was then stored at 4 ◦C.

4.5. Analysis of Tannic Acid, Gallic Acid, and Tannin Enzyme Activities Tannic acid was analyzed using Folin–Ciocalteau on the basis of the standard curve generated with tannic acid standard [9–11]. HPLC was used to assay tannic acid and gallic acid. The HPLC conditions were as follows: C18 (150 mm × Φ 4.6 mm × 5 µm) chromatographic column, PDA detector, methanol–water (0.5%) = 5:95 of mobile phase, 270-nm wavelength determination, 1 mL min−1 of flow speed. The degradation rate of tannic acid was calculated with the formula: R = 1 − TAt , where R is TA0 degradation rate of tannic acid, TA0 is the concentration of tannic acid in the TAA medium, and TAt is the concentration of tannic acid at the time of T(t). A small amount of nutrient solution was taken, and the mycelium filtered as a crude enzyme liquid. Three tubes were marked as the blank, test, and control tubes, respectively. Gallic acid accumulation and tannase activities were assayed by the spectrophotometric method of methanolic rhodanine [15]. One unit (U) of tannase was defined as one micromole of gallic acid formed per minute. Tannin enzyme activities were determined according to the content of TA and GA.

4.6. The Box–Behnken Experiment on GWE Biodegradation For determining the optimum parameters of GWE biodegradation with the LHS-1 strain, a Box–Behnken experiment was designed with three factors and three levels (Table4). The parameters were studied with ranges as follows: temperature of 25~35 ◦C, initial pH of 4~6, and incubation period of 36~60 h. Molecules 2017, 22, 2253 10 of 11

Table 4. Factors and levels in response surface analysis.

Levels Factors Code −1 0 1 ◦ A: temperature ( C) X1 25 30 35 B: initial pH X2 4 5 6 C: incubation period (h) X3 36 48 60

4.7. Conditions of the Py-GC/MS Experiments Py-GC/MS was performed using a double-shot pyrolyzer (Frontier Laboratories, model 2020i) attached to a GC/MS system Agilent 6890N (Santa Clara, CA, USA). TAS, GWE, and FGWE samples were analyzed by Py-GC/MS in accordance with procedures. Samples of 0.4 mg of TAS, GWE, and FGWE were placed in their respective sample cups. Sufficient methylated derivatives were added with a microsyringe (TMAH, 25% methanol), and then the samples were placed in small crucible capsules and introduced into the furnace, which was preheated at 500 ◦C for 1 min. GC-MS analysis with the Agilent 6890 N system was performed. The GC was equipped with a low-to-mid polarity-fused silica capillary column of HP-5 of 30 m × 250 µm × 25 µm film thickness. Column temperature: initial temperature 50 ◦C for 5 min, with a subsequent increase rate of 10 ◦C/min until 280 ◦C, maintained for 15 min. The vaporizing chamber temperature was 300 ◦C and the carrier gas was helium. There was a constant pressure mode (6.0 kPa), and a split ratio of 20:1. Pyrolysis temperature was 380 ◦C for 5 s. The detector consisted of an Agilent 5975 mass selective detector and the electrical energy acquired was 70 eV. The compounds were identified by comparing their mass spectra with reference compounds from the NISTO2 and Wiley libraries. Traces corresponding to selected homologous series of chemical families were obtained by single ion monitoring (SIM) of characteristic ions.

5. Conclusions TAS, GWE, and FGWE samples were analyzed using HPLC and Py-GC/MS. The optimization technology of the LHS-1 strain degrading GWE was determined by a response surface method. The optimum fermentation conditions of the LHS-1 strain were as follows: temperature of 31 ◦C, pH of 5, and a 50-h fermentation time. The tannase activity was 1.17 U·mL−1. Through comparing the mass spectra of analytes in NITS02 libraries and the retention times with the corresponding standard references, over 20 kinds of compounds were determined and annotated on the chromatogram. The pyrolytic products of all three samples were shown to be composed of linear and aromatic compounds. Among these, the total abundance of benzene ring derivatives of FGWE was about 48.97%. The metabolites of the LHS-1 strain contained almost no high polymerized tannic acid, giving a high degradation rate of tannic acid resulting in the formation of gallic acid and small amounts of phenolic compounds and glucose. Py-GC/MS is a convenient and efﬁcient method for tracing tannic acid and gallic acid in fermentation broth, and has signiﬁcant applicable value.

Acknowledgments: This work was supported by the work was supported by the National Key Research & Development Program (No. 2016YFD0600805) and National Natural Science Foundation of China (No. 31570564). Author Contributions: Chengzhang Wang and Wenjun Li conceived and designed the experiments; Wenjun Li performed the experiments; Wenjun Li analyzed the data; Chengzhang Wang contributed reagents/materials/analysis tools; Chengzhang Wang and Wenjun Li wrote the paper. Conﬂicts of Interest: The authors declare no conﬂict of interest. Molecules 2017, 22, 2253 11 of 11

References

1. Aguilar, C.N.; Rodríguez, R.; Gutiérrez-Sánchez, G.; Augur, C.; Favela-Torres, E.; Prado-Barragan, L.A.; Ramírez-Coronel, A.; Contreras-Esquivel, J.C. Microbial tannase: Advances and perspectives. Appl. Microbiol. Biotechnol. 2007, 76, 47–59. [CrossRef][PubMed] 2. Chávez-González, M.; Rodríguez-Durán, L.V.; Balagurusamy, N.; Prado-Barragán, A.; Rodríguez, R.; Contreras, J.C.; Aguilar, C.N. Biotechnological advances and challenges of tannase: An overview. Food Bioprocess Technol. 2012, 5, 445–459. [CrossRef] 3. Van Diepeningen, A.D.; Debets, A.J.M.; Varga, J. Efﬁcient degradation of tannic acid by black Aspergillus species. Mycol. Res. 2004, 108, 919–925. [CrossRef][PubMed] 4. Wang, W.W. Preparation of gallic acid by high conversion rate biological method. For. Chem. Commun. 1996, 1, 3–6. 5. Yang, X.K. Enzymatic Synthesis of Gallic Acid from Tannic Acid. Fine Spec. Chem. 2005, 5, 12–15. 6. Rintu, B.; Gargi, M.; Krushan, C.P. Microbial transformation of tannin-rich substrate to gallic acid through co-culture method. Bioresour. Technol. 2005, 96, 949–953. 7. Tieghem, P. Sur la Fermentation Gallique. Comptes Rendus Hebdomadaires des Seances de I’Academie des Sciences. 1867, Volume 65, pp. 1091–1094. Available online: http://gallica.bnf.fr/ark: /12148/bpt6k3021f.image.r=Comptes++Rendus+Hebdomadaires+des+S%C3%A9ances+de+l%E2%80% 99Acad%C3%A9mie+des+Sciences.f837.langFR (accessed on 16 December 2017). 8. Garnier, N.; Richardin, P.; Cheynier, V.; Regert, M. Characterization of thermally assisted hydrolysis and methylation products of polyphenols from modern and archaeological vine derivatives using gas chromatography–mass spectrometry. Anal. Chim. Acta 2003, 493, 137–157. [CrossRef] 9. Kuroda, K.; Nakagawa-izumi, A.; Mazumder, B.; Ohtani, Y.; Sameshima, K. Evaluation of chemical composition of the core and bast lignins of variety Chinpi-3 kenaf (Hibiscus cannabinus L.) by pyrolysis-gas chromatography/mass spectrometry and cupric oxide oxidation. Ind. Crops Prod. 2005, 22, 223–232. [CrossRef] 10. Wang, L.L.; Qian, J.; Hu, Z.C.; Zheng, Y.G.; Hu, W. Determination of dihydroxyacetone and glycerol in fermentation broth by pyrolytic methylation/gas chromatography. Anal. Chim. Acta 2006, 557, 262–266. 11. Cai, X.H.; Liu, Y.R.; Tian, S.B.; Zhou, J.; Liu, Z.L. Direct ﬁngerprinting polycyclic aromatic hydrocarbons in heavy oils by PY-GC/MS and its application to hydrotreating processes. China Pet. Process. Petrochem. Technol. 2012, 14, 8–14. 12. Min, F.Q. Screening of Tannic Acid Degradation Strains and Metabolites Research. Master’s Thesis, Chinese Academy Forestry, Beijing, China, 2014. 13. Zhang, L.L.; Xu, M.; Wang, Y.M.; Wu, D.M.; Chen, J.H. Optimization of Ultrasonic Extraction Conditions of Ellagic Acid from Infructescence of Platycarya strobilacea Using Response Surface Method. Chem. Ind. For. Prod. 2011, 31, 19–24. 14. Liu, S.P.; Li, Y. Study on the Extraction of Tannic acid from Chinese Gallnut. He Bei Chem. Eng. Ind. 2007, 20, 44–45. 15. Bao, Y.X.; Qiu, S.Y.; Li, Y.Z.; Huang, Y.G. A Method for Detecting Activity of Extracellular Tannase. Fine Chem. 2008, 25, 621–624.

Sample Availability: Samples of the compounds are available from the authors.

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