Influence of Geosmin-Producing Streptomyces on the Growth and

Dec 9, 2014 - ABSTRACT: Diverse Streptomyces species act as geosmin producers in the Chinese liquor-making process, causing an earthy, off-odor ...
0 downloads 0 Views 1MB Size
Article pubs.acs.org/JAFC

Influence of Geosmin-Producing Streptomyces on the Growth and Volatile Metabolites of Yeasts during Chinese Liquor Fermentation Hai Du, Hu Lu, and Yan Xu* Synergetic Innovation Center of Food Safety and Nutrition, State Key Laboratory of Food Science and Technology, Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu 214122, People’s Republic of China ABSTRACT: Diverse Streptomyces species act as geosmin producers in the Chinese liquor-making process, causing an earthy, off-odor containment. Through microbiological and metabolite analyses, this paper investigates the influence of several geosminproducing Streptomyces on the microbial community of a brewing system. The antifungal activity against functional liquorbrewing microbes was assayed by an agar diffusion method. Several Streptomyces, most notably Streptomyces sampsonii QC-2, inhibited the growth of the brewing functional yeasts and molds in pure culture. In a simulated coculture, Streptomyces spp. reduced the flavor compounds (alcohols and esters) contributed by yeasts. Nine components in Streptomyces sampsonii QC-2 broth were detected by ultraperformance liquid chromatography coupled with photo diode array (UPLC−PDA), with characteristic ultraviolet absorptions at 360, 380, and 400 nm. The main products of Streptomyces sampsonii QC-2 were identified by ultraperformance liquid chromatography−quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF−MS/MS), and confirmed by standard mass spectrometry. The antifungal active components were revealed as a series of heptaene macrolide antibiotics. KEYWORDS: Streptomyces, geosmin, antifungal activity, heptaene macrolide antibiotics, Chinese liquor



yomyces hansenii),5 which may potentially improve flavor diversity and reduce the alcohol content.9,10 When the fermented grains are removed from the opened fermenter, they become contaminated by a variety of microorganisms derived from the surrounding environment, many of which contribute to subsequent fermentation. In our previous studies, we found that if Streptomyces-sourced geosmin is introduced to the fermentation process, it becomes concentrated in the liquor after distillation.2,11,12 Geosmin presence usually indicates that off-flavor producers have become well-established in the Chinese liquor-making process. Geosmin-producing Streptomyces have been identified in certain high- and low-temperature Daqu.2 Streptomyces spp. are ubiquitous soil bacteria noted for their ability to produce a vast array of antibiotics.13 Indeed, 90% of Streptomyces produce antibiotics that inhibit the growth of other microbes.13−15 Antibiotic-mediated species interactions are believed to enhance Streptomyces fitness and have been exploited as a plant disease biocontrol in soil.16 The present study investigates the extent to which introducing geosmin-producing Streptomyces affects the microbial community and flavor development in the fermentation processes of Chinese liquor. Adopting an analytical chemistry approach, it also proposes a mechanism by which geosminproducing Streptomyces influences the brewing-functional microbes in the Chinese liquor-making process.

INTRODUCTION

Chinese liquor is fermented from grains (rice, sticky rice, sorghum, wheat, and corn) in multistrain solid-state culture. Liquor fermentation spontaneously occurs in pottery vats or mud pits over several months. The microbial counts and metabolite concentrations in heterogeneous natural liquor fermentations are widely variable, affecting the flavor and composition of Chinese liquor.1 Successful Chinese liquor fermentation requires a succession of particular microbial activities. The main microbial source in Chinese liquor making is the fermentation starter Daqu, which provides nutrients for microbes that produce an array of enzymes with various biochemical properties. Therefore, the fermentation activities of yeasts, bacteria and filamentous fungi in Daqu largely determine the typical characteristics of the resulting fermented brans and fresh liquor.2−5 The Daqu fungi, including molds and yeasts, play an important role in the saccharification and alcoholic fermentation of Chinese liquor. Molds appear to influence the evolution of flavors and the activities of enzymes such as amylases and proteinases,5,6 which are required for alcoholic fermentation of cooked sorghum. The predominant Daqu yeasts have been reported as Saccharomyces cerevisiae, Pichia anomala, and Saccharomycopsis f ibuligera, which constitute the functional microbes in other fermented alcoholic beverages. S. cerevisiae and P. anomala mainly contribute flower-like and fruit-like volatile compounds, such as alcohols and esters, to the liquor flavor.1,7 S. f ibuligera secretes high-activity α-amylase and glucoamylase that can degrade raw starch.8 Culture-independent methods have also identified non-Saccharomyces species (Issatchenkia orientalis, Hanseniaspora guilliermondii, and Debar© 2014 American Chemical Society

Received: Revised: Accepted: Published: 290

July 15, 2014 November 27, 2014 December 9, 2014 December 9, 2014 DOI: 10.1021/jf503351w J. Agric. Food Chem. 2015, 63, 290−296

Article

Journal of Agricultural and Food Chemistry



antifungal activity were subjected to UPLC−PDA and ultraperformance liquid chromatography−quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF−MS/MS) for compound identification. Identifying Antifungal Active Substances. The active antifungal substances were identified by UPLC/Q-TOF−MS/MS. UPLC was performed in a Waters Acquity UPLC system (Milford, MA, U.S.A.) equipped with a binary solvent delivery system and an auto sampler. Chromatography was performed on a Waters Acquity C18 column (5 cm × 2.1 mm, 1.7-μm particles). The mobile phase was a gradient prepared from 0.1% formic acid (component A) and 100% acetonitrile (component B). Elution started with 5% B for 0.5 min; the proportion of A was then linearly increased to 100% over 10 min, reduced to 95% in the next 1 min and maintained isocratic for 2 min. Total run time, including conditioning of the column to the initial conditions, was 12 min. The flow rate of the eluting solvent was 0.3 mL/min. During the analyses, the column was maintained at 30 °C. The injection volume was 5 μL. Mass spectrometry was performed with a Waters Synapt Q-TOF system in positive and negative ion modes according to the manufacturer’s instructions. The masses and compositions of the precursor and fragment ions were accurately calculated by the MassLynx 4.1 software supplied with the instrument, which calculates all possible elemental compositions consistent with the accurate mass. The relative concentrations of antifungal compounds to the external standard (amphotericin B 200 μg/L) were semiquantified on the basis of comparing their peak areas to that of the external standard on UPLC-PDA chromatograms at 380 nm. Analysis of Volatile Metabolites. Analytes were extracted by solid-phase microextraction (SPME), using an automatic headspace sampling system (Multi Purpose Sample MPS 2 with an SPME adapter (GERSTEL Inc., Baltimore, MD, U.S.A.) equipped with a 50/ 30 μm DVB/CAR/PDMS fiber (2 cm, Supelco Inc., Bellefonte, PA, U.S.A.). Prior to analysis, the fiber was conditioned by insertion into the GC injector at 250 °C for 2 h to prevent contamination. A solidstate sample (3 g) was transferred to a screw-capped, straight-sided headspace vial (volume = 15 mL) and spiked with 10 μL of L(−)-menthol (internal standard, IS), 4 mg/L in ethanol. After saturating the diluted solution with NaCl, the vial was tightly capped with a Teflon-faced silicone septum. The samples were equilibrated at 60 °C for 5 min and extracted for 45 min at the same temperature with stirring (250 rpm). Following extraction, the fiber was inserted into the injection port of the GC (250 °C) for 5 min to desorb the analytes. The GC−MS analysis was performed in an Agilent 6890N GC coupled to an Agilent 5975 mass selective detector (MSD). The capillary column was a CP-Wax column (length = 60 m, i.d. = 0.25 mm, film thickness = 0.25 μm; Varian Inc., Palo Alto, CA, U.S.A.). The injector temperature was 250 °C, and the analysis was conducted in splitless mode. The operating conditions were as follows: temperature was started at 80 °C (holding for 2 min), then raised to 230 °C at 8 °C/min, and held at 230 °C for 10 min. The column carrier gas was helium (99.9995% purity) supplied at a constant flow rate of 2 mL/ min. The electron impact energy was 70 eV, and the ion source temperature was set to 230 °C. The compounds were characterized by full-scan acquisition over an appropriate mass range (30−350 amu). The compounds were clearly identified by comparison to reference spectra (NIST05a.L, Agilent Technologies, Inc.) and pure standards. The compounds were quantified by their main mass fragments. A calibration curve was derived by plotting the peak area ratios against the concentration ratios of main mass fragments to IS (m/z 81) as described in our previously published paper.12 Each compound concentration in the sample was quantified by comparing the ratios of the peak areas with the calibration curve. Each determination was triplicated. Statistical Analysis. To determine the effect of geosminproducing strains on the flavors produced by the functional liquorbrewing fungus, the volatile metabolites were quantified. The obtained results were visualized on a heat map computed by R (version 3.0.2), in which cells were colored according to their Z scores (where Z = [observed value − mean]/standard deviation).

MATERIALS AND METHODS

Strains and Assays of Antifungal Activity. All strains in this study were isolated from the making process of Daqu-based Chinese light aromatic liquor. The isolated strains were identified by molecular taxonology based on their conservative rDNA sequences. Four geosmin-producing Streptomyces strains (Streptomyces albus FXJ, Streptomyces f radiae HX, Streptomyces radiopugnansQC-1, and Streptomyces sampsonii QC-2) were isolated from Daqu, and geosmin production was confirmed by gas chromatography−mass spectrometry (GC−MS). Antifungal activity was determined by the modified agar diffusion assay described in Coda et al.17 Petri plates (90 mm diameter) containing 10 mL of potato dextrose agar (PDA) medium were inoculated with geosmin-producing strains. The plates were incubated at 30 °C for 72 h. Once the colony had developed, it was excised from the plate as an agar cylinder (9 mm diameter) and placed on a plate spread with functional liquor-brewing microbes at an appropriate concentration of cell suspension. The spread plates were then incubated at 30 °C for 72 h. All experiments were run in triplicate. The inhibitory activity of geosmin-producing strains was quantified as the diameter ratio of inhibition zone to agar cylinder (Rinhibition zone/Rcolony). The inhibitory effect of QC-2 broth sterilized by filtration was also assayed by modified agar diffusion assay. Strain QC-2 was inoculated in potato dextrose medium at 30 °C with shaking (250 rpm). At different incubation times, the QC-2 broths were filtered through a 0.22 μm membrane filter (Millipore Corporation, Bedford, MA). A plate inoculated with the test fungus (S. f ibuligera FJ-4) at an optimal cell concentration was then dotted with 5 μL of the sterilized broth. All assays were performed in triplicate. The plate was incubated at 30 °C for 72 h, and the diameter (in mm) of the inhibition zone was measured. Co-cultivation and Fermentation Experiments. The fermentation system of Chinese liquor was simulated as a cocultured system of the main brewing microorganisms. The solid-state fermentation medium was prepared as follows: 250 g sorghum was added to 350 mL hot water (70−80 °C), soaked for 24 h, and sterilized at 121 °C for 20 min. Rhizopus oryzae G4 (R. oryzae G4) was introduced to the simulated fermentation system at 104 CFU/g spore suspension, and incubated at 30 °C for 1 day to allow saccharification. The saccharified cultures were then inoculated with 105 CFU/g each of four yeast species (S. cerevisiae FJ-7, P. anomala FJ-3, I. orientalis FJ-2, and S. f ibuligera FJ-4). Following 2-days of incubation at 30 °C, the spore or cell concentrations of the yeasts were adjusted to match the results of plate counts on PDA medium. Streptomyces sampsonii QC-2 (spore suspension 104 CFU/g) was inoculated into potato dextrose broth, and shaken at 250 rpm. The broths at different growth stages (lag phase (I), exponential phase (II), and stationary phase (III)) were sterilized by filtering through a 0.22 μm nylon membrane. Fivemilliliters of the filtered QC-2 broths were added to the yeast cocultures. Medium without broth addition was prepared as a control reference sample. Analysis of Yeast Growth. Yeast growth was measured by spreadplating onto Wallerstein Laboratory Nutrient (WLN) medium. Yeast cells from the simulation fermentation system were collected, 10-fold serially diluted in saline (0.85%, wt/vol, NaCl), and spread on triplicate WLN medium plates. Following incubation for 3 days at 30 °C, the yeast colony-forming units (CFUs) were identified by their morphological characteristics and counted. Purifying Antifungal Active Compounds. The culture broth of strain QC-2 was prepared under the conditions of the cocultivation and fermentation experiments. The broth was incubated for 5 days, then centrifuged at 10 000 rpm for 15 min at 4 °C. The supernatant (approximately 40 mL) was purified and concentrated by a solid phase extraction (SPE) column (Welchrom C18E, Welch materials, Inc., MD, U.S.A.), and then successively eluted by 10 mL water, 50% acetonitrile/water, and 100% acetonitrile. The three eluents were separately collected and concentrated to 2 mL under a jet of nitrogen gas. After filtration through 0.22 μm membrane filters, 5 μL of each eluted fraction was retained for agar diffusion assay. Fractions showing 291

DOI: 10.1021/jf503351w J. Agric. Food Chem. 2015, 63, 290−296

Article

Journal of Agricultural and Food Chemistry

Table 1. Inhibition of Four Geosmin-Producing Strains on the Brewing Functional Microbes in a Chinese Liquor-Making Process inhibition resultsa (Rinhibition zone/Rcolony)b

tested microorganisms origin

strain

species

FXJ

Qingcha Daqu from Fenjiu Gongan Daqu Qingcha Daqu from Fenjiu Hongxin Daqu from Fenjiu Qingcha Daqu from Fenjiu Qingcha Daqu from Fenjiu Gongan Daqu Qingcha Daqu from Fenjiu Gongan Daqu Qingcha Daqu from Fenjiu Qingcha Daqu from Fenjiu Qingcha Daqu from Fenjiu Yangchun Daqu Qingcha Daqu from Fenjiu Qingcha Daqu from Laobaigan

FJ-4 Y4 GS-1 GS-2 FJ-7 FJ-2 Y8m FJ-3 Y6 GS-4 GS-5 GS-6 G4 GS-3 GS-7

Saccharomycopsis f ibuligera Saccharomycopsis f ibuligera Hanseniaspora sp. Saccharomyces cerevisiae Saccharomyces cerevisiae Issatchenkia orientalis Issatchenkia orientalis Pichia anomala Pichia anomala Bacillus amyloliquefaciens Absidia corymbifera Mucor circinelloides Rhizopus oryzae Penicillium chrysogenum Trichoderma viride

c              1.63 ± 0.09

HX 1.22 1.10          1.63   2.50

± 0.16 ± 0.03

± 0.33

± 0.27

QC-1 1.33 1.11 1.88 1.13 1.10 1.11 1.01 1.03 1.02 1.38   1.38  2.63

± ± ± ± ± ± ± ± ± ±

0.07 0.11 0.12 0.02 0.06 0.18 0.19 0.06 0.22 0.11

± 0.27 ± 0.15

QC-2 1.39 1.56 1.50 1.25 1.44 1.56 1.39 1.01 1.01 1.13 1.13 1.02  1.56 2.25

± ± ± ± ± ± ± ± ± ± ± ±

0.07 0.21 0.19 0.08 0.13 0.17 0.11 0.27 0.14 0.15 0.11 0.07

± 0.26 ± 0.12

a

Inhibition results are expressed as the mean diameter ratio of inhibition zone to agar cylinder in triplicate experiments. bR denotes the diameter of an inhibition zone or colony. c indicates no inhibition zone.



RESULTS AND DISCUSSION Influence of Geosmin-Producing Streptomyces On The Growth Of Microbes And Dynamics Of Yeast Populations Involved In Chinese Liquor Fermentation. Four Streptomyces strains (Streptomyces albus FXJ, Streptomyces f radiae HX, Streptomyces radiopugnans QC-1, and Streptomyces sampsonii QC-2) were identified as geosmin producers in our previous studies.11 Communities of geosmin-producing Streptomyces have been studied by denaturing gradient gel electrophoresis (DGGE) and quantitative PCR.2 In the present study, the antibacterial activity of geosmin-producing Streptomyces was determined by the agar diffusion method. Singlecolony agar cylinders of the four geosmin-producing strains were placed on agar previously spread with functional brewing strains isolated from the liquor-making process, which provide flavor and enzymes. The inhibitions of geosmin-producing Streptomyces on the main functional strains were determined from the inhibition zones. As shown in Table 1, strain FXJ exerted a very weak antifungal activity, whereas strain HX inhibited only a portion of the fungus. Strain QC-1 inhibited a fraction of the bacteria, yeasts and molds. The strongest effect was exhibited by strain QC-2, which inhibited not only yeasts, molds and other fungi, but also (to some extent) bacteria under the same conditions. Importantly, the transparent inhibition zones around S. f ibuligera FJ-4 and Y4 show that QC-2 strongly inhibits both of these strains. S. f ibuligera, also known as Endomycopsis f ibuligera, expresses liquefied raw starch enzyme and saccharifying enzyme. S. f ibuligera ranks among the best yeast producers of starch decomposition enzymes.21,22 The sterilized potato dextrose broths, in which S. sampsonii QC-2 had been incubated up to different stages (QC-2 I (lag phase); QC-2 II (exponential phase), and QC-2 III (stationary phase)), were introduced to mixed cultures of the four yeasts, S. cerevisiae FJ-7, P. anomala FJ-3, I. orientalis FJ-2, and S. f ibuligera FJ-4. According to Figure 1, the populations of the four yeasts were reduced by different degrees, reflecting their different inhibitions by strain QC-2. The QC-2 III results conspicuously differed from those of the controlled experiment, implying that Streptomyces can unbalance the microbial

Figure 1. Populations of four brewing functional yeasts in a mixedculture fermentation (s.c, Saccharomyces cerevisiae FJ-7; p.a, Pichia anomala FJ-3; i.o, Issatchenkia orientalis FJ-2; and sm.f, Saccharomycopsis f ibuligera FJ-4) exposed to Streptomyces sampsonii QC-2 at different incubation stages (I, lag phase; II, exponential phase; and III, stationary phase). Numbers show the percentage of the yeast populations.

community of the brewing system. In contrast, the QC-2 I results varied only slightly from those of the control. Kinetics of Biomass, Geosmin and Antifungal Activity during Strain QC-2 Fermentation. During fermentation in potato dextrose medium, strain QC-2 reached a maximum dry cell weight of 3.75 g/L after 5 days (stage II). The dry cell weight slightly decreased at later times, stabilizing at 2.65−3.68 g/L after prolonged fermentation (7−8 days; stage III). The concentration of the geosmin produced by strain QC-2 followed similar trends to the biomass (Figure 2). The activities of the sterile QC-2 broths were assayed against S. f ibuligera FJ-4 from Daqu. Sterile broths of strain QC-2 incubated at different stages were pipetted onto PDA spread with S. f ibuligera FJ-4, and the inhibition zone was measured after 3 days at 30 °C. As shown in Figure 3, the QC-2 broth of cells incubated for 2 days exerted no obvious antifungal effect. After 3−9 days, the inhibition zones became obvious and enlarged at later fermentation times. 292

DOI: 10.1021/jf503351w J. Agric. Food Chem. 2015, 63, 290−296

Article

Journal of Agricultural and Food Chemistry

Figure 2. Biomasses, geosmin contents and antifungal activities of broths exposed to QC-2 at different fermentation stages. The test fungus is Saccharomycopsis fibuligera FJ-4.

Figure 3. Antifungal activities of filter-sterilized broths of Streptomyces sampsoniiQC-2 fermented for different periods. Shown are the inhibition zones against Saccharomycopsis f ibuligera FJ-4 after 3 days of incubation at 30 °C. A, 2 days; B, 3 days; C, 4 days; D, 5 days; E, 6 days; F, 7 days; G, 8 days; and H, 9 days.

Influence of Strain QC-2 on the Volatile Metabolites in Mixed-Culture System. The mixed-culture system contained four brewing functional yeasts (S. cerevisiae FJ-7, P. anomala FJ-3, I. orientalis FJ-2, and S. fibuligera FJ-4) isolated from a Chinese liquor-making environment. The sterilized broths of geosmin-producing strain QC-2 at different incubation stages were introduced to the system. The volatile compounds in these experimental batches were quantified by HS−SPME−GC−MS, and a heat map of their Z scores was prepared. As shown in Figure 4, the main products of the mixed-yeast culture were esters, alcohols and acids. The geosmin concentration increased as the QC-2 incubation period lengthened before spiking into the mixed-culture system. The flavor of the mixed-culture brewing system was most obviously decreased by the QC-2 broth of cells incubated for 5 days (stage III). This decline was chiefly due to degradation of Group I and II alcohols and esters, such as 3-octanol, 3-methyl butanol, ethyl octanoate, and ethyl decanote. Meanwhile, the geosmin produced by Streptomyces has been recognized as an off-flavor volatile.23,24 The results imply that geosminproducing Streptomyces secretes antifungal compounds against the functional yeasts.

Figure 4. Volatile metabolites of four brewing functional yeasts in a mixed-culture fermentation (s.c, Saccharomyces cerevisiae FJ-7; p.a, Pichia anomala FJ-3; i.o, Issatchenkia orientalis FJ-2; sm.f, Saccharomycopsis f ibuligera FJ-4) exposed to Streptomyces sampsonii QC-2 at different incubation stages (I, lag phase; II, exponential phase; and III, stationary phase).

Identifying the Antifungal Active Substances Produced by Strain QC-2. The QC-2 broth was purified and concentrated in a solid phase extraction (SPE) column. Three fractions were collected from three different eluents (see Materials and Methods). The ultraviolet absorption spectrum of the 50% acetonitrile/water fraction exhibited three characteristic peaks at 360, 380, and 400 nm. These peaks, detected by a photodiode array (PDA) detector at 6.5−7.6 min retention time (Figure 5B), characterize the chromophores of conjugate heptaene macrolides.18,19 Nine peaks appeared in the spectrum of potato dextrose broth incubated with strain QC-2. However, 293

DOI: 10.1021/jf503351w J. Agric. Food Chem. 2015, 63, 290−296

Article

Journal of Agricultural and Food Chemistry

16−25. The most likely formula was identified by analyzing the chemical element composition, based on the [M − H]− peak in negative ion mode. This analysis was performed by MassLynx application management software. The molecular formulas of the nine target compounds are listed in Table 2. To confirm the structural formulas of these separate products, we directly imported the parent fragment ions from the raw data file, along with their structural formulas, into the Massfragment software, thus providing the fragment “soft spots”. Finally, the proposed structural formulas were elucidated by Massfragment to assess their match to the corresponding fragment information. The formulas input to Massfragment were obtained from the accurate masses, which deviated from the theoretical values by 1.5 ppm at most. When the candicidin mixture standard was spiked into the broth of strain QC-2, the peaks of components B, C, E, and I were enhanced (see Figure 6A). The retention time of component C in QC-2 broth was similar to that of candicidin D, a heptaene macrolide of known chemical structure.19 The contents of different components produced during fermentation were also quantified. Antifungal compound production in the QC-2 broth was maximized aat the seventh day of fermentation (Figure 6B), and was dominated by component C (whose concentration reached 504.83 μg/L). The changing trends of the antibiotics in the fermenting broth were consistent with those of the antifungal effect (inhibition zone diameter; see Figure 3). These results further confirm the active antifungal materials as heptaene macrolide antibiotics. Jørgensen and colleagues reported that most of their Streptomyces isolates produced candicidin, while all of them contained the can gene cluster responsible for candicidin biosynthesis.19 The plasmid containing the genes can be conjugatively transferred to other streptomycetes.19 Linear plasmids have also been identified as temperature-sensitive and easily lost,25 and are commonly transferred to other species.26 Although the can plasmid is not transferable to other Streptomyces strains in the laboratory, it may be responsible for spreading the candicidin biosynthesis gene cluster to other strains in natural environments such as Chinese liquor-making plants. In conclusion, there were significant influences of diverse Streptomyces spp. acting as geosmin producers on the brewing functional microbials in a Chinese liquor making system. Some geosmin-producing Streptomyces could secrete antifungal compounds, heptaene macrolide antibiotics, against the normal fungi microbiota. In view of our results, the presence of Streptomyces may cause the imbalance of the microbial community of a brewing system. Accordingly, the contribution

Figure 5. Photodiode array UV spectra of (A) standard sample amphotericin B and (B) peak A-I extracted from Streptomyces sampsonii QC-2 fermentation broth, precipitated on a C18 UPLC column (5 cm × 2.1 mm, 1.7-μm particles).

comparison with the retention time of amphotericin B in the PDA chromatogram reveals no amphotericin B production by strain QC-2 (Figure 5A). The main components produced by strain QC-2 were identified and confirmed by UPLC−Q-TOF−MS/MS, standard spiking and mass spectrometry. The masses of these components were accurately determined by TOF−MS using negative electron spray ionization,20 and the molecular ions are listed in Table 2. The approximate molecular weights of the main components in QC-2 broth were 1110.4, 1108.4, 1092.4, and 1176.4. This method also determines the abundances of the ion fragments. The molecular structures, derived from the mass spectrogram and compared with the PDA chromatogram, confirm the compounds as heptaene macrolide antibiotics. The number of double-bond equivalents (DBE) reported by the elemental composition tool were also consistent with the heptaene basic skeleton structure of the macrolide antibiotic class: C: 45, N: 1, O: 16, and DBE: 12. Error 0.1 Da. The ranges of possible elements and atomic numbers were as follows: C: 45−100, H: 0−100, N: 1−3, O: 16−20, and DBE:

Table 2. Antibiotic Contents in QC-2 Broth and Their Molecular Formulas, Hypothesized from Mass Spectrometry Information [M − H]−

a

M

component

retention time (min)

content (mg/L)

ion weight

formula

DBEa

formula

DBEa

A B C D E F G H I

6.60 6.72 6.90 7.03 7.18 7.25 7.41 7.51 7.61

0.06 0.12 1.43 3.77 1.60 0.56 0.54 0.15 0.08

1109.4296 1107.4186 1109.4172 1107.4064 1091.4081 1107.4064 1091.4203 1075.4175 1107.4064

C57H77N2O20 C57H75N2O20 C57H77N2O20 C57H75N2O20 C57H75N2O19 C57H75N2O20 C57H75N2O19 C57H75N2O18 C57H75N2O20

20.5 21.5 20.5 21.5 20.5 21.5 21.5 21.5 21.5

C57H78N2O20 C57H76N2O20 C57H78N2O20 C57H76N2O20 C57H76N2O19 C57H76N2O20 C57H76N2O19 C57H76N2O18 C57H76N2O20

20 21 20 21 20 21 21 21 21

DBE = double-bond equivalents. 294

DOI: 10.1021/jf503351w J. Agric. Food Chem. 2015, 63, 290−296

Article

Journal of Agricultural and Food Chemistry

Figure 6. (A) Comparison of UV chromatogram (at 380 nm) of QC-2-produced antifungal active substances and that spiked with candicidin standard, precipitated on a C18 UPLC column (10 cm × 2.1 mm, 1.7-μm particles) and (B) concentrations of antibiotic component A-I produced by Streptomyces sampsonii QC-2 at different fermentation stages.



of yeasts to liquor flavor were decreased, such as alcohols and esters. Therefore, liquor makers should keep a close eye on the geosmin-producing Streptomyces.



AUTHOR INFORMATION

Corresponding Author

*Tel: +86 510 85918197; fax: +86 510 85918201; e-mail: yxu@ jiangnan.edu.cn. Notes

The authors declare no competing financial interest.



REFERENCES

(1) Zhang, W. X.; Wu, Z. Y.; Zhang, Q. S.; Wang, R.; Li, H. Combination of newly developed high quality Fuqu with traditional Daqu for Luzhou-flavor liquor brewing. World J. Microb. Biot. 2009, 25, 1721−1726. (2) Du, H.; Lu, H.; Xu, Y.; Du, X. Community of environmental Streptomyces related to geosmin development in Chinese liquors. J. Agric. Food Chem. 2013, 61, 1343−1348. (3) Zheng, X. W.; Tabrizi, M. R.; Nout, M. J. R.; Han, B. Z. DaquA traditional Chinese liquor fermentation starter. J. I. Brewing 2011, 117, 82−90. (4) Wu, Z. Y.; Zhang, W. X.; Zhang, Q. S.; Hu, C.; Wang, R.; Liu, Z. H. Developing new sacchariferous starters for liquor production based on functional strains isolated from the pits of several famous Luzhouflavor liquor brewers. J. I. Brewing 2009, 115, 111−115. (5) Wang, H. Y.; Gao, Y. B.; Fan, Q. W.; Xu, Y. Characterization and comparison of microbial community of different typical Chinese liquor Daqus by PCR-DGGE. Lett. Appl. Microbiol. 2011, 53, 134−140. (6) Zheng, X.-W.; Yan, Z.; Han, B.-Z.; Zwietering, M. H.; Samson, R. A.; Boekhout, T.; Nout, M. J. R. Complex microbiota of a Chinese ″Fen″ liquor fermentation starter (Fen-Daqu), revealed by culturedependent and culture-independent methods. Food Microbiol. 2012, 31, 293−300. (7) Li, X.-R.; Ma, E.-B.; Yan, L.-Z.; Meng, H.; Du, X.-W.; Zhang, S.W.; Quan, Z.-X. Bacterial and fungal diversity in the traditional Chinese liquor fermentation process. Int. J. Food Microbiol. 2011, 146, 31−37.

ACKNOWLEDGMENTS

We are grateful to the National High Technology Research and Development Program of China (2013AA102108, 2012AA021301), the National Natural Science Foundation of China (31371822, 31271921), the Program of Introducing Talents of Discipline to Universities (111 Project) (111-2-06), the Cooperation Project of Jiangsu Province among Industries, Universities, and Institutes (BY2010116) and the Fundamental Research Funds for the Central Universities (JUDCF10016). Special thanks also go to the “169” plan of Chinese liquor for its financial support and supply of Daqu samples. 295

DOI: 10.1021/jf503351w J. Agric. Food Chem. 2015, 63, 290−296

Article

Journal of Agricultural and Food Chemistry (8) Van Rensburg, P.; Van Zyl, W. H.; Pretorius, I. S. Engineering yeast for efficient cellulose degradation. Yeast 1998, 14, 67−76. (9) Medina, K.; Boido, E.; Farina, L.; Gioia, O.; Gomez, M. E.; Barquet, M.; Gaggero, C.; Dellacassa, E.; Carrau, F. Increased flavour diversity of Chardonnay wines by spontaneous fermentation and cofermentation with Hanseniaspora vineae. Food Chem. 2013, 141, 2513− 2521. (10) Contreras, A.; Hidalgo, C.; Henschke, P. A.; Chambers, P. J.; Curtin, C.; Varela, C. Evaluation of non-Saccharomyces yeasts for the reduction of alcohol content in wine. Appl. Environ. Microb. 2014, 80, 1670−1678. (11) Du, H.; Xu, Y. Determination of the microbial origin of geosmin in Chinese liquor. J. Agric. Food Chem. 2012, 60, 2288−2292. (12) Du, H.; Fan, W.; Xu, Y. Characterization of geosmin as source of earthy odor in different aroma type Chinese liquors. J. Agric. Food Chem. 2011, 59, 8331−8337. (13) Schrempf, H. Streptomycetaceae: Life Style, Genome, Metabolism and Habitats; John Wiley & Sons, Ltd: New York, 2001. (14) Li, Q. L.; Ning, P.; Zheng, L.; Huang, J. B.; Li, G. Q.; Hsiang, T. Fumigant activity of volatiles of Streptomyces globisporus JK-1 against Penicillium italicum on Citrus microcarpa. Postharvest. Biol. Tec. 2010, 58, 157−165. (15) Jain, P. K.; Jain, P. C. Isolation, characterization and antifungal activity of Streptomyces sampsonii GS 1322. Indian J. Exp. Biol. 2007, 45, 203−206. (16) McNeal, K. S.; Herbert, B. E. Volatile organic metabolites as indicators of soil microbial activity and community composition shifts. Soil Sci. Soc. Am. J. 2009, 73, 579−588. (17) Coda, R.; Cassone, A.; Rizzello, C. G.; Nionelli, L.; Cardinali, G.; Gobbetti, M. Antifungal activity of Wickerhamomyces anomalus and Lactobacillus plantarum during sourdough fermentation: Identification of novel compounds and long-term effect during storage of wheat bread. Appl. Environ. Microb. 2011, 77, 3484−3492. (18) Parekh, A. C.; Dave, C. V. HamycinSeparation of toxicity and antifungal activity in mice. Life Sci. 1976, 19, 1737−1742. (19) Jørgensen, H.; Fjaervik, E.; Hakvag, S.; Bruheim, P.; Bredholt, H.; Klinkenberg, G.; Ellingsen, T. E.; Zotchev, S. B. Candicidin biosynthesis gene cluster is widely distributed among Streptomyces spp. isolated from the sediments and the neuston layer of the Trondheim fjord, Norway. Appl. Environ. Microb. 2009, 75, 3296−303. (20) Cao, W.; Zhang, K.; Tao, G.; Wang, X.; Liu, Y. Identification of the fatty acyl residues composition and molecular species of phosphatidylcholines in soy lecithin powder by UPLC−ESI-MS/MS. Chromatographia 2012, 75, 1271−1278. (21) Lv, X.-C.; Huang, X.-L.; Zhang, W.; Rao, P.-F.; Ni, L. Yeast diversity of traditional alcohol fermentation starters for Hong Qu glutinous rice wine brewing, revealed by culture-dependent and culture-independent methods. Food Control 2013, 34, 183−190. (22) Ismaya, W. T.; Hasan, K.; Kardi, I.; Zainuri, A.; Rahmawaty, R. I.; Permanahadi, S.; El Viera, B. V.; Harinanto, G.; Gaffar, S.; Natalia, D.; Subroto, T.; Soemitro, S. Chemical modification of Saccharomycopsis f ibuligera R64 α-Amylase to improve its stability against thermal, chelator, and proteolytic inactivation. Appl. Biochem. Biotechnol. 2013, 170, 44−57. (23) Jachymova, J.; Votruba, J.; Viden, I.; Rezanka, T. Identification of Streptomyces odor spectrum. Folia Microbiol. 2002, 47, 37−41. (24) Scholler, C. E. G.; Gurtler, H.; Pedersen, R.; Molin, S.; Wilkins, K. Volatile metabolites from actinomycetes. J. Agric. Food Chem. 2002, 50, 2615−2621. (25) Pang, X.; Sun, Y.; Liu, J.; Zhou, X.; Deng, Z. A linear plasmid temperature-sensitive for replication in Streptomyces hygroscopicus 10− 22. FEMS Microbiol. Lett. 2002, 208, 25−28. (26) Ravel, J.; Wellington, E. M. H.; Hill, R. T. Interspecific transfer of Streptomycesgiant linear plasmids in sterile amended soil microcosms. Appl. Environ. Microb. 2000, 66, 529−534.

296

DOI: 10.1021/jf503351w J. Agric. Food Chem. 2015, 63, 290−296