- Email: [email protected]
Multistarter fermentation of glutinous rice with Fu brick tea: Effects on microbial, chemical, and volatile compositions
Food Chemistry xxx (xxxx) xxxx
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Multistarter fermentation of glutinous rice with Fu brick tea: Effects on microbial, chemical, and volatile compositions Xiao Xua,b,c, , Siduo Zhoub, David Julian McClementsc, Lu Huangb,e, Ling Mengb, Xiudong Xiad, ⁎ Mingsheng Dongb, ⁎
a
College of Life Science, Shaoxing University, Shaoxing, Zhejiang 312000, China College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China c Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA d Institute of Agro-Product Processing, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu 210014, China e Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu 210014, China b
ARTICLE INFO
ABSTRACT
Keywords: Fermented glutinous rice Fu brick tea Enzyme activity HS-SPME-GC-MS E-nose
A higher fermentation efficiency was achieved, using multistarter fermentation of glutinous rice supplemented with Fu brick tea (FGR-FBT), than when using traditional fermentation. The effects of multistarter fermentation on the microbial, chemical, and volatile compositions were determined. When FBT was incorporated during glutinous rice fermentation, increased population of yeasts and fungi, as well as enhanced α-amylase, proteinase and β-glucosidase activities, were observed. Specific fungi were isolated and identified as Aspergillus spp., which are known to secrete extracellular enzymes that modify the chemical properties, including ethanol levels, pH, total acids, and total soluble solids. The aroma profile of fermented glutinous rice was studied in the absence and presence of FBT, using HS-SPME-GC-MS and the electronic-nose. This analysis indicated that 35 characteristic volatile compounds were only found in FGR-FBT. The results show that FBT can be added during the fermentation of food products to enhance microbial biotransformation and modify flavour metabolism.
1. Introduction In Asia, glutinous rice-based fermented products are commonly consumed as foods and beverages, including products such as Indian dosa and babru (Mondal, 2016), Korean koji (Lee et al., 2017) and Chinese rice wine (Yang et al., 2018). Throughout their thousands of years of history, glutinous rice products have been fermented using various starter cultures, each leading to their own distinctive physicochemical and sensory properties (Wu et al., 2016). Over the past few decades, novel starters and microbial ratios have been developed, including species in the Rhizopus, Aspergillus and Saccharomyces genera (Lv, Weng, Zhang, Rao, & Li, 2012), to achieve improvements of fermentation efficiency, juice yield, and food palatability (Yang et al., 2018; Yang, Xia, Wang, Yu, & Ai, 2017). Fu brick tea (FBT) has a long history that can be dated as far back as 1500 CE This traditional post-fermentation tea was originally created in southwestern China, but then brought to West Asia and Europe via the
silk trade route (Zhang, Zhang, Zhou, Ling, & Wan, 2013). In recent years, FBT has attracted considerable attention from researchers due to its unique microbial profile and functional compounds generated, which lead to unique sensory properties (Xu, Hu, Wang, Wan, & Bao, 2015). Cao, Guo, Liu, Song, Ho, Hou, Zhang & Wan, reported that the volatile compounds of FBT, which contribute to its characteristic flavour profile, are the result of fermentation by specific microorganisms (Cao et al., 2018). For instance, the generation of α-terpineol, β-ionone, and cis-jasmone was attributed to the metabolism of Eurotium cristatum, while the accumulation of linalool oxides, β-ionone, and geraniol was related to the metabolism of Aspergillus niger. In addition to the dominant Eurotium spp., dozens of other microorganisms were identified belonging to the Aspergillus, Penicillium, and Saccharomyces genera (Xu et al., 2011). Several extracellular enzymes, such as amylase, glucosidase and proteinase, have been reported to be secreted during the growing and fermentation process (Cao et al., 2018; Su, Xia, Gao, Dai, & Zhang, 2010). The enzymes generated by the fungi in FBT have the
Abbreviations: FGR, fermented glutinous rice; FBT, Fu brick tea; FGR-FBT, fermented glutinous rice supplemented with Fu brick tea; HS-SPME-GC-MS, headspace solid-phase microextraction and gas chromatography combined with mass spectrometry ⁎ Corresponding authors at: College of Life Science, Shaoxing University, Shaoxing, Zhejiang 312000, China (X. Xu, Lead contact). College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China (M. Dong). E-mail addresses: [email protected] (X. Xu), [email protected] (M. Dong). https://doi.org/10.1016/j.foodchem.2019.125790 Received 4 July 2019; Received in revised form 21 October 2019; Accepted 23 October 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Xiao Xu, et al., Food Chemistry, https://doi.org/10.1016/j.foodchem.2019.125790
Food Chemistry xxx (xxxx) xxxx
X. Xu, et al.
ability to degrade starch and protein. For this reason, the activity of the natural microbial community found in FBT during the fermentation of glutinous rice was investigated in this study. The constituents and interactions of microorganisms play an important role in fermentation efficiency, food physiochemical properties, and sensory attributes. Consequently, fermentation processes can be modulated by using mixed microbial strains to carry out multistarter fermentation. Previously, we developed a fermented glutinous rice supplemented with FBT (FGR-FBT) and found that it had increased levels of bioactive phenolic compounds and enhanced antioxidant activities (Xu et al., 2019). However, there is currently a lack of understanding of the effects of multistarter fermentation on the chemical properties and microbial constituents, as well as the flavour profile, when FGR is fermented with FBT. The natural polyphenol-rich community of FBT and its associated microorganisms, was utilized as one part of the starter culture to carry out multistarter fermentation. In this study, the effects of FBT supplementation on the fermentation of glutinous rice was systematically analyzed, the populations of yeasts and fungi were determined, and the fungi in FGR-FBT were isolated and identified. Moreover, extracellular enzyme activities were determined during the fermentation process, including α-amylase, proteinase, lipase, and β-glucosidase. The chemical properties of the fermented rice were also determined, including ethanol content, pH, total acid content, and total soluble solid content. In addition, headspace solid-phase microextraction and gas chromatography combined with mass spectrometry were used to analyze volatile compounds, and an electronic-nose was used to characterize the aroma differences between FGR-FBT and FGR. This study provides valuable information that could promote the utilization of FBT in the production of traditional fermented foods.
2.3. Isolation and identification of special culture-dependent fungi in FGRGBT FGR and FGR-FBT (5 g) were mixed separately with 45 ml of 0.85% (w/v) NaCl solution and shaken at 150 rpm for 30 min at room temperature. Then, the supernatant was serially diluted, ten-fold, and plated on potato-dextrose agar medium. After incubation at 28 °C for 3–5 days, the well-isolated colonies were sub-cultured on dichloran glycerol agar medium and further sub-cultured on czapek dox agar. The isolates were separated according to their morphologies. The DNAs of all isolates with distinct morphologies were extracted using a DNA Extraction Kit (Sangon Biotech Inc., China). The PCR amplification of internal transcribed spacer (ITS) gene region was carried out using primers ITS1/ITS4, covering forward primer (5′-TCCGT AGGTGAACCTCGG-3′) and reverse primer (5′-TCCTCCGCTTATTGATA TGC-3′) (Marsh, O'Sullivan, Hill, Ross, & Cotter, 2014). A 20 μl PCR reaction system was performed with SanTaq Plus PCR Kit (Sangon Biotech Inc., China), using the following programme: 94 °C for 5 min; followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 68 °C for 60 s; and an additional extension at 68 °C for 10 min. The sequence similarities were searched in the NCBI-GenBank, using the BLAST method. Phylogenetic analysis was carried out to establish datasets, which were based on partial DNA sequences of the isolates and reference strains closely related to the isolates. The neighbour-joining trees were obtained by statistical analysis and a maximum likelihood substitution model, using the MEGA 7.0 software (Kurtzman, Fell, Boekhout, & Robert, 2011). 2.4. Estimation of major enzyme activities during fermentation progress The major extracellular enzymes produced during FGR and FGRFBT fermentation were amylase, protease, lipase, and β-glucosidase. To estimate the enzyme activities, crude extracts were analyzed as follows: samples (10 g) were mixed with 90 ml of deionized water and then shaken at 150 rpm for 30 min at room temperature. Then, the supernatant was collected and centrifuged at 5000 g at 4 °C for 10 min. The amylase activity was determined, using a method described previously, where 1 unit (U) of activity was defined as the amount of enzyme required to produce 1 μmole of reducing sugar from starch hydrolyzation in 1 min (Gupta, Gigras, Mohapatra, Goswami, & Chauhan, 2003). The β-glucosidase activity was determined in a buffer solution (pH 5.0) with p-nitrophenyl-β-D-glucopyranoside. In this case, 1 U of activity was defined as the amount of enzyme required to produce 1 μmole of pnitrophenol in 1 min (de Lima, Kurozawa, & Ida, 2014). The protease activity was determined in an acidic buffer solution (pH 3.0), using casein as a substrate by a method described previously (Germano, Pandey, Osaku, Rocha, & Soccol, 2003). The lipase activity was determined using 4-nitrophenol palmitate as a substrate, according to another method described previously (McDougall, Kulkarni, & Stewart, 2009). In this case, 1 U of activity was defined as the amount of protease and lipase required to generate 1 μmole of tyrosine and p-nitrophenol from the substrate in 1 min.
2. Materials and methods 2.1. Fermentation and sampling of FGR and FGR-FBT Fermented glutinous rice (FGR) and fermented glutinous rice with Fu brick tea (FGR-FBT) were produced according to our previous study (Xu et al., 2019). Each sample, at different fermentation times, was collected every 3 h from 0 h to 48 h. All the samples were immediately submitted to further microbial and chemical analyses. 2.2. Yeast and fungi enumeration (yeast counting and fungi quantification) in FGR and FGR-FBT The enumeration of yeasts was carried out by colony counting (log10 CFU/g sample), based on a method described previously (Li, Hu, Huang, & Xu, 2018). Briefly, each sample was weighed and placed in a sterile physiological solution for 30 min at room temperature. Then, the suspension was diluted serially and plated on Yeast extract peptone dextrose agar that contained chloramphenicol for inhibiting bacterial growth. After incubation at 28 °C for 48–72 h, the yeast colonies were counted after they could be clearly distinguished by the morphology of the colony types. The quantification of fungi was determined using quantitative realtime PCR (qPCR) analysis. First, a genomic DNA Kit (TransGen Biotech, Shanghai, China) was used to extract the DNA of the fungi from the samples. After being purified, DNA was suspended in double-distilled water as a template. The qPCR mixture contained 10 μl of 2× SYBR Premix Ex Taq (Takara Bio, Japan), template DNA (2 μl) at 10 ng/ml, and 0.5 μl of each primer at 10 μM in a final volume (20 μl). The amplification was performed with universal primer for ITS1 amplicons in an ABI 7500 real-time PCR system (Applied Biosystems, USA). The reaction was initiated at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 40 s. The copy numbers of the samples were calculated using a standard curve.
2.5. Chemical analysis The pH values were measured using a digital pH meter (Schott Lab 850, Mainz, Germany). The total acid content (grammes per kilogramme of sample) was measured, using a method described previously (Jung, Lee, Lim, Kim, & Park, 2014). The total soluble solid content was expressed as Brix, measured using a refractometer (Makroo, Prabhakar, Rastogi, & Srivastava, 2019). The total alcohol content was measured according to the People's Republic of China National Standard for fermented glutinous rice (GB/T 13662-2018). 2
Food Chemistry xxx (xxxx) xxxx
X. Xu, et al.
2.6. HS-SPME extraction and GC-MS analysis
sample. During the following exponential growth phase from 9 h to 30 h, the total yeast counts increased from 5.22 ± 0.03 to 6.59 ± 0.01 log10 CFU/g for FGR, and from 5.23 ± 0.03 to 6.68 ± 0.00 log10 CFU/g for FGR-FBT. The maximum yeast populations in both samples were obtained after 42 h of fermentation in the stationary phase. However, the fermentation time (30 h) was optimized as the end of FGR-FBT fermentation, as described in our previous study (Xu et al., 2019). During the first 30 h, the yeast count in FGR-FBT was significantly higher than that in FGR. The fungi population was obtained by quantitative real-time PCR analysis. The cycle threshold values (copy numbers) and amplification curves were acquired and then the results were used to quantify the fungi levels in FGR and FGR-FBT at the beginning and end of fermentation. As shown in Fig. 1b, the fungi quantification of FGR and FGRFBT were 9.43 ± 0.09 and 9.80 ± 0.01 log10 copy number/g. An increased production of yeasts and fungi could have a positive effect on microbial metabolism, which would promote fermentation efficiency and flavour development. Fermenting FGR with FBT is therefore a potential approach to enhance the quality of traditional fermented foods by increasing the level of beneficial bioactive components, such as phenolics (Xu et al., 2019). Moreover, this multi-strain approach shortens the fermentation time, enhances the biotransformation efficiency, and leads to the creation of novel fermented foods.
The volatile compounds were extracted and qualitatively analyzed by head space solid-phase micro-extraction and gas chromatography coupled with mass spectrometry (HS-SPME-GC-MS). The parameters for HS-SPME-GC-MS were based on previous studies (Huang et al., 2018; Huang et al., 2019). Samples were prepared with 1:5 dilution and salted out with 2 g of sodium chloride. Then, SPME, coated with 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane fibre (Supelco, Inc., Bellefonte, PA, USA), was used to extract volatile compounds in a 20-ml hermetical vial with 50 °C water bath and stirring for 45 min, followed by incubation in the GC inlet for 5 min at 250 °C, and desorbing in pulsed splitless mode. A fused silica capillary column DB-5MS (60 m × 0.25 mm) was used for compound separations, as previously reported (Schueuermann et al., 2019). Briefly, the following temperature–time profile was utilized: 40 °C for 3 min; 40 to 125 °C at 3 °C/min; 125 °C for 4 min; 125 to 180 °C at 3 °C/min; 180 °C for 2 min; 180 to 220 °C at 6 °C/min; and finally, 220 °C for 3 min. The carrier gas was helium with a flow-rate of 2 ml/min. The temperature of the mass spectrometer quadrupole was set at 230 °C, the source temperature was set at 230 °C, and the transfer line temperature was set at 250 °C. All mass spectra were acquired in electron impact (EI) mode at 70 eV, using full scan mode with a scan range of 30–550 amu. The identification of all compounds was confirmed by mass spectra and retention indices. All analyses were performed in triplicate.
3.2. Isolation and identification of special culture-dependent fungi in FGRFBT
2.7. Electronic-nose data acquisition
In total, 24 fungi isolates were collected during each culture-dependent isolation from FGR-FBT and FGR. Based on the morphological and microscopical characteristics, a total of 3 special fungi was found only in FGR-FBT. These isolated strains were identified as Aspergillus cristatum, A. niger and A. oryzae through comparative analysis of physiology and molecules. As shown in Fig. 2a–c, the asexual spore-forming structure was considered as a distinct characteristic of the Aspergillus family. A reliable resolution at the species level of the isolates was carried out by the phylogenetical analysis of the ITS sequences (Fig. 2d). It is likely that the three dominant Aspergillus species identified in FBT participated in the fermentation of FGR-FBT. Previously, it has been reported that the Aspergillus spp. occupied over 90% relative abundance of all the microorganisms in post-fermented teas (Mao, Wei, Teng, Huang, & Xia, 2017). An enhanced population of fungi was also present in the FGR-FBT after 30 h of fermentation, including A. cristatum, A. niger and A. oryzae. Microorganisms in the genus Aspergillus are capable of secreting extracellular enzymes, such as amylase, cellulose and glucosidase (Handa, Couto, Vicensoti, Georgetti, & Ida, 2014). Hence, an enhanced production of yeasts and fungi has the potential for higher extracellular enzyme activities and greater fermentation efficiency.
A commercial PEN3 E-nose (Airsense Analytics GmbH, Schwerin, Germany) instrument was used to acquire information about the aromatic profile of the samples (Benedetti, Buratti, Spinardi, Mannino, & Mignani, 2008). The gas detector system consisted of 10 different metal-oxide sensors that were sensitive to aromatic compounds (W1C), broad range of sensitivity and nitrogen oxides (W5S), aromatic ammoniacal compounds (W3C), hydrogen (W6S), alkanes (W5C), methane (W1S), terpenes and sulphur compounds covering organic compounds (W1W), alcohol (W2S), sulfur organic compounds (W2W) and methanealiphatic compounds (W3S). The volatile compositions of the samples were reflected by their response values (G/G0), in which G and G0 are conductivity values of metaloxide gas sensors connected with sample gas and clean gas, respectively. Each sample (50 g) was taken and sealed in a flask with foil paper. Then, the headspace was equilibrated at 25 °C for 30 min to minimize sensor drift. After that, the headspace gas was pumped into the sensor chamber at a flow rate of 120 ml/min. The response values were then recorded for 60 s at intervals of 1 s, until reaching a stable state. Finally, the probe was cleaned for 120 s and the baseline was reset in 5 s. All the tests were performed at 25 °C. 2.8. Statistical analysis
3.3. Change of major enzyme activities during the fermentation of FGR and FGR-FBT
All the results are presented as means ± standard deviation. Oneway analysis of variance, Duncan’s multiple range tests and t-test were carried out to analyze significant differences, using SPSS version 19.0 (SPSS Inc., Chicago, IL, USA) software for Windows, where p < 0.05 was considered statistically significant.
The enzyme activities were analyzed during the whole fermentation period (Fig. 3). As shown in Fig. 3a, the highest α-amylase activities were found to be around 85.6 ± 0.9 U/g FGR-FBT at 33 h and 79.4 ± 1.2 U/g FGR at 36 h. The enzyme activity of FGR was significantly different from that exhibited by FGR-FBT, with a higher αamylase activity at each phase during the first 30 h of fermentation. After 30 h of fermentation, the α-amylase activities in FGR-FBT and FGR were 78.3 ± 1.0 U/g and 75.6 ± 1.2 U/g. The α-amylase activity impacts the efficiency of starch digestion of rice-based foodstuffs during fermentation. Previous researchers have reported that the starch structure was degraded by semi-solid fermentation and starch liquefaction was attributed to α-amylase (Liu et al., 2017). Similarly, the protease activity impacts the efficiency of protein digestion and
3. Results and discussion 3.1. Total yeast counts and fungi quantification during fermentation The evolution of the total yeast counts during fermentation of the FGR and FGR-FBT samples was measured (Fig. 1a). The yeast population was divided into three phases, ranging from the initial to terminal fermentation. During the first 9 h, the adaptive phase displayed a shortened tendency in the FGR-FBT sample compared to the FGR 3
Food Chemistry xxx (xxxx) xxxx
X. Xu, et al.
Fig. 1. Total yeast counts (a) and fungi population (b) during the fermentation progress of fermented glutinous rice with Fu brick tea (FGR-FBT) and traditional fermented glutinous rice (FGR).
biotransformation in these products, which influences the quality attributes of the fermented foods produced (Sanjukta & Rai, 2016). In our study, FGR-FBT and FGR achieved their highest protease activities of 138 ± 2.3 U/g at 24 h and 109 ± 3.7 U/g at 30 h, respectively (Fig. 3b). During the first 30 h of fermentation, the proteinase activity in FGR-FBT was much higher than that in FGR. It is noteworthy, that β-glucosidase activity was detected in FGRFBT, but not in FGR (Fig. 3d). Previous studies have reported that A. cristatum, A. niger and A. oryzae could produce β-glucosidase (Abdella, Mazeed, El-Baz, & Yang, 2016; Cao et al., 2018; Handa et al., 2014). βGlucosidase is an extracellular enzyme that can hydrolyze β-glycosidic bonds, thus leading to the release of polyphenols, as well as the modification of phenolic chemicals (Hur, Lee, Kim, Choi, & Kim, 2014). In this study, we found that there was a close relationship between the total phenolic content and β-glucosidase activity. After 15 h and 30 h of fermentation, the β-glucosidase activities in FGR-FBT were 9.73 ± 0.77 U/g and 21.9 ± 1.0 U/g, respectively. Furthermore, as shown in Fig. 3c, the lipase activities in FGR-FBT and FGR were fairly
similar, with no significant differences, which may be attributed to the low lipid content of glutinous rice. Together, α-amylase, proteinase and β-glucosidase activities in FGR-FBT were much higher than those in FGR, especially during the first 30 h of fermentation. These results suggest that the biotransformation efficiency could be enhanced by the microorganisms in FBT. This result is in agreement with a recent study, which found that a tannase that was prepared from Aspergillus niger by a solid-state fermentation of tea byproducts could effectively transform the catechin in tea infusion and improve its antioxidant activity (Ni et al., 2015). 3.4. Chemical character analysis during the fermentation of FGR and FGRFBT 3.4.1. General The quality of fermented food products depends on the metabolic processes carried out by the microorganisms present (Wang et al., 2014). In our study, three novel strains were found in FGR-FBT that
Fig. 2. The morphological and microscopial characteristics of Aspergillus niger (a), Aspergillus oryzae (b) and Aspergillus cristatellus (c) isolated from fermented glutinous rice with Fu brick tea (FGR-FBT). The neighbour joining trees (d) based on the dataset covering the sequences of ITS gene of isolates and reference strains. 4
Food Chemistry xxx (xxxx) xxxx
X. Xu, et al.
Fig. 2. (continued)
exhibited different enzyme activities. The presence of different microorganisms with different enzymatic activities would be expected to lead to fermented products with different chemical compositions. Thus, the pH, total acid contents, total soluble solids content, and alcohol content of the FGR and FGR-FBT were analyzed during the whole fermentation process.
3.4.2. pH and total titrable acidity Microbial bilateral fermentation and metabolism is known to lead to the production of several kinds of organic acids, which results in a decline in pH and an increase in total acid content. As shown in Fig. 4a and b, the production of acid occurred during the initiation period. After 15 h of fermentation, FGR-FBT and FGR had pH values of 5
Food Chemistry xxx (xxxx) xxxx
X. Xu, et al.
Fig. 3. The activities of α-amylase (a), proteinase (b), lipase (c) and β-glucosidase (d) in fermented glutinous rice with Fu brick tea (FGR-FBT) and traditional fermented glutinous rice (FGR) during the whole fermentation progress (from 12 h to 48 h).
3.77 ± 0.01 and 3.78 ± 0.01, and total titratable acid contents of 2.85 ± 0.20 and 2.53 ± 0.22 g/kg, respectively. In the first 27 h of fermentation, the acid accumulation in FGR-FBT was slightly higher than that in FGR. In contrast to the first 27 h of fermentation, the efficiency of acid production was higher in FGR during the terminal fermentation phase. Nevertheless, after 30 h of fermentation, there were no significant statistical differences in the pH values or total titrable acidity of FGR-FBT and FGR.
respectively. 3.5. Volatile compounds analyzed by HS-SPME-GC-MS In this study, referring to the retention indices and mass spectrometer analysis, 94 volatile compounds were identified in FGR-FBT, including 16 alcohols, 11 acids, 32 esters, 11 aldehydes, 3 ketones, 2 ethers, 3 furans, 2 terpenes, 4 hydrocarbons, 6 phenolic compounds, 3 aromatics and 1 nitrogenous compound, while only 59 volatile compounds were found in FGR (Table S1). Several kinds of volatile compounds (35) were only found in FGR-FBT and accounted for 8.34% of the total content, including 3 acids (3.05%), 8 esters (1.16%), 8 alcohols (1.06%) and 5 aldehydes (0.27%). This result indicates that a richer aroma with more distinctive volatile compounds was generated in FGR-FBT than in FGR. For example, linalool and linalool oxides provided a special sweet odour (like lily), as well as contributing to the other characteristic flavour notes of fermented teas. Ionone provided a unique wood-like and violet-like scent. Valencene and farnesol provided a citrus-like scent. The 59 volatile compounds common to both FGR-FBT and FGR were present at different levels in the two samples. In FGR-FBT, 2-methoxy-4vinylphenol accounted for the highest relative content (27.9%) with a roasted peanut-like and clove-like scent, followed by palmitic acid ethyl ester (25.5%) with a cheese-like scent and 4-ethyl-2-methoxyphenol (6.0%) with a sweet grass-like scent. Also, the predominant volatile compounds that contributed over 1% relative content included phenethyl alcohol (3.16%), oxalic acid (2.68%), ethanol (2.65%), ethyl oleate (2.46%), ethyl linoleate (2.40%), ethyl acetate (2.30%), p-tolualdehyde (1.77%), 3-methyl-1-butanol (1.70%), glycolic acid
3.4.3. Total soluble solid content The total soluble solid content provides valuable information about the hydrolyzation efficiency of rice starch. The reduced carbohydrates in FGR-FBT and FGR reached a peak and then decreased, by the consumption of microorganisms, after 36 h of fermentation. As shown in Fig. 4c, it took 21 h to reach the rapid growth phase for FGR-FBT, but 24 h for FGR. Moreover, the highest total soluble solid content was obtained after 30 h of fermentation for FGR-FBT (41.5 ± 0.3 °Brix), but after 36 h for FGR (41.0 ± 0.4 °Brix). The faster starch degradation and more reducing sugar accumulation of FGR-FBT suggested that an enhanced biotransformation efficiency could be obtained when fermenting FGR with FBT. 3.4.4. Ethanol content As shown in Fig. 4d, the production of ethanol mainly occurred at the beginning of the fermentation process. FGR-FBT had a higher efficiency than FBT. For instance, during the first 21 h, the ethanol accumulation for FGR-FBT was 3.33-fold greater than for FBT, indicating different microbial metabolisms. After 30 h of fermentation, the ethanol contents in FGR-FBT and FGR were 1.82 ± 0.10 and 1.22 ± 0.11, 6
Food Chemistry xxx (xxxx) xxxx
X. Xu, et al.
Fig. 4. Chemical properties analysis of pH (a), total acid content (b), total soluble solid content (c) and ethanol content (d) in fermented glutinous rice with Fu brick tea (FGR-FBT) and traditional fermented glutinous rice (FGR) during the whole fermentation progress (from 15 h to 48 h).
presence of these volatile compounds has been attributed to the metabolism by the genus Aspergillus, which accounts for a considerable proportion of the fungi in fermentation starters (Xu et al., 2015). Ethanol accounted for 2.42% in FGR and 2.65% in FGR-FBT. Furthermore, butyl alcohol and hexyl alcohol accounted for 4.12% and 0.58% in FGR, while only 0.10% and 0.05% of them were detected in FGRFBT. Therefore, a softer aroma would be expected in FGR-FBT because lower levels of the irritants butyl alcohol and hexyl alcohol were generated. In addition, a wider range of alcohols was formed in FGR-FBT, e.g. as 2-methyl-1-pentanol and linalool, which generate grass-like and rose-like fresh scents. Moreover, eight kinds of volatile acids were detected in FGR (7.08% of total) and 11 kinds of volatile acids were detected in FGR-FBT (6.21% of total). Besides, higher relative contents of furans (burnt sugar-like and grass-like scents) and terpenes (a citrus-like scent) were detected in FGR-FBT. In addition, the relative contents of saturated hydrocarbons in FGR-FBT and FGR presented no significant differences (p > 0.01), while the relative content of unsaturated hydrocarbons was significantly higher in FGR-FBT, with a fresh tea-like scent (p < 0.01). Together, distinctive aromatic properties of FGR-FBT may derive from the special volatile compounds from FBT and various microorganism metabolisms. Hence, the fermentation with FBT could improve the sensory properties of FGR.
(1.68%), lactamide (1.50%) and ethyl laurate (1.12%). In FGR, the most abundant compound was palmitic acid ethyl ester, accounting for a relative content of 34.0%, which was higher than that in FGR-FBT (25.5%). The predominant volatile compounds found in FGR included phenethyl alcohol, ethyl myristate, 4-hydroxy-3-methoxystyrene and pentanol (4.12%). Esters were crucial because of their highest total relative content and wide variety. There were 24 esters in FGR (occupying 59.4%) and 32 esters in FGR-FBT (occupying 43.5%). Among them, palmitic acid ethyl ester was abundant in FGR and FGR-FBT, followed by ethyl myristate, ethyl oleate, octyl 4-meth-oxycinnamate, ethyl acetate, ethyl laurate, pentadecanoic acid ethyl ester and isoamyl acetate, which were considered as metabolites biotransformed by bacteria and yeasts at the terminal fermentation phase. Esters are considered to contribute an irreplaceable part of the fruity and flowery aroma of Chinese rice wine (Yang et al., 2017). Moreover, the composition of the esters formed in fermented foods has been reported to be distinctive of the nature of the microbes present (Yu, Xie, Xie, Ai, & Tian, 2019). Esters present at relatively low concentrations would also be expected to contribute to the rich aroma of fermented foods. The methyl salicylate, phenylacetic acid ethyl ester, and dihydroactinidiolide were detected at levels of 0.15%, 0.02% and 0.10% of FGR-FBT, which produced honey-like sweet and kiwifruit-like fruity aromas. Phenolic compounds occupied the second highest total relative content (16.7% in FGR and 37.6% in FGR-FBT), followed by alcohols and acids. Several phenolic compounds in FBT were metabolized by microorganisms. The relative contents of phenolic compounds in FGRFBT were higher than those in FGR, such as 4-butyl-2,6-ditert-butylphenol. In this study, a total of 8 alcohols (10.5%) and 16 alcohols (6.36%) were identified in FGR and FGR-FBT, respectively. The
3.6. E-nose analysis The signals of ten sensors in response to aromatic compounds of FGR and FGR-FBT were expressed by G0/G values (Fig. 5a and b). All the signals reached a stable state after 50 s, and so the response curves were represented as the averages of multiple values measured at 55 s. Sensors W2S, W1S, W2W and W1W had the highest response to the 7
Food Chemistry xxx (xxxx) xxxx
X. Xu, et al.
Fig. 5. G/G0 values of ten sensors detected by electronic nose (E-nose) in response to the odour of fermented glutinous rice with Fu brick tea (FGR-FBT) (a) and traditional fermented glutinous rice (FGR) (b). Principal component analysis (c) and linear discriminant analysis (d) were performed, based on the E-nose detection and analysis.
volatile compounds in FGR and FGR-FBT. The signals detected by these sensors were higher for FGR-FBT than for FGR, which is indicative of high levels of volatile alcohol and alkane compounds in the aroma profile. FGR-FBT also gave higher G0/G responses for the W2W, W1C and W5C sensors, which suggests that there were more aromatic, sulfurchlorine, and alkene compounds generated. Based on a loading analysis of the correlations of ten sensors, the W2S and W1S sensors were able to differentiate the volatile compositions at the highest sensitivity, followed by W2W and W1W, which responded to aromatic and organic compounds. According to principal component analysis and linear discriminant analysis of the FGR and FGR-FBT profiles, there were significant aromatic differences between the samples, which would be expected to lead to different sensory properties (Fig. 5c and d). Differences in the nature of the volatile compounds formed when FBT was present during fermentation can be attributed to differences in microbial metabolism. Previous researchers reported that the quality of Baijiu flavour was influenced by microbial interactions, and using mixed cultures improved the flavour metabolism by yeasts (Zha, Sun, Wu, Yin, & Wang, 2018). Co-culture yeasts have also been reported to provide a synergistic effect on metabolic pathways, leading to increased aroma complexity (Sadoudi et al., 2012). Also, mixed inoculation with various ratios of different strains has been shown to reduce the content of tannin-protein complexes formed, thereby leading to decreased astringency (Kang, Kang, Lee, & Chang, 2018). The metabolism of Saccharomyces cerevisiae was changed by lactic acid bacteria during the fermentation of Chinese liquor, thus resulting in altered levels of various kinds of flavour compounds (Meng et al., 2015). Combining chemical analysis, GC-MS and E-nose, we verified that FGR-FBT had a higher ethanol content, acidity, and more abundant and diverse aromatic compounds, along with special microbial properties and a higher population of yeasts and fungi.
4. Conclusion In this study, fermented glutinous rice (FGR) was produced using a multi-starter fermentation technique that relied on the diverse microbial cultures naturally found in Fu brick tea (FBT). This innovative fermentation approach led to the production of a novel functional food product (FGR-FBT) with unique sensory properties. Different fungi were isolated from the FGR-FBT and identified as Aspergillus cristatum, A. niger and A. oryzae. The increased levels of yeast and fungi present in FGR-FBT led to enhanced α-amylase, proteinase and β-glucosidase activities, which increased the fermentation efficiency. Moreover, the utilization of the multi-starter fermentation technique led to differences in the chemical composition and aroma profile of the final product. In particular, a richer sensory profile was found for the FGR-FBT compared to the FGR. This study may therefore lead to a new range of highquality fermented food products based on the use of Fu brick tea and glutinous rice. Author contributions Mingsheng Dong conceived the project idea and obtained funding. Xiao Xu was assigned the project, conducted the research, wrote and revised this manuscript. David Julian McClements gave constructive suggestions on the content and revised the manuscript. Siduo Zhou and other authors participated in the method optimization of chemical analysis and revised the manuscript. All the authors approved the final manuscript drafts. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 8
Food Chemistry xxx (xxxx) xxxx
X. Xu, et al.
Acknowledgement
Makroo, H. A., Prabhakar, P. K., Rastogi, N. K., & Srivastava, B. (2019). Characterization of mango puree based on total soluble solids and acid content: Effect on physicochemical, rheological, thermal and ohmic heating behavior. LWT-Food Science and Technology, 103, 316–324. Mao, Y., Wei, B., Teng, J., Huang, L., & Xia, N. (2017). Analyses of fungal community by Illumina MiSeq platforms and characterization of Eurotium species on Liupao tea, a distinctive post-fermented tea from China. Food Research International, 99, 641–649. Marsh, A. J., O'Sullivan, O., Hill, C., Ross, R. P., & Cotter, P. D. (2014). Sequence-based analysis of the bacterial and fungal compositions of multiple kombucha (tea fungus) samples. Food Microbiology, 38(2), 171–178. McDougall, G. J., Kulkarni, N. N., & Stewart, D. (2009). Berry polyphenols inhibit pancreatic lipase activity in vitro. Food Chemistry, 115(1), 193–199. Meng, X., Wu, Q., Wang, L., Wang, D., Chen, L., & Xu, Y. (2015). Improving flavor metabolism of Saccharomyces cerevisiae by mixed culture with Bacillus licheniformis for Chinese Maotai-flavor liquor making. Journal of Industrial Microbiology & Biotechnology, 42(12), 1601–1608. Mondal, K. C. (2016). Folk to functional: An explorative overview of rice-based fermented foods and beverages in India. Journal of Ethnic Foods, 3(1), 5–18. Ni, H., Chen, F., Jiang, D., Cai, M., Yang, Y., Xiao, A., & Cai, H. (2015). Biotransformation of tea catechins using Aspergillus niger tannase prepared by solid state fermentation on tea byproduct. LWT – Food Science and Technology, 60(2), 1206–1213. Sadoudi, M., Tourdot-Marechal, R., Rousseaux, S., Steyer, D., Gallardo-Chacon, J.-J., Ballester, J., ... Alexandre, H. (2012). Yeast-yeast interactions revealed by aromatic profile analysis of Sauvignon Blanc wine fermented by single or co-culture of nonSaccharomyces and Saccharomyces yeasts. Food Microbiology, 32(2), 243–253. Sanjukta, S., & Rai, A. K. (2016). Production of bioactive peptides during soybean fermentation and their potential health benefits. Trends in Food Science & Technology, 50, 1–10. Schueuermann, C., Steel, C. C., Blackman, J. W., Clark, A. C., Schwarz, L. J., Moraga, J., ... Schmidtke, L. M. (2019). A GC-MS untargeted metabolomics approach for the classification of chemical differences in grape juices based on fungal pathogen. Food Chemistry, 270, 375–384. Su, E., Xia, T., Gao, L., Dai, Q., & Zhang, Z. (2010). Immobilization of beta-glucosidase and its aroma-increasing effect on tea beverage. Food and Bioproducts Processing, 88(C2–3), 83–89. Wang, P., Mao, J., Meng, X., Li, X., Liu, Y., & Feng, H. (2014). Changes in flavour characteristics and bacterial diversity during the traditional fermentation of Chinese rice wines from Shaoxing region. Food Control, 44, 58–63. Wu, Z., Xu, E., Long, J., Pan, X., Xu, X., Jin, Z., & Jiao, A. (2016). Comparison between ATR-IR, Raman, concatenated ATR-IR and Raman spectroscopy for the determination of total antioxidant capacity and total phenolic content of Chinese rice wine. Food Chemistry, 194, 671–679. Xu, A., Wang, Y., Wen, J., Liu, P., Liu, Z., & Li, Z. (2011). Fungal community associated with fermentation and storage of Fuzhuan brick-tea. International Journal of Food Microbiology, 146(1), 14–22. Xu, E., Long, J., Wu, Z., Li, H., Wang, F., Xu, X., ... Jiao, A. (2015). Characterization of volatile flavor compounds in Chinese rice wine fermented from enzymatic extruded rice. Journal of Food Science, 80(7), 1476–1489. Xu, J., Hu, F.-L., Wang, W., Wan, X.-C., & Bao, G.-H. (2015). Investigation on biochemical compositional changes during the microbial fermentation process of Fu brick tea by LC–MS based metabolomics. Food Chemistry, 186, 176–184. Xu, X., Hu, W., Zhou, S., Tu, C., Xia, X., Zhang, J., & Dong, M. (2019). Increased phenolic content and enhanced antioxidant activity in fermented glutinous rice supplemented with Fu brick tea. Molecules, 24(4), 671. Yang, Y., Xia, Y., Lin, X., Wang, G., Zhang, H., Xiong, Z., ... Ai, L. (2018). Improvement of flavor profiles in Chinese rice wine by creating fermenting yeast with superior ethanol tolerance and fermentation activity. Food Research International, 108, 83–92. Yang, Y., Xia, Y., Wang, G., Yu, J., & Ai, L. (2017). Effect of mixed yeast starter on volatile flavor compounds in Chinese rice wine during different brewing stages. LWT-Food Science and Technology, 78, 373–381. Yu, H., Xie, T., Xie, J., Ai, L., & Tian, H. (2019). Characterization of key aroma compounds in Chinese rice wine using gas chromatography-mass spectrometry and gas chromatography-olfactometry. Food Chemistry, 293, 8–14. Zha, M., Sun, B., Wu, Y., Yin, S., & Wang, C. (2018). Improving flavor metabolism of Saccharomyces cerevisiae by mixed culture with Wickerhamomyces anomalus for Chinese Baijiu making. Journal of Bioscience and Bioengineering, 126(2), 189–195. Zhang, L., Zhang, Z.-Z., Zhou, Y.-B., Ling, T.-J., & Wan, X.-C. (2013). Chinese dark teas: Postfermentation, chemistry and biological activities. Food Research International, 53(2), 600–607.
This work was supported by the Earmarked Fund for Jiangsu Agricultural Industry Technology System (No. JATS-2018-296). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.125790. References Abdella, A., Mazeed, T. E.-S., El-Baz, A. F., & Yang, S.-T. (2016). Production of β-glucosidase from wheat bran and glycerol by Aspergillus niger in stirred tank and rotating fibrous bed bioreactors. Process Biochemistry, 51(10), 1331–1337. Benedetti, S., Buratti, S., Spinardi, A., Mannino, S., & Mignani, I. (2008). Electronic nose as a non-destructive tool to characterise peach cultivars and to monitor their ripening stage during shelf-life. Postharvest Biology & Technology, 47(2), 181–188. Cao, L., Guo, X., Liu, G., Song, Y., Ho, C.-T., Hou, R., ... Wan, X. (2018). A comparative analysis for the volatile compounds of various Chinese dark teas using combinatory metabolomics and fungal solid-state fermentation. Journal of Food and Drug Analysis, 26(1), 112–123. de Lima, F. S., Kurozawa, L. E., & Ida, E. I. (2014). The effects of soybean soaking on grain properties and isoflavones loss. LWT-Food Science and Technology, 59(2), 1274–1282. Germano, S., Pandey, A., Osaku, C. A., Rocha, S. N., & Soccol, C. R. (2003). Characterization and stability of proteases from Penicillium sp produced by solid-state fermentation. Enzyme and Microbial Technology, 32(2), 246–251. Gupta, R., Gigras, P., Mohapatra, H., Goswami, V. K., & Chauhan, B. (2003). Microbial alpha-amylases: A biotechnological perspective. Process Biochemistry, 38(11), 1599–1616. Handa, C. L., Couto, U. R., Vicensoti, A. H., Georgetti, S. R., & Ida, E. I. (2014). Optimisation of soy flour fermentation parameters to produce beta-glucosidase for bioconversion into aglycones. Food Chemistry, 152, 56–65. Huang, Z. R., Guo, W. L., Zhou, W. B., Li, L., Xu, J. X., Hong, J. L., ... Lv, X. C. (2019). Microbial communities and volatile metabolites in different traditional fermentation starters used for Hong Qu glutinous rice wine. Food Research International, 121, 593–603. Huang, Z. R., Hong, J. L., Xu, J. X., Li, L., Guo, W. L., Pan, Y. Y., ... Lv, X. C. (2018). Exploring core functional microbiota responsible for the production of volatile flavour during the traditional brewing of Wuyi Hong Qu glutinous rice wine. Food Microbiology, 76, 487–496. Hur, S. J., Lee, S. Y., Kim, Y.-C., Choi, I., & Kim, G.-B. (2014). Effect of fermentation on the antioxidant activity in plant-based foods. Food Chemistry, 160, 346–356. Jung, H., Lee, S. J., Lim, J. H., Kim, B. K., & Park, K. J. (2014). Chemical and sensory profiles of makgeolli, Korean commercial rice wine, from descriptive, chemical, and volatile compound analyses. Food Chemistry, 152(2), 624–632. Kang, M.-S., Kang, Y.-R., Lee, Y., & Chang, Y. H. (2018). Effect of mixed culture inoculation on chemical and sensory properties of aronia (Aronia melanocarpa). LWTFood Science and Technology, 98, 418–423. Kurtzman, C. P., Fell, J. W., Boekhout, T., & Robert, V. (2011). Chapter 7 - Methods for Isolation, Phenotypic Characterization and Maintenance of Yeasts. In: C. P. Kurtzman, J. W. Fell & T. Boekhout (Eds.), The Yeasts (Fifth Edition), (pp. 87–110). Lee, D. E., Lee, S., Singh, D., Jang, E. S., Shin, H. W., Moon, B. S., & Lee, C. H. (2017). Time-resolved comparative metabolomes for Koji fermentation with brown-, white-, and giant embryo-rice. Food Chemistry, 231, 258–266. Li, J., Hu, W., Huang, X., & Xu, Y. (2018). Investigation of yeast population diversity and dynamics in spontaneous fermentation of Vidal blanc icewine by traditional culturedependent and high-throughput sequencing methods. Food Research International, 112, 66–77. Liu, L., Zhang, R., Deng, Y., Zhang, Y., Xiao, J., Huang, F., ... Zhang, M. (2017). Fermentation and complex enzyme hydrolysis enhance total phenolics and antioxidant activity of aqueous solution from rice bran pretreated by steaming with alpha-amylase. Food Chemistry, 221, 636–643. Lv, X. C., Weng, X., Zhang, W., Rao, P. F., & Li, N. (2012). Microbial diversity of traditional fermentation starters for Hong Qu glutinous rice wine as determined by PCRmediated DGGE. Food Control, 28(2), 426–434.
9
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Multistarter fermentation of glutinous rice with Fu brick tea: Effects on microbial, chemical, and volatile compositions Xiao Xua,b,c, , Siduo Zhoub, David Julian McClementsc, Lu Huangb,e, Ling Mengb, Xiudong Xiad, ⁎ Mingsheng Dongb, ⁎
a
College of Life Science, Shaoxing University, Shaoxing, Zhejiang 312000, China College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China c Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA d Institute of Agro-Product Processing, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu 210014, China e Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences, Nanjing, Jiangsu 210014, China b
ARTICLE INFO
ABSTRACT
Keywords: Fermented glutinous rice Fu brick tea Enzyme activity HS-SPME-GC-MS E-nose
A higher fermentation efficiency was achieved, using multistarter fermentation of glutinous rice supplemented with Fu brick tea (FGR-FBT), than when using traditional fermentation. The effects of multistarter fermentation on the microbial, chemical, and volatile compositions were determined. When FBT was incorporated during glutinous rice fermentation, increased population of yeasts and fungi, as well as enhanced α-amylase, proteinase and β-glucosidase activities, were observed. Specific fungi were isolated and identified as Aspergillus spp., which are known to secrete extracellular enzymes that modify the chemical properties, including ethanol levels, pH, total acids, and total soluble solids. The aroma profile of fermented glutinous rice was studied in the absence and presence of FBT, using HS-SPME-GC-MS and the electronic-nose. This analysis indicated that 35 characteristic volatile compounds were only found in FGR-FBT. The results show that FBT can be added during the fermentation of food products to enhance microbial biotransformation and modify flavour metabolism.
1. Introduction In Asia, glutinous rice-based fermented products are commonly consumed as foods and beverages, including products such as Indian dosa and babru (Mondal, 2016), Korean koji (Lee et al., 2017) and Chinese rice wine (Yang et al., 2018). Throughout their thousands of years of history, glutinous rice products have been fermented using various starter cultures, each leading to their own distinctive physicochemical and sensory properties (Wu et al., 2016). Over the past few decades, novel starters and microbial ratios have been developed, including species in the Rhizopus, Aspergillus and Saccharomyces genera (Lv, Weng, Zhang, Rao, & Li, 2012), to achieve improvements of fermentation efficiency, juice yield, and food palatability (Yang et al., 2018; Yang, Xia, Wang, Yu, & Ai, 2017). Fu brick tea (FBT) has a long history that can be dated as far back as 1500 CE This traditional post-fermentation tea was originally created in southwestern China, but then brought to West Asia and Europe via the
silk trade route (Zhang, Zhang, Zhou, Ling, & Wan, 2013). In recent years, FBT has attracted considerable attention from researchers due to its unique microbial profile and functional compounds generated, which lead to unique sensory properties (Xu, Hu, Wang, Wan, & Bao, 2015). Cao, Guo, Liu, Song, Ho, Hou, Zhang & Wan, reported that the volatile compounds of FBT, which contribute to its characteristic flavour profile, are the result of fermentation by specific microorganisms (Cao et al., 2018). For instance, the generation of α-terpineol, β-ionone, and cis-jasmone was attributed to the metabolism of Eurotium cristatum, while the accumulation of linalool oxides, β-ionone, and geraniol was related to the metabolism of Aspergillus niger. In addition to the dominant Eurotium spp., dozens of other microorganisms were identified belonging to the Aspergillus, Penicillium, and Saccharomyces genera (Xu et al., 2011). Several extracellular enzymes, such as amylase, glucosidase and proteinase, have been reported to be secreted during the growing and fermentation process (Cao et al., 2018; Su, Xia, Gao, Dai, & Zhang, 2010). The enzymes generated by the fungi in FBT have the
Abbreviations: FGR, fermented glutinous rice; FBT, Fu brick tea; FGR-FBT, fermented glutinous rice supplemented with Fu brick tea; HS-SPME-GC-MS, headspace solid-phase microextraction and gas chromatography combined with mass spectrometry ⁎ Corresponding authors at: College of Life Science, Shaoxing University, Shaoxing, Zhejiang 312000, China (X. Xu, Lead contact). College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China (M. Dong). E-mail addresses: [email protected] (X. Xu), [email protected] (M. Dong). https://doi.org/10.1016/j.foodchem.2019.125790 Received 4 July 2019; Received in revised form 21 October 2019; Accepted 23 October 2019 0308-8146/ © 2019 Elsevier Ltd. All rights reserved.
Please cite this article as: Xiao Xu, et al., Food Chemistry, https://doi.org/10.1016/j.foodchem.2019.125790
Food Chemistry xxx (xxxx) xxxx
X. Xu, et al.
ability to degrade starch and protein. For this reason, the activity of the natural microbial community found in FBT during the fermentation of glutinous rice was investigated in this study. The constituents and interactions of microorganisms play an important role in fermentation efficiency, food physiochemical properties, and sensory attributes. Consequently, fermentation processes can be modulated by using mixed microbial strains to carry out multistarter fermentation. Previously, we developed a fermented glutinous rice supplemented with FBT (FGR-FBT) and found that it had increased levels of bioactive phenolic compounds and enhanced antioxidant activities (Xu et al., 2019). However, there is currently a lack of understanding of the effects of multistarter fermentation on the chemical properties and microbial constituents, as well as the flavour profile, when FGR is fermented with FBT. The natural polyphenol-rich community of FBT and its associated microorganisms, was utilized as one part of the starter culture to carry out multistarter fermentation. In this study, the effects of FBT supplementation on the fermentation of glutinous rice was systematically analyzed, the populations of yeasts and fungi were determined, and the fungi in FGR-FBT were isolated and identified. Moreover, extracellular enzyme activities were determined during the fermentation process, including α-amylase, proteinase, lipase, and β-glucosidase. The chemical properties of the fermented rice were also determined, including ethanol content, pH, total acid content, and total soluble solid content. In addition, headspace solid-phase microextraction and gas chromatography combined with mass spectrometry were used to analyze volatile compounds, and an electronic-nose was used to characterize the aroma differences between FGR-FBT and FGR. This study provides valuable information that could promote the utilization of FBT in the production of traditional fermented foods.
2.3. Isolation and identification of special culture-dependent fungi in FGRGBT FGR and FGR-FBT (5 g) were mixed separately with 45 ml of 0.85% (w/v) NaCl solution and shaken at 150 rpm for 30 min at room temperature. Then, the supernatant was serially diluted, ten-fold, and plated on potato-dextrose agar medium. After incubation at 28 °C for 3–5 days, the well-isolated colonies were sub-cultured on dichloran glycerol agar medium and further sub-cultured on czapek dox agar. The isolates were separated according to their morphologies. The DNAs of all isolates with distinct morphologies were extracted using a DNA Extraction Kit (Sangon Biotech Inc., China). The PCR amplification of internal transcribed spacer (ITS) gene region was carried out using primers ITS1/ITS4, covering forward primer (5′-TCCGT AGGTGAACCTCGG-3′) and reverse primer (5′-TCCTCCGCTTATTGATA TGC-3′) (Marsh, O'Sullivan, Hill, Ross, & Cotter, 2014). A 20 μl PCR reaction system was performed with SanTaq Plus PCR Kit (Sangon Biotech Inc., China), using the following programme: 94 °C for 5 min; followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 68 °C for 60 s; and an additional extension at 68 °C for 10 min. The sequence similarities were searched in the NCBI-GenBank, using the BLAST method. Phylogenetic analysis was carried out to establish datasets, which were based on partial DNA sequences of the isolates and reference strains closely related to the isolates. The neighbour-joining trees were obtained by statistical analysis and a maximum likelihood substitution model, using the MEGA 7.0 software (Kurtzman, Fell, Boekhout, & Robert, 2011). 2.4. Estimation of major enzyme activities during fermentation progress The major extracellular enzymes produced during FGR and FGRFBT fermentation were amylase, protease, lipase, and β-glucosidase. To estimate the enzyme activities, crude extracts were analyzed as follows: samples (10 g) were mixed with 90 ml of deionized water and then shaken at 150 rpm for 30 min at room temperature. Then, the supernatant was collected and centrifuged at 5000 g at 4 °C for 10 min. The amylase activity was determined, using a method described previously, where 1 unit (U) of activity was defined as the amount of enzyme required to produce 1 μmole of reducing sugar from starch hydrolyzation in 1 min (Gupta, Gigras, Mohapatra, Goswami, & Chauhan, 2003). The β-glucosidase activity was determined in a buffer solution (pH 5.0) with p-nitrophenyl-β-D-glucopyranoside. In this case, 1 U of activity was defined as the amount of enzyme required to produce 1 μmole of pnitrophenol in 1 min (de Lima, Kurozawa, & Ida, 2014). The protease activity was determined in an acidic buffer solution (pH 3.0), using casein as a substrate by a method described previously (Germano, Pandey, Osaku, Rocha, & Soccol, 2003). The lipase activity was determined using 4-nitrophenol palmitate as a substrate, according to another method described previously (McDougall, Kulkarni, & Stewart, 2009). In this case, 1 U of activity was defined as the amount of protease and lipase required to generate 1 μmole of tyrosine and p-nitrophenol from the substrate in 1 min.
2. Materials and methods 2.1. Fermentation and sampling of FGR and FGR-FBT Fermented glutinous rice (FGR) and fermented glutinous rice with Fu brick tea (FGR-FBT) were produced according to our previous study (Xu et al., 2019). Each sample, at different fermentation times, was collected every 3 h from 0 h to 48 h. All the samples were immediately submitted to further microbial and chemical analyses. 2.2. Yeast and fungi enumeration (yeast counting and fungi quantification) in FGR and FGR-FBT The enumeration of yeasts was carried out by colony counting (log10 CFU/g sample), based on a method described previously (Li, Hu, Huang, & Xu, 2018). Briefly, each sample was weighed and placed in a sterile physiological solution for 30 min at room temperature. Then, the suspension was diluted serially and plated on Yeast extract peptone dextrose agar that contained chloramphenicol for inhibiting bacterial growth. After incubation at 28 °C for 48–72 h, the yeast colonies were counted after they could be clearly distinguished by the morphology of the colony types. The quantification of fungi was determined using quantitative realtime PCR (qPCR) analysis. First, a genomic DNA Kit (TransGen Biotech, Shanghai, China) was used to extract the DNA of the fungi from the samples. After being purified, DNA was suspended in double-distilled water as a template. The qPCR mixture contained 10 μl of 2× SYBR Premix Ex Taq (Takara Bio, Japan), template DNA (2 μl) at 10 ng/ml, and 0.5 μl of each primer at 10 μM in a final volume (20 μl). The amplification was performed with universal primer for ITS1 amplicons in an ABI 7500 real-time PCR system (Applied Biosystems, USA). The reaction was initiated at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 40 s. The copy numbers of the samples were calculated using a standard curve.
2.5. Chemical analysis The pH values were measured using a digital pH meter (Schott Lab 850, Mainz, Germany). The total acid content (grammes per kilogramme of sample) was measured, using a method described previously (Jung, Lee, Lim, Kim, & Park, 2014). The total soluble solid content was expressed as Brix, measured using a refractometer (Makroo, Prabhakar, Rastogi, & Srivastava, 2019). The total alcohol content was measured according to the People's Republic of China National Standard for fermented glutinous rice (GB/T 13662-2018). 2
Food Chemistry xxx (xxxx) xxxx
X. Xu, et al.
2.6. HS-SPME extraction and GC-MS analysis
sample. During the following exponential growth phase from 9 h to 30 h, the total yeast counts increased from 5.22 ± 0.03 to 6.59 ± 0.01 log10 CFU/g for FGR, and from 5.23 ± 0.03 to 6.68 ± 0.00 log10 CFU/g for FGR-FBT. The maximum yeast populations in both samples were obtained after 42 h of fermentation in the stationary phase. However, the fermentation time (30 h) was optimized as the end of FGR-FBT fermentation, as described in our previous study (Xu et al., 2019). During the first 30 h, the yeast count in FGR-FBT was significantly higher than that in FGR. The fungi population was obtained by quantitative real-time PCR analysis. The cycle threshold values (copy numbers) and amplification curves were acquired and then the results were used to quantify the fungi levels in FGR and FGR-FBT at the beginning and end of fermentation. As shown in Fig. 1b, the fungi quantification of FGR and FGRFBT were 9.43 ± 0.09 and 9.80 ± 0.01 log10 copy number/g. An increased production of yeasts and fungi could have a positive effect on microbial metabolism, which would promote fermentation efficiency and flavour development. Fermenting FGR with FBT is therefore a potential approach to enhance the quality of traditional fermented foods by increasing the level of beneficial bioactive components, such as phenolics (Xu et al., 2019). Moreover, this multi-strain approach shortens the fermentation time, enhances the biotransformation efficiency, and leads to the creation of novel fermented foods.
The volatile compounds were extracted and qualitatively analyzed by head space solid-phase micro-extraction and gas chromatography coupled with mass spectrometry (HS-SPME-GC-MS). The parameters for HS-SPME-GC-MS were based on previous studies (Huang et al., 2018; Huang et al., 2019). Samples were prepared with 1:5 dilution and salted out with 2 g of sodium chloride. Then, SPME, coated with 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane fibre (Supelco, Inc., Bellefonte, PA, USA), was used to extract volatile compounds in a 20-ml hermetical vial with 50 °C water bath and stirring for 45 min, followed by incubation in the GC inlet for 5 min at 250 °C, and desorbing in pulsed splitless mode. A fused silica capillary column DB-5MS (60 m × 0.25 mm) was used for compound separations, as previously reported (Schueuermann et al., 2019). Briefly, the following temperature–time profile was utilized: 40 °C for 3 min; 40 to 125 °C at 3 °C/min; 125 °C for 4 min; 125 to 180 °C at 3 °C/min; 180 °C for 2 min; 180 to 220 °C at 6 °C/min; and finally, 220 °C for 3 min. The carrier gas was helium with a flow-rate of 2 ml/min. The temperature of the mass spectrometer quadrupole was set at 230 °C, the source temperature was set at 230 °C, and the transfer line temperature was set at 250 °C. All mass spectra were acquired in electron impact (EI) mode at 70 eV, using full scan mode with a scan range of 30–550 amu. The identification of all compounds was confirmed by mass spectra and retention indices. All analyses were performed in triplicate.
3.2. Isolation and identification of special culture-dependent fungi in FGRFBT
2.7. Electronic-nose data acquisition
In total, 24 fungi isolates were collected during each culture-dependent isolation from FGR-FBT and FGR. Based on the morphological and microscopical characteristics, a total of 3 special fungi was found only in FGR-FBT. These isolated strains were identified as Aspergillus cristatum, A. niger and A. oryzae through comparative analysis of physiology and molecules. As shown in Fig. 2a–c, the asexual spore-forming structure was considered as a distinct characteristic of the Aspergillus family. A reliable resolution at the species level of the isolates was carried out by the phylogenetical analysis of the ITS sequences (Fig. 2d). It is likely that the three dominant Aspergillus species identified in FBT participated in the fermentation of FGR-FBT. Previously, it has been reported that the Aspergillus spp. occupied over 90% relative abundance of all the microorganisms in post-fermented teas (Mao, Wei, Teng, Huang, & Xia, 2017). An enhanced population of fungi was also present in the FGR-FBT after 30 h of fermentation, including A. cristatum, A. niger and A. oryzae. Microorganisms in the genus Aspergillus are capable of secreting extracellular enzymes, such as amylase, cellulose and glucosidase (Handa, Couto, Vicensoti, Georgetti, & Ida, 2014). Hence, an enhanced production of yeasts and fungi has the potential for higher extracellular enzyme activities and greater fermentation efficiency.
A commercial PEN3 E-nose (Airsense Analytics GmbH, Schwerin, Germany) instrument was used to acquire information about the aromatic profile of the samples (Benedetti, Buratti, Spinardi, Mannino, & Mignani, 2008). The gas detector system consisted of 10 different metal-oxide sensors that were sensitive to aromatic compounds (W1C), broad range of sensitivity and nitrogen oxides (W5S), aromatic ammoniacal compounds (W3C), hydrogen (W6S), alkanes (W5C), methane (W1S), terpenes and sulphur compounds covering organic compounds (W1W), alcohol (W2S), sulfur organic compounds (W2W) and methanealiphatic compounds (W3S). The volatile compositions of the samples were reflected by their response values (G/G0), in which G and G0 are conductivity values of metaloxide gas sensors connected with sample gas and clean gas, respectively. Each sample (50 g) was taken and sealed in a flask with foil paper. Then, the headspace was equilibrated at 25 °C for 30 min to minimize sensor drift. After that, the headspace gas was pumped into the sensor chamber at a flow rate of 120 ml/min. The response values were then recorded for 60 s at intervals of 1 s, until reaching a stable state. Finally, the probe was cleaned for 120 s and the baseline was reset in 5 s. All the tests were performed at 25 °C. 2.8. Statistical analysis
3.3. Change of major enzyme activities during the fermentation of FGR and FGR-FBT
All the results are presented as means ± standard deviation. Oneway analysis of variance, Duncan’s multiple range tests and t-test were carried out to analyze significant differences, using SPSS version 19.0 (SPSS Inc., Chicago, IL, USA) software for Windows, where p < 0.05 was considered statistically significant.
The enzyme activities were analyzed during the whole fermentation period (Fig. 3). As shown in Fig. 3a, the highest α-amylase activities were found to be around 85.6 ± 0.9 U/g FGR-FBT at 33 h and 79.4 ± 1.2 U/g FGR at 36 h. The enzyme activity of FGR was significantly different from that exhibited by FGR-FBT, with a higher αamylase activity at each phase during the first 30 h of fermentation. After 30 h of fermentation, the α-amylase activities in FGR-FBT and FGR were 78.3 ± 1.0 U/g and 75.6 ± 1.2 U/g. The α-amylase activity impacts the efficiency of starch digestion of rice-based foodstuffs during fermentation. Previous researchers have reported that the starch structure was degraded by semi-solid fermentation and starch liquefaction was attributed to α-amylase (Liu et al., 2017). Similarly, the protease activity impacts the efficiency of protein digestion and
3. Results and discussion 3.1. Total yeast counts and fungi quantification during fermentation The evolution of the total yeast counts during fermentation of the FGR and FGR-FBT samples was measured (Fig. 1a). The yeast population was divided into three phases, ranging from the initial to terminal fermentation. During the first 9 h, the adaptive phase displayed a shortened tendency in the FGR-FBT sample compared to the FGR 3
Food Chemistry xxx (xxxx) xxxx
X. Xu, et al.
Fig. 1. Total yeast counts (a) and fungi population (b) during the fermentation progress of fermented glutinous rice with Fu brick tea (FGR-FBT) and traditional fermented glutinous rice (FGR).
biotransformation in these products, which influences the quality attributes of the fermented foods produced (Sanjukta & Rai, 2016). In our study, FGR-FBT and FGR achieved their highest protease activities of 138 ± 2.3 U/g at 24 h and 109 ± 3.7 U/g at 30 h, respectively (Fig. 3b). During the first 30 h of fermentation, the proteinase activity in FGR-FBT was much higher than that in FGR. It is noteworthy, that β-glucosidase activity was detected in FGRFBT, but not in FGR (Fig. 3d). Previous studies have reported that A. cristatum, A. niger and A. oryzae could produce β-glucosidase (Abdella, Mazeed, El-Baz, & Yang, 2016; Cao et al., 2018; Handa et al., 2014). βGlucosidase is an extracellular enzyme that can hydrolyze β-glycosidic bonds, thus leading to the release of polyphenols, as well as the modification of phenolic chemicals (Hur, Lee, Kim, Choi, & Kim, 2014). In this study, we found that there was a close relationship between the total phenolic content and β-glucosidase activity. After 15 h and 30 h of fermentation, the β-glucosidase activities in FGR-FBT were 9.73 ± 0.77 U/g and 21.9 ± 1.0 U/g, respectively. Furthermore, as shown in Fig. 3c, the lipase activities in FGR-FBT and FGR were fairly
similar, with no significant differences, which may be attributed to the low lipid content of glutinous rice. Together, α-amylase, proteinase and β-glucosidase activities in FGR-FBT were much higher than those in FGR, especially during the first 30 h of fermentation. These results suggest that the biotransformation efficiency could be enhanced by the microorganisms in FBT. This result is in agreement with a recent study, which found that a tannase that was prepared from Aspergillus niger by a solid-state fermentation of tea byproducts could effectively transform the catechin in tea infusion and improve its antioxidant activity (Ni et al., 2015). 3.4. Chemical character analysis during the fermentation of FGR and FGRFBT 3.4.1. General The quality of fermented food products depends on the metabolic processes carried out by the microorganisms present (Wang et al., 2014). In our study, three novel strains were found in FGR-FBT that
Fig. 2. The morphological and microscopial characteristics of Aspergillus niger (a), Aspergillus oryzae (b) and Aspergillus cristatellus (c) isolated from fermented glutinous rice with Fu brick tea (FGR-FBT). The neighbour joining trees (d) based on the dataset covering the sequences of ITS gene of isolates and reference strains. 4
Food Chemistry xxx (xxxx) xxxx
X. Xu, et al.
Fig. 2. (continued)
exhibited different enzyme activities. The presence of different microorganisms with different enzymatic activities would be expected to lead to fermented products with different chemical compositions. Thus, the pH, total acid contents, total soluble solids content, and alcohol content of the FGR and FGR-FBT were analyzed during the whole fermentation process.
3.4.2. pH and total titrable acidity Microbial bilateral fermentation and metabolism is known to lead to the production of several kinds of organic acids, which results in a decline in pH and an increase in total acid content. As shown in Fig. 4a and b, the production of acid occurred during the initiation period. After 15 h of fermentation, FGR-FBT and FGR had pH values of 5
Food Chemistry xxx (xxxx) xxxx
X. Xu, et al.
Fig. 3. The activities of α-amylase (a), proteinase (b), lipase (c) and β-glucosidase (d) in fermented glutinous rice with Fu brick tea (FGR-FBT) and traditional fermented glutinous rice (FGR) during the whole fermentation progress (from 12 h to 48 h).
3.77 ± 0.01 and 3.78 ± 0.01, and total titratable acid contents of 2.85 ± 0.20 and 2.53 ± 0.22 g/kg, respectively. In the first 27 h of fermentation, the acid accumulation in FGR-FBT was slightly higher than that in FGR. In contrast to the first 27 h of fermentation, the efficiency of acid production was higher in FGR during the terminal fermentation phase. Nevertheless, after 30 h of fermentation, there were no significant statistical differences in the pH values or total titrable acidity of FGR-FBT and FGR.
respectively. 3.5. Volatile compounds analyzed by HS-SPME-GC-MS In this study, referring to the retention indices and mass spectrometer analysis, 94 volatile compounds were identified in FGR-FBT, including 16 alcohols, 11 acids, 32 esters, 11 aldehydes, 3 ketones, 2 ethers, 3 furans, 2 terpenes, 4 hydrocarbons, 6 phenolic compounds, 3 aromatics and 1 nitrogenous compound, while only 59 volatile compounds were found in FGR (Table S1). Several kinds of volatile compounds (35) were only found in FGR-FBT and accounted for 8.34% of the total content, including 3 acids (3.05%), 8 esters (1.16%), 8 alcohols (1.06%) and 5 aldehydes (0.27%). This result indicates that a richer aroma with more distinctive volatile compounds was generated in FGR-FBT than in FGR. For example, linalool and linalool oxides provided a special sweet odour (like lily), as well as contributing to the other characteristic flavour notes of fermented teas. Ionone provided a unique wood-like and violet-like scent. Valencene and farnesol provided a citrus-like scent. The 59 volatile compounds common to both FGR-FBT and FGR were present at different levels in the two samples. In FGR-FBT, 2-methoxy-4vinylphenol accounted for the highest relative content (27.9%) with a roasted peanut-like and clove-like scent, followed by palmitic acid ethyl ester (25.5%) with a cheese-like scent and 4-ethyl-2-methoxyphenol (6.0%) with a sweet grass-like scent. Also, the predominant volatile compounds that contributed over 1% relative content included phenethyl alcohol (3.16%), oxalic acid (2.68%), ethanol (2.65%), ethyl oleate (2.46%), ethyl linoleate (2.40%), ethyl acetate (2.30%), p-tolualdehyde (1.77%), 3-methyl-1-butanol (1.70%), glycolic acid
3.4.3. Total soluble solid content The total soluble solid content provides valuable information about the hydrolyzation efficiency of rice starch. The reduced carbohydrates in FGR-FBT and FGR reached a peak and then decreased, by the consumption of microorganisms, after 36 h of fermentation. As shown in Fig. 4c, it took 21 h to reach the rapid growth phase for FGR-FBT, but 24 h for FGR. Moreover, the highest total soluble solid content was obtained after 30 h of fermentation for FGR-FBT (41.5 ± 0.3 °Brix), but after 36 h for FGR (41.0 ± 0.4 °Brix). The faster starch degradation and more reducing sugar accumulation of FGR-FBT suggested that an enhanced biotransformation efficiency could be obtained when fermenting FGR with FBT. 3.4.4. Ethanol content As shown in Fig. 4d, the production of ethanol mainly occurred at the beginning of the fermentation process. FGR-FBT had a higher efficiency than FBT. For instance, during the first 21 h, the ethanol accumulation for FGR-FBT was 3.33-fold greater than for FBT, indicating different microbial metabolisms. After 30 h of fermentation, the ethanol contents in FGR-FBT and FGR were 1.82 ± 0.10 and 1.22 ± 0.11, 6
Food Chemistry xxx (xxxx) xxxx
X. Xu, et al.
Fig. 4. Chemical properties analysis of pH (a), total acid content (b), total soluble solid content (c) and ethanol content (d) in fermented glutinous rice with Fu brick tea (FGR-FBT) and traditional fermented glutinous rice (FGR) during the whole fermentation progress (from 15 h to 48 h).
presence of these volatile compounds has been attributed to the metabolism by the genus Aspergillus, which accounts for a considerable proportion of the fungi in fermentation starters (Xu et al., 2015). Ethanol accounted for 2.42% in FGR and 2.65% in FGR-FBT. Furthermore, butyl alcohol and hexyl alcohol accounted for 4.12% and 0.58% in FGR, while only 0.10% and 0.05% of them were detected in FGRFBT. Therefore, a softer aroma would be expected in FGR-FBT because lower levels of the irritants butyl alcohol and hexyl alcohol were generated. In addition, a wider range of alcohols was formed in FGR-FBT, e.g. as 2-methyl-1-pentanol and linalool, which generate grass-like and rose-like fresh scents. Moreover, eight kinds of volatile acids were detected in FGR (7.08% of total) and 11 kinds of volatile acids were detected in FGR-FBT (6.21% of total). Besides, higher relative contents of furans (burnt sugar-like and grass-like scents) and terpenes (a citrus-like scent) were detected in FGR-FBT. In addition, the relative contents of saturated hydrocarbons in FGR-FBT and FGR presented no significant differences (p > 0.01), while the relative content of unsaturated hydrocarbons was significantly higher in FGR-FBT, with a fresh tea-like scent (p < 0.01). Together, distinctive aromatic properties of FGR-FBT may derive from the special volatile compounds from FBT and various microorganism metabolisms. Hence, the fermentation with FBT could improve the sensory properties of FGR.
(1.68%), lactamide (1.50%) and ethyl laurate (1.12%). In FGR, the most abundant compound was palmitic acid ethyl ester, accounting for a relative content of 34.0%, which was higher than that in FGR-FBT (25.5%). The predominant volatile compounds found in FGR included phenethyl alcohol, ethyl myristate, 4-hydroxy-3-methoxystyrene and pentanol (4.12%). Esters were crucial because of their highest total relative content and wide variety. There were 24 esters in FGR (occupying 59.4%) and 32 esters in FGR-FBT (occupying 43.5%). Among them, palmitic acid ethyl ester was abundant in FGR and FGR-FBT, followed by ethyl myristate, ethyl oleate, octyl 4-meth-oxycinnamate, ethyl acetate, ethyl laurate, pentadecanoic acid ethyl ester and isoamyl acetate, which were considered as metabolites biotransformed by bacteria and yeasts at the terminal fermentation phase. Esters are considered to contribute an irreplaceable part of the fruity and flowery aroma of Chinese rice wine (Yang et al., 2017). Moreover, the composition of the esters formed in fermented foods has been reported to be distinctive of the nature of the microbes present (Yu, Xie, Xie, Ai, & Tian, 2019). Esters present at relatively low concentrations would also be expected to contribute to the rich aroma of fermented foods. The methyl salicylate, phenylacetic acid ethyl ester, and dihydroactinidiolide were detected at levels of 0.15%, 0.02% and 0.10% of FGR-FBT, which produced honey-like sweet and kiwifruit-like fruity aromas. Phenolic compounds occupied the second highest total relative content (16.7% in FGR and 37.6% in FGR-FBT), followed by alcohols and acids. Several phenolic compounds in FBT were metabolized by microorganisms. The relative contents of phenolic compounds in FGRFBT were higher than those in FGR, such as 4-butyl-2,6-ditert-butylphenol. In this study, a total of 8 alcohols (10.5%) and 16 alcohols (6.36%) were identified in FGR and FGR-FBT, respectively. The
3.6. E-nose analysis The signals of ten sensors in response to aromatic compounds of FGR and FGR-FBT were expressed by G0/G values (Fig. 5a and b). All the signals reached a stable state after 50 s, and so the response curves were represented as the averages of multiple values measured at 55 s. Sensors W2S, W1S, W2W and W1W had the highest response to the 7
Food Chemistry xxx (xxxx) xxxx
X. Xu, et al.
Fig. 5. G/G0 values of ten sensors detected by electronic nose (E-nose) in response to the odour of fermented glutinous rice with Fu brick tea (FGR-FBT) (a) and traditional fermented glutinous rice (FGR) (b). Principal component analysis (c) and linear discriminant analysis (d) were performed, based on the E-nose detection and analysis.
volatile compounds in FGR and FGR-FBT. The signals detected by these sensors were higher for FGR-FBT than for FGR, which is indicative of high levels of volatile alcohol and alkane compounds in the aroma profile. FGR-FBT also gave higher G0/G responses for the W2W, W1C and W5C sensors, which suggests that there were more aromatic, sulfurchlorine, and alkene compounds generated. Based on a loading analysis of the correlations of ten sensors, the W2S and W1S sensors were able to differentiate the volatile compositions at the highest sensitivity, followed by W2W and W1W, which responded to aromatic and organic compounds. According to principal component analysis and linear discriminant analysis of the FGR and FGR-FBT profiles, there were significant aromatic differences between the samples, which would be expected to lead to different sensory properties (Fig. 5c and d). Differences in the nature of the volatile compounds formed when FBT was present during fermentation can be attributed to differences in microbial metabolism. Previous researchers reported that the quality of Baijiu flavour was influenced by microbial interactions, and using mixed cultures improved the flavour metabolism by yeasts (Zha, Sun, Wu, Yin, & Wang, 2018). Co-culture yeasts have also been reported to provide a synergistic effect on metabolic pathways, leading to increased aroma complexity (Sadoudi et al., 2012). Also, mixed inoculation with various ratios of different strains has been shown to reduce the content of tannin-protein complexes formed, thereby leading to decreased astringency (Kang, Kang, Lee, & Chang, 2018). The metabolism of Saccharomyces cerevisiae was changed by lactic acid bacteria during the fermentation of Chinese liquor, thus resulting in altered levels of various kinds of flavour compounds (Meng et al., 2015). Combining chemical analysis, GC-MS and E-nose, we verified that FGR-FBT had a higher ethanol content, acidity, and more abundant and diverse aromatic compounds, along with special microbial properties and a higher population of yeasts and fungi.
4. Conclusion In this study, fermented glutinous rice (FGR) was produced using a multi-starter fermentation technique that relied on the diverse microbial cultures naturally found in Fu brick tea (FBT). This innovative fermentation approach led to the production of a novel functional food product (FGR-FBT) with unique sensory properties. Different fungi were isolated from the FGR-FBT and identified as Aspergillus cristatum, A. niger and A. oryzae. The increased levels of yeast and fungi present in FGR-FBT led to enhanced α-amylase, proteinase and β-glucosidase activities, which increased the fermentation efficiency. Moreover, the utilization of the multi-starter fermentation technique led to differences in the chemical composition and aroma profile of the final product. In particular, a richer sensory profile was found for the FGR-FBT compared to the FGR. This study may therefore lead to a new range of highquality fermented food products based on the use of Fu brick tea and glutinous rice. Author contributions Mingsheng Dong conceived the project idea and obtained funding. Xiao Xu was assigned the project, conducted the research, wrote and revised this manuscript. David Julian McClements gave constructive suggestions on the content and revised the manuscript. Siduo Zhou and other authors participated in the method optimization of chemical analysis and revised the manuscript. All the authors approved the final manuscript drafts. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 8
Food Chemistry xxx (xxxx) xxxx
X. Xu, et al.
Acknowledgement
Makroo, H. A., Prabhakar, P. K., Rastogi, N. K., & Srivastava, B. (2019). Characterization of mango puree based on total soluble solids and acid content: Effect on physicochemical, rheological, thermal and ohmic heating behavior. LWT-Food Science and Technology, 103, 316–324. Mao, Y., Wei, B., Teng, J., Huang, L., & Xia, N. (2017). Analyses of fungal community by Illumina MiSeq platforms and characterization of Eurotium species on Liupao tea, a distinctive post-fermented tea from China. Food Research International, 99, 641–649. Marsh, A. J., O'Sullivan, O., Hill, C., Ross, R. P., & Cotter, P. D. (2014). Sequence-based analysis of the bacterial and fungal compositions of multiple kombucha (tea fungus) samples. Food Microbiology, 38(2), 171–178. McDougall, G. J., Kulkarni, N. N., & Stewart, D. (2009). Berry polyphenols inhibit pancreatic lipase activity in vitro. Food Chemistry, 115(1), 193–199. Meng, X., Wu, Q., Wang, L., Wang, D., Chen, L., & Xu, Y. (2015). Improving flavor metabolism of Saccharomyces cerevisiae by mixed culture with Bacillus licheniformis for Chinese Maotai-flavor liquor making. Journal of Industrial Microbiology & Biotechnology, 42(12), 1601–1608. Mondal, K. C. (2016). Folk to functional: An explorative overview of rice-based fermented foods and beverages in India. Journal of Ethnic Foods, 3(1), 5–18. Ni, H., Chen, F., Jiang, D., Cai, M., Yang, Y., Xiao, A., & Cai, H. (2015). Biotransformation of tea catechins using Aspergillus niger tannase prepared by solid state fermentation on tea byproduct. LWT – Food Science and Technology, 60(2), 1206–1213. Sadoudi, M., Tourdot-Marechal, R., Rousseaux, S., Steyer, D., Gallardo-Chacon, J.-J., Ballester, J., ... Alexandre, H. (2012). Yeast-yeast interactions revealed by aromatic profile analysis of Sauvignon Blanc wine fermented by single or co-culture of nonSaccharomyces and Saccharomyces yeasts. Food Microbiology, 32(2), 243–253. Sanjukta, S., & Rai, A. K. (2016). Production of bioactive peptides during soybean fermentation and their potential health benefits. Trends in Food Science & Technology, 50, 1–10. Schueuermann, C., Steel, C. C., Blackman, J. W., Clark, A. C., Schwarz, L. J., Moraga, J., ... Schmidtke, L. M. (2019). A GC-MS untargeted metabolomics approach for the classification of chemical differences in grape juices based on fungal pathogen. Food Chemistry, 270, 375–384. Su, E., Xia, T., Gao, L., Dai, Q., & Zhang, Z. (2010). Immobilization of beta-glucosidase and its aroma-increasing effect on tea beverage. Food and Bioproducts Processing, 88(C2–3), 83–89. Wang, P., Mao, J., Meng, X., Li, X., Liu, Y., & Feng, H. (2014). Changes in flavour characteristics and bacterial diversity during the traditional fermentation of Chinese rice wines from Shaoxing region. Food Control, 44, 58–63. Wu, Z., Xu, E., Long, J., Pan, X., Xu, X., Jin, Z., & Jiao, A. (2016). Comparison between ATR-IR, Raman, concatenated ATR-IR and Raman spectroscopy for the determination of total antioxidant capacity and total phenolic content of Chinese rice wine. Food Chemistry, 194, 671–679. Xu, A., Wang, Y., Wen, J., Liu, P., Liu, Z., & Li, Z. (2011). Fungal community associated with fermentation and storage of Fuzhuan brick-tea. International Journal of Food Microbiology, 146(1), 14–22. Xu, E., Long, J., Wu, Z., Li, H., Wang, F., Xu, X., ... Jiao, A. (2015). Characterization of volatile flavor compounds in Chinese rice wine fermented from enzymatic extruded rice. Journal of Food Science, 80(7), 1476–1489. Xu, J., Hu, F.-L., Wang, W., Wan, X.-C., & Bao, G.-H. (2015). Investigation on biochemical compositional changes during the microbial fermentation process of Fu brick tea by LC–MS based metabolomics. Food Chemistry, 186, 176–184. Xu, X., Hu, W., Zhou, S., Tu, C., Xia, X., Zhang, J., & Dong, M. (2019). Increased phenolic content and enhanced antioxidant activity in fermented glutinous rice supplemented with Fu brick tea. Molecules, 24(4), 671. Yang, Y., Xia, Y., Lin, X., Wang, G., Zhang, H., Xiong, Z., ... Ai, L. (2018). Improvement of flavor profiles in Chinese rice wine by creating fermenting yeast with superior ethanol tolerance and fermentation activity. Food Research International, 108, 83–92. Yang, Y., Xia, Y., Wang, G., Yu, J., & Ai, L. (2017). Effect of mixed yeast starter on volatile flavor compounds in Chinese rice wine during different brewing stages. LWT-Food Science and Technology, 78, 373–381. Yu, H., Xie, T., Xie, J., Ai, L., & Tian, H. (2019). Characterization of key aroma compounds in Chinese rice wine using gas chromatography-mass spectrometry and gas chromatography-olfactometry. Food Chemistry, 293, 8–14. Zha, M., Sun, B., Wu, Y., Yin, S., & Wang, C. (2018). Improving flavor metabolism of Saccharomyces cerevisiae by mixed culture with Wickerhamomyces anomalus for Chinese Baijiu making. Journal of Bioscience and Bioengineering, 126(2), 189–195. Zhang, L., Zhang, Z.-Z., Zhou, Y.-B., Ling, T.-J., & Wan, X.-C. (2013). Chinese dark teas: Postfermentation, chemistry and biological activities. Food Research International, 53(2), 600–607.
This work was supported by the Earmarked Fund for Jiangsu Agricultural Industry Technology System (No. JATS-2018-296). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2019.125790. References Abdella, A., Mazeed, T. E.-S., El-Baz, A. F., & Yang, S.-T. (2016). Production of β-glucosidase from wheat bran and glycerol by Aspergillus niger in stirred tank and rotating fibrous bed bioreactors. Process Biochemistry, 51(10), 1331–1337. Benedetti, S., Buratti, S., Spinardi, A., Mannino, S., & Mignani, I. (2008). Electronic nose as a non-destructive tool to characterise peach cultivars and to monitor their ripening stage during shelf-life. Postharvest Biology & Technology, 47(2), 181–188. Cao, L., Guo, X., Liu, G., Song, Y., Ho, C.-T., Hou, R., ... Wan, X. (2018). A comparative analysis for the volatile compounds of various Chinese dark teas using combinatory metabolomics and fungal solid-state fermentation. Journal of Food and Drug Analysis, 26(1), 112–123. de Lima, F. S., Kurozawa, L. E., & Ida, E. I. (2014). The effects of soybean soaking on grain properties and isoflavones loss. LWT-Food Science and Technology, 59(2), 1274–1282. Germano, S., Pandey, A., Osaku, C. A., Rocha, S. N., & Soccol, C. R. (2003). Characterization and stability of proteases from Penicillium sp produced by solid-state fermentation. Enzyme and Microbial Technology, 32(2), 246–251. Gupta, R., Gigras, P., Mohapatra, H., Goswami, V. K., & Chauhan, B. (2003). Microbial alpha-amylases: A biotechnological perspective. Process Biochemistry, 38(11), 1599–1616. Handa, C. L., Couto, U. R., Vicensoti, A. H., Georgetti, S. R., & Ida, E. I. (2014). Optimisation of soy flour fermentation parameters to produce beta-glucosidase for bioconversion into aglycones. Food Chemistry, 152, 56–65. Huang, Z. R., Guo, W. L., Zhou, W. B., Li, L., Xu, J. X., Hong, J. L., ... Lv, X. C. (2019). Microbial communities and volatile metabolites in different traditional fermentation starters used for Hong Qu glutinous rice wine. Food Research International, 121, 593–603. Huang, Z. R., Hong, J. L., Xu, J. X., Li, L., Guo, W. L., Pan, Y. Y., ... Lv, X. C. (2018). Exploring core functional microbiota responsible for the production of volatile flavour during the traditional brewing of Wuyi Hong Qu glutinous rice wine. Food Microbiology, 76, 487–496. Hur, S. J., Lee, S. Y., Kim, Y.-C., Choi, I., & Kim, G.-B. (2014). Effect of fermentation on the antioxidant activity in plant-based foods. Food Chemistry, 160, 346–356. Jung, H., Lee, S. J., Lim, J. H., Kim, B. K., & Park, K. J. (2014). Chemical and sensory profiles of makgeolli, Korean commercial rice wine, from descriptive, chemical, and volatile compound analyses. Food Chemistry, 152(2), 624–632. Kang, M.-S., Kang, Y.-R., Lee, Y., & Chang, Y. H. (2018). Effect of mixed culture inoculation on chemical and sensory properties of aronia (Aronia melanocarpa). LWTFood Science and Technology, 98, 418–423. Kurtzman, C. P., Fell, J. W., Boekhout, T., & Robert, V. (2011). Chapter 7 - Methods for Isolation, Phenotypic Characterization and Maintenance of Yeasts. In: C. P. Kurtzman, J. W. Fell & T. Boekhout (Eds.), The Yeasts (Fifth Edition), (pp. 87–110). Lee, D. E., Lee, S., Singh, D., Jang, E. S., Shin, H. W., Moon, B. S., & Lee, C. H. (2017). Time-resolved comparative metabolomes for Koji fermentation with brown-, white-, and giant embryo-rice. Food Chemistry, 231, 258–266. Li, J., Hu, W., Huang, X., & Xu, Y. (2018). Investigation of yeast population diversity and dynamics in spontaneous fermentation of Vidal blanc icewine by traditional culturedependent and high-throughput sequencing methods. Food Research International, 112, 66–77. Liu, L., Zhang, R., Deng, Y., Zhang, Y., Xiao, J., Huang, F., ... Zhang, M. (2017). Fermentation and complex enzyme hydrolysis enhance total phenolics and antioxidant activity of aqueous solution from rice bran pretreated by steaming with alpha-amylase. Food Chemistry, 221, 636–643. Lv, X. C., Weng, X., Zhang, W., Rao, P. F., & Li, N. (2012). Microbial diversity of traditional fermentation starters for Hong Qu glutinous rice wine as determined by PCRmediated DGGE. Food Control, 28(2), 426–434.
9