A study revealing volatile aroma produced by Pediococcus pentosaceus in dough fermentation

Abstract Pediococcus pentosaceus is important probiotics in Chinese Laomian. Its role in meat and fermented vegetable has been largely demonstrated, but few studies have investigated the role of P. pentosaceus in Chinese Laomian. For this purpose, we simulated Laomian fermentation using Saccharomyces cerevisiae and P. pentosaceus. Volatile aroma was detected by headspace solid‐phase microextraction gas–chromatography‐mass spectrometry. Real‐time fluorescent quantitative polymerase chain reaction was used to determine dynamic growth of S. cerevisiae and P. pentosaceus in fermentation. Extracellular proteins were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Folin–Ciocalteu method was used to detect extracellular protease activity in different pH values. Owing to addition of P. pentosaceus, the types and contents of esters increase, the relative contents of acetic acid hexyl ester, formic acid octyl ester, and heptanoic acid ethyl ester rise obliviously; especially, the relative content of hexanoic acid ethyl ester was highly correlated with P. pentosaceus by increasing 20.61%. As the gel electrophoresis results display, due to mixed fermentation of S. cerevisiae and P. pentosaceus, the 25k Da and 51k Da proteins expression quantity of P. pentosaceus clearly increased. Under neutral and alkaline culture conditions, the extracellular protease activity of P. pentosaceus is higher. This research benefits to gain insight into the fermentation actions of P. pentosaceus in Chinese Laomian.

eight samples from 13 Chinese traditional Laomian). Pediococcus pentosaceus were commonly found in dairy products (Yu, Chen, Tao, Bo, & Mu, 2015), meat products (Anastasiadou, Papagianni, Filiousis, Ambrosiadis, & Koidis, 2008), and vegetable fermentation products (Xu, Wenshui, Fang, & Xiaohua, 2010), and mainly helpful to the quality, safety, flavor, and shelf life of fermented products (Kwon, Jo, & Park, 2018). But few studies reported action of P. pentosaceus in the dough fermentation. In this study, we systematically studied the action mechanism of P. pentosaceus in the fermentation dough by adding S. cerevisiae (S. cerevisiae) alone or S. cerevisiae and P. pentosaceus joint as a starter to simulate the dough fermentation. Headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS) was used as a detection method of volatile aroma substances to analyze the contribution of P. pentosaceus to the fermentation aroma. Realtime fluorescent quantitative polymerase chain reaction (RT-qPCR) was used to detect the dynamic changes of S. cerevisiae and P. pentosaceus during the simulated fermentation process to explore their interaction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) protein gel electrophoresis was used to analyze extracellular proteins, and Folin-Ciocalteu method was used to detect extracellular protease activity. Therefore, this study benefits to explore the interaction between S. cerevisiae and P. pentosaceus, and gain insight of the fermentation characteristics of P. pentosaceus.

| Materials
Saccharomyces cerevisiae and P. pentosaceus were obtained from our laboratory isolated from Chinese Laomian (sourdoughs), their cultures were maintained and revived as described by Handbook of Culture Media for Food Microbiology ("de man, rogosa, and sharpe (MRS) agar," 2003). Saccharomyces cerevisiae and P. pentosaceus were cultured to mid-log growth phase by yeast extract peptone dextrose (YPD) and de Man, Rogosa and Sharpe (MRS) liquid media, the cells were collected by centrifuge, washed twice with normal saline, resuspended in normal saline, and respectively adjusted by spectrophotometry to 1.00/0.40 (OD 600nm ), and final cell quantities were confirmed by plate culture.

| Simulated dough fermentation
To simulate fermentation process of the Chinese traditional Laomian, we prepared 10% (w/v) sterile flour solution (SFS) as flour fermented system, 10% (w/v) SFS preparation are following: 100 g of flour added into 1,000 ml distilled water, stirred evenly and sterilized at 121°C for 20 min. The simulated fermentation group consisted of 20 ml of SFS and 1 ml strains solution including S. cerevisiae alone or S. cerevisiae and P. pentosaceus joint and then incubated to double start volume. Each test was repeated three times independently, and each time has three repeats.

| Determination of aroma in simulated fermentation group
Volatile aroma of fermented dough was detected by HS-SPME-GC-MS. Firstly, a Supelco fiber was conditioned according to the manufacturer's instructions. Secondly, after equilibrating at 40°C for 20 min, the fiber was exposed to 5.00 g of simulated fermentation group and 5 ml sterile water in a 20-ml glass vial containing a microstirring bar for 40-min extraction. In the end, after volatiles were extracted, the fiber was inserted into the gas chromatography (GC) injector at 250°C to desorb the analytes for 5 min (Qiao et al., 2012).
Volatile aroma substances were subjected to GC analysis coupled to a mass spectrometer. The mass spectrometer was operated in an electron ionization mode at a voltage of 70 eV. The flow rate of helium on the HP-5MS column was 1.0 ml/min. Analysis was conducted in a splitless mode. The column was initially maintained at 40°C for 3 min; temperature was then increased from 40°C to 160°C at 3°C/min speed and finally increased to 220°C at a rate of 8°C/ min. Temperature was maintained at 220°C for 3 min. The retention index of each chromatographic peak was calculated using the retention time of the C6-C25 n-alkanes. The analysis was performed by using computer spectra (NIST05/WILE7.0) for preliminary search and data analysis, combined with the retention index of the literature for comparison and manual spectrometer analysis to confirm the composition of volatile substances.

| The population dynamic change of S. cerevisiae and P. pentosaceus during fermentation
The cell dynamic concentrations were detected by RT-qPCR to explore the interaction between S. cerevisiae and P. pentosaceus during the simulated fermentation process. RT-qPCR and standard curves were carried out using the previous count methods with some small modification (Pontonio et al., 2017). The standard curves of S. cerevisiae and P. pentosaceus were constructed using the quantification cycle (Ct) versus different cell numbers of strains. During fermentation progress, each 4 hr, 10 g of simulated fermentation dough was removed into 90 ml of sterile distilled water, followed by mixing and centrifuging at 300 × g for 10 min, 1 ml of supernatant was transferred to a new tube and centrifuged for 10 min (4°C, 10,000 × g), the pellets were collected and washed twice by sterile water for DNA extraction. Each strain concentration was calculated by RT-qPCR assay, each test was repeated three times independently, and each time has three repeats.

| Extracellular protein composition of S. cerevisiae and P. pentosaceus by SDS-PAGE
To investigate the roles of extracellular protein during dough fermentation, deoxycholate-trichloroacetic acid(TCA-DOC) method was used to extract extracellular proteins with some light improvement (Wang, He, Huang, & Qingmei, 2014). In brief, the strains were cultured in 400 ml MRS broth at 5% inoculum (w/w) overnight at 37°C, centrifuged (3,000 × g, 10 min) to collect the supernatant, and 40 ml 2% (w/w) DOC solution was added and kept for 30 min at 4°C. TCA was added to form final 6% TCA solution, and the solution was allowed to stand at 4°C for 4 hr and centrifuged (4°C, 10,000 × g, 15 min) to collect the precipitate. The precipitate was ultrasonically washed three times with 10 ml ice acetone (stored at −20°C), naturally air-dried at room temperature in the end. All samples were adjusted to a uniform total concentration by quick start bradford protein assay kit (Bio-Rad) using bovine serum albumin (BSA) as a standard. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried using 12% resolving gel and 5% stacking gel. Standard molecular weight makers (Solarbio) were used to estimate the molecular weight of the extracellular protein (Yanshun, Dai, Zang, Jiang, & Xia, 2015). Each test was repeated three times independently, and each time has three repeats.

| Extractcellular protease activity of P. pentosaceus
The extracellular protease activity was quantified under different pH conditions as described by Bhaskar et al. with minor modification (Bhaskar, Sudeepa, Rashmi, & Tamil Selvi, 2007). In brief, protease activity of P. pentosaceus was determined using casein as substrate at 40°C according to the instructions of lowry protein concentration determination kit. One unit of proteolytic activity (U) was defined as microgram tyrosine liberated per min of the enzyme extract. Specific activity was expressed as units per miligram protein (U/mg protein) of the enzyme extract. The standard curve of extracellular protease activity was generated by plotting the absorbance (OD 680nm ) versus different concentrations of casein (μg/mL). The P. pentosaceus cells were washed twice with normal saline and adjusted to 10 9 CFU/mL as inoculum. 500 μl of P. pentosaceus was respectively inoculated into 10 ml MRS medium in pH 5, 6, 7 and 8) and incubated at 37°C for 24 hr, followed by centrifuging at 10,000 × g for 15 min to separate the biomass from supernatant. The supernatant was then collected and filtered through a 0.2-μm cellulose acetate membrane to obtain cell-free supernatant (CFS), which was used as extracellular enzyme for the determination of protease activity. Each test was repeated three times independently, and each time has three repeats.

| Statistical analysis
Microbiological data were log transformed before statistical treatment. The data were expressed as the means ± standard error of biological triplicates. A completely randomized design was used to plan the flavor study.

| Aroma analysis produced by simulated fermentation groups
In order to detect the contribution of P. pentosaceus to aroma substances in the fermented dough, we simulated fermentation dough by S. cerevisiae alone or S. cerevisiae and P. pentosaceus joint as starter, and HS-SPME-GC-MS was performed to detect the aroma substances. The ( Figure S1) showed the 2-pentylfuran, a fruit-aroma substance (Kati, Kaisa, & Karin, 2007), was detected at extremely high level in S. cerevisiae alone dough, the content of hexanoic acid ethyl ester and 1-pentanol increased obviously in S. cerevisiae and P. pentosaceus joint fermentation dough. The detailed results were shown in Table 1, there were total 19 kinds of aroma substances detected in S. cerevisiae alone fermented dough, and 34 volatile aroma substances in jointly fermented dough. Firstly, alcohols include 1-pentanol, 1-hexanol, 1-octen-3-ol, 1-octanol and phenylethyl alcohol, which appear in the both fermented doughs. However, the relative content of 1-pentanol obviously increased and the others lessen due to the addition of P. pentosaceus. Secondly, three kinds of organic acids were also detected in both, and the relative contents of volatile organic acids decreased slightly because of the addition of P. pentosaceus. Thirdly, the main differences of volatile aroma were the contents and types of esters, hydrocarbons, and other substances. In S. cerevisiae alone fermentation dough, only six kinds of esters were detected and their contents are relatively low, with the addition of P. pentosaceus, additional eight kinds of esters were detected. To our surprise, the relative content of hexanoic acid ethyl ester dramatically increases from 9.12% to 29.73%, while 2-pentylfuran significantly decreases from 26.71% to 2.8%. Generally, due to the addition of P. pentosaceus, the contents and types of volatile compounds esters increased and organic acids contents weakly declined. 2-pentylfuran is a characteristic aroma substance of S. cerevisiae, and hexanoic acid ethyl ester is a characteristic aroma of P. pentosaceus.
TA B L E 1 The aroma compounds detected by HS-SPME-GC-MS

| The population dynamic change of S. cerevisiae and P. pentosaceus during fermentation
In order to gain insights into the kinetic interaction between S. cerevisiae and P. pentosaceus during the simulated fermentation, RT-qPCR was used to monitor the cell quantities of S. cerevisiae and P. pentosaceus. For this propose, DNA was prepared from similar amounts of sourdough taken at different time points during fermentation. Amplification curves showed reproducible differences between the amount of strains special nucleic acid fragments, and the calculation results show that the correlation coefficients (r 2 ) of the standard curves were 0.96 for S. cerevisiae and 0.97 for P. pentosaceus, which prove good correlations. Their growth trends were plotted. As showed in Figure 1a, when only S. cerevisiae was used as fermentation agent, the cell concentrations of S. cerevisiae con-

| Extracellular protein composition S. cerevisiae and P. pentosaceus
To investigate the aroma forming mechanism by P. pentosaceus, extracellular protease activity is a very important indicator. To detect extracellular proteins of P. pentosaceus, we extracted the whole extracellular proteins and used SDS-PAGE to isolate the protein spectra, the protein gel electrophoresis results were shown in Figure 2. Pediococcus pentosaceus usually has two important functions in the food field, one is to extent shelf life of food by producing antimicrobial peptides such as bacteriocin or pediocin and others (Kwon et al., 2018;Michalska et al., 2007), and another is to produce protease to improve flavors. Our study displays that P. pentosaceus can produce more extracellular proteins of 51.1k Da due to being induced by S. cerevisiae, which may be consistent with LysM peptidoglycan binding domain proteins based on the molecular weight, can inhibit the adhesion of pathogenic bacteria (Sun, Chen, & Wang, 2019 (Yanshun et al., 2015). Therefore, we speculate that the 25 k Da of extracellular protein in this study may be same type of protease. It is certain that the secondary metabolites produced by S. cerevisiae caused P. pentosaceus to increase the 25k Da and 51.1k Da protein expression of or increase the transport efficiency, but mechanisms in details need to further study using proteomics.

| Extracellular protease activity of P. pentosaceus in different pH conditions
Dough fermentation often appeared acidity; in order to research extracellular protease activity of P. pentosaceus in different pH conditions, we used Folin-Ciocalteu method to detect extracellular protease activity. The result exhibits that correlation coefficients (r 2 ) of the standard curve is 0.9994, which means an excellent linear relationship. Figure 3  Traditional Laomian is a kind of fermentation agent naturally selected by environmental microorganisms, mixed with dough in the amount of 10%-30%, generally. The selection of sourdough microbiota occurs spontaneously during daily refreshment (also called backslopping) (Taccari et al., 2016). Therefore, the whole system is weakly alkaline in the initial stage of traditional Laomian fermentation. Our research results show that the activity of P. pentosaceus extracellular protease was higher in both neutral and weakly alkaline environments, which also reveals the reason for its increasing cell concentration in the early stages of fermentation. As the fermentation time increases, P. pentosaceus constantly produces acid, and the accumulation of organic acids causes the overall pH to decrease continuously. As the pH value decreases, protease activity continues to decrease, especially at pH 6. The occurrence of P. pentosaceus reflects its adaptability to the prevailing Laomian fermentation conditions. Pediococcus pentosaceus extracellular proteases play a role in breaking down the macromolecules proteins into amino acids which is also precursor of esters (Li & Wang, 2013), so that the fermentation dough with S. cerevisiae and P. pentosaceus has richer volatile aroma substances. China is a great country of bread consumption (Huang, 2014), to our knowledge, it is the first time to study the aroma substances associated with mechanism of P. pentosaceus in fermentation dough by researching dynamic changes, extracellular proteins and extracellular protease activity, and our research will benefit to explore the fermentation characteristics of P. pentosaceus from Chinese traditional Laomian and develop some new type joint fermentation starter.

| CON CLUS ION
Pediococcus pentosaceus is important probiotics and widely exists in Chinese Laomian. Simulated fermentation shows that addition of P. pentosaceus results in significant increase in the type and content of esters in fermented dough. Additional eight kinds of esters were produced, and among them, the concentrations of acetic acid hexyl ester, formic acid octyl ester, and heptanoic acid ethyl ester rise drastically. Especially, the most dramatic changes in content are hexanoic acid ethyl ester, and the concentration increases from 9.12% to 29.73%. Due to nutritional competition and microbial growth habits, the cell concentration of S. cerevisiae decreases slightly by 0.4 log 10 CFU/ml; the cell concentration of P. pentosaceus increases by 1.9 log 10 CFU/ml in the end of the simulated fermentation, the metabolic activity of S. cerevisiae provided nutrients for the growth of P. pentosaceus. With the addition of S. cerevisiae, the 25 k Da and 51 k Da extracellular proteins quantities of P. pentosaceus increase significantly. The extracellular protease activity of P. pentosaceus was best at pH 7, which was 1,039.71 U/ml. As the pH value decreases, the extracellular protease activity continues to decrease. However, the decrease rate is fast. In conclusion, with the addition of S. cerevisiae, the expression of P. pentosaceus extracellular proteases increases during the fermentation process, resulting in a dramatic increase in the volatile aroma substances. As the fermentation continues, the cell concentration of S. cerevisiae decreases slightly, and the secondary metabolites of S. cerevisiae provide nutrients for the growth of P. pentosaceus. This research systematically explores the fermentation characteristics of P. pentosaceus in Chinese traditional Laomian and benefits the development of Chinese traditional Laomian bread.

AUTH O R CO NTR I B UTI O N S
Yunxia Gong involved on experimental research and writing-original draft preparation. Xiaobao Qi involved in design, formal analysis and writing-review and editing.

E THI C AL S TATEMENT
The authors declare that they had no competing interests. The authors declare that there is no study involving human/animals subjects.