Accepted Manuscript Analysis of proteomic responses of freeze-dried Oenococcus oeni to access the molecular mechanism of acid acclimation on cell freeze-drying resistance Kun Yang, Yang Zhu, Yiman Qi, Tingjing Zhang, Miaomiao Liu, Jie Zhang, Xinyuan Wei, Mingtao Fan, Guoqiang Zhang PII: S0308-8146(19)30188-8 DOI: https://doi.org/10.1016/j.foodchem.2019.01.120 Reference: FOCH 24215 To appear in: Food Chemistry Received Date: 2 October 2018 Revised Date: 24 December 2018 Accepted Date: 17 January 2019 Please cite this article as: Yang, K., Zhu, Y., Qi, Y., Zhang, T., Liu, M., Zhang, J., Wei, X., Fan, M., Zhang, G., Analysis of proteomic responses of freeze-dried Oenococcus oeni to access the molecular mechanism of acid acclimation on cell freeze-drying resistance, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem. 2019.01.120 This is a PDF file of an unedited manuscript that has been accepted for publication. 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Analysis of proteomic responses of freeze-dried Oenococcus oeni to access the molecular mechanism of acid acclimation on cell freeze-drying resistance Kun Yang1,2, Yang Zhu3, Yiman Qi2,Tingjing Zhang4, Miaomiao Liu2, Jie Zhang2, Xinyuan Wei2, Mingtao Fan2,*, Guoqiang Zhang1,* 1 College of Biological and Chemical Engineering, Anhui Polytechnic University, Wuhu, 241000, China 2 College of Food Science and Engineering, Northwest A ＆ F University, Yangling, 712100, China 3 School of Agriculture and Food Sciences, University of Queensland, QLD, 4046, Australia 4 College of Food Science and Technology, Henan University of Technology, Zhenzhou, 450001, China * Corresponding author: 1. Tel.: +86-13618940517; E-Mail: [email protected] (Guoqiang Zhang). * Co-Corresponding author: 2. Tel.: +86-13892877726; E-Mail: [email protected] (Mingtao Fan) 1 Abstract Malolactic fermentation (MLF), usually induced by Oenococcus oeni (O. oeni), is an important process to improve wine quality. Acid acclimation has been proven to be useful for enhancing the viability of lyophilized O. oeni. To explain the involved mechanisms, cell integrity, morphology and protein patterns of lyophilized O. oeni SD-2a were investigated with acid acclimation. After lyophilization, improvement of cell integrity and more extracellular polymeric substances (EPS) were observed in acid acclimated cells. Combined with GO and KEGG analysis, different abundant proteins were noticeably enriched in the carbohydrate metabolism process, especially amino sugar and nucleotide sugar metabolism. The most significant result was the over-expression of proteins participating in cell wall biosynthesis, EPS production, ATP binding and the bacterial secretion system. This result indicated the important role of acid acclimation on cell envelope properties. In addition, protein response to stress and arginine deiminase pathway were also proven to be over- expressed. Keywords: Oenococcus oeni; Acid acclimation; Freeze-drying resistance; Cross-protection; Comparative proteomics. 1. Introduction During wine making, the alcoholic fermentation (AF) by yeast and the following process of malolactic fermentation (MLF) are usually promoted with starter cultures to make sure a controlled and efficient process. MLF is crucial for wine making, as it deacidifies wine by the conversion of malate to lactate, prevents the growth of spoilage microbes and contributes wine flavor. Generally, MLF is mainly driven by O. oeni, though members of other lactic acid bacteria (LAB) such as 2 Lactobacillus and Leuconostoc are also found in wine and may contribute to wine quality. Nonetheless, the growth of O. oeni is often inhibited in wine, leading to the delay of MLF. This failure was usually attributed to two aspects. Firstly, the growth of LAB for MLF was inhibited in wine conditions owing to its harsh environment, such as high content of ethanol, low pH, cold and SO2 concentrations (Spano & Massa, 2006). Based on this, strains with better tolerance performance in wine could be regarded as the better candidate of MLF starters. Secondly, strains chosen for industrial application should withstand preservation processes (freeze-drying, freezing, or spray-drying). However, these processes might generate cell membrane damage, protein and DNA denaturation, thereby significantly reducing cell viability (Santivarangkna, Kulozik, & Foerst, 2008). Taking these into consideration, the improvement of preservation processes plays an important role in MLF performance. The low pH (< 3.5) in wines is one of the main parameters that decreases LAB viability. To overcome this extreme condition, wine LAB have generated several mechanisms to escape or tolerate acid stress: (a) alteration of membrane fatty acid composition and modification of membrane fluidity (Grandvalet et al., 2008); (b) induction of stress protein synthesis (Liu et al., 2017); (c) activation of the proton extruding ATPase to keep proton motive force (PMF) maintenance and pH homeostasis (Tourdot-Marechal, Fortier, Guzzo, Lee, & Divies, 1999). More and more studies reported that pre-adaptation by treating strains to a lethal or sub-lethal stress for a limited time could generate cross-protection (Wang, Cui, & Qu, 2018). Correspondingly, acid pretreatment was proven to be useful for improving the viability of freeze-dried O. oeni SD-2a in our previous study (Zhang et al., 2013). Currently, proteomics is often applied to clarify molecular mechanisms related to stress responses. Briz-Cid et al. (2016) reported the proteome changes of Garnacha Tintorera red grapes 3 during harvest drying and indicated that the changes are important for the quality of sweet wine. As for O. oeni, most proteome studies have focused on its adaptation to wine stress conditions (Costantini et al., 2015; Margalef-Catala, Araque, Bordons, Reguant, & Bautista-Gallego, 2016), and there was little proteome research concerning acid stress. Though adaptation of O. oeni to acid stress has been researched via transcriptome analysis and traditional methods (Liu et al., 2017), mechanisms of its effects on freeze-drying resistance are yet to be well understood. Therefore, cell integrity and cell morphology of freeze-dried O. oeni SD-2a were evaluated by flow cytometry (FCM) and scanning electron microscope (SEM) in the present study. Furthermore, two- dimensional gel electrophoresis (2-DE) was used to compare the protein patterns of freeze-dried O. oeni SD-2a to provide molecular information of acid adaptation treatment on the cell lyophilized resistance. 2. Materials and methods 2.1. Bacterial culture and growth conditions MLF strain O. oeni SD-2a (Patent number, 02123444.2), originally isolated from wine in Shandong province (China), was selected as a MLF starter due to its good performance in deacidification during MLF (Liu et al., 2017). The cultivation of O. oeni SD-2a was performed in acidic tomato broth (ATB medium) at pH4.8 for 80 h to attain the early stationary phase and the preparation of ATB medium was according to the method of Zhang et al. (2013). 2.2. Acclimation conditions and lyoprotection Cells grown to the early stationary phase (80 h, 1×109 CFU/ml) were divided into four equal parts and washed with sterile saline (0.85%, m/v) twice. Three cell pellets were suspended in the same volume of acid acclimation ATB medium (pH3.2, 3.5 and 4.0 respectively) for 2 h, and the 4 pellet suspended in fresh ATB medium (pH4.8) as a control. After incubation at 25 °C for 2 h, O. oeni SD-2a were centrifuged at 3000×g for 10 min and washed with sterile saline (0.85%, m/v) twice. Then cell pellets were resuspended in 2.5% (m/v) monosodium glutamate solution (MSG solution) and homogenized slightly at room temperature (15 min) for freeze-drying. 2.3. Freeze-drying procedure and rehydration The lyophilization procedure was conducted in accordance with the method of Zhang et al. (2013). Aliquots of 10 ml cell suspension (MSG solution) in 50-ml sterile vials were frozen at −20 °C overnight, then vacuum drying was performed in a freeze dryer (Labogene, Denmark) for 28 h. The condenser temperature was −50 °C and the chamber pressure was lower than 0.06 mbar during this process. After freeze-drying, ATB medium with the original volume was applied to rehydrate the lyophilized cells. They were mixed gently and incubated at room temperature for 15 min for protein extraction, FCM and SEM analysis. 2.4. Bacterial damage and cell surface properties Bacterial damage was evaluated by FCM on the basis of the method of Pan et al. (2014). Samples prepared previously were diluted according to the instruction of the LIVE/DEAD BacLight bacterial viability kit (Invitrogen, USA). Then cell suspensions were stained with 5 μM SYTO 9 green fluorescent nucleic acid dye and 30μM propidium iodide (PI) and incubated at 25 °C for 15 min in the dark. Cells without stain were used as background control and single stained samples with PI and SYTO9 respectively were used for instrument adjustment. Then bacterial cell damage assay was conducted on a FACS Calibur flow cytometer (BD, USA). Data were analyzed using the FlowJo software and populations of live, damaged and dead cells were gated for analysis of different acid treatments. Rehydrated cell morphology was detected using SEM (Hitachi, Ltd., Japan). Samples were 5 prepared according to the study described by Bastard et al. (2016). Briefly, rehydrated bacteria were fixed with 2.5% (v/v) glutaraldehyde for 3 h at room temperature and washed twice with phosphate buffer (pH7.0). Next, samples were dehydrated with a graded series of ethanol solutions (30%, 50%, 70%, 90%, 100% (v/v)) every 10 min. Finally, samples were dried for 4 h in a critical point dryer to obtain absolute dry samples, sputter-coated with gold and viewed by SEM.