Article pubs.acs.org/JAFC
Comparative Proteomic Analysis of Dan’er Malts Produced from Distinct Malting Processes by Two-Dimensional Fluorescence Difference in Gel Electrophoresis (2D-DIGE) Xiaomin Li,†,‡,#,∥ Zhao Jin,†,‡,#,∥ Fei Gao,†,‡,# Jian Lu,*,†,‡,#,⊥ Guolin Cai,†,‡,# Jianjun Dong,§ Junhong Yu,§ and Mei Yang§ †
The Key Laboratory of Industrial Biotechnology, Ministry of Education, ‡National Engineering Laboratory for Cereal Fermentation Technology, and #School of Biotechnology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, People’s Republic of China ⊥ Industrial Technology Research Institute of Jiangnan University in Suqian, 888 Renmin Road, Suqian 223800, People’s Republic of China § State Key Laboratory of Biological Fermentation Engineering of Beer, Tsingtao Brewery Company, Ltd., Qingdao 266100, People’s Republic of China ABSTRACT: The malting process is the controlled germination, followed by drying, of the barley grain. For brewing beer, the malting process is modified according to the features of the barley variety being malted. In China, there are two schedules routinely used for malting the widely grown Dan’er cultivar, processes I and II. The quality of malt produced with process II is considered to be superior to that from process I for Dan’er by maltsters and brewers. In the present study, comparative proteomic analysis was performed between Dan’er malts produced by malting processes I and II. The data showed that enzymes and proteins responsible for cell wall polysaccharide degradation and starch and protein hydrolysis were more abundant in malt produced by process II, leading to improved quality, especially for the commercially important filterability, saccharification time, and diastatic power (DP) quality traits. In addition, to verify the proteomic results, the activities of several key enzymes (αamylase, β-amylase, and limit dextrinase) were compared between the two malts. This enabled the influence of malting process on malt quality to be determined and suggested malting process schedule changes to optimize the malting process for the Dan’er cultivar, especially for improving filterability, which is often deemed as suboptimal by maltsters and brewers. KEYWORDS: barley cultivars, malting process, filterability, saccharification time, diastatic power, comparative proteomic analysis
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INTRODUCTION Barley (Hordeum vulgare ssp. vulgare) is the preferred cereal for the production of malt,1 which is the primary raw material for beer and whiskey production. The soluble extract produced from malt by the mashing process provides most, if not all, of the nutrients needed for yeast growth during the fermentation process. As summarized by Gupta et al.,2 during fermentation, yeast requires sugar to grow and to produce alcohol. The amount of available sugar is influenced by barley cultivar traits including starch content and composition, starch hydrolysis, and diastatic power (DP) enzymes involved in starch breakdown of the grain. Therefore, the level and balance of the malt starch-degrading DP enzymes in barley malt is considered to be one of the key malt quality traits.3 Inefficient degradation of barley macromolecules, such as cell wall components (e.g., β-glucan), may cause filtration and viscosity problems, leading to increased production time and cost and even poor quality of beer. Various factors including hormones combine to ensure the accumulation and release of the hydrolytic enzymes in their active form during malting.4,5 Yeast growth also requires proteins to be either partially or fully hydrolyzed into amino acids by a range of proteolytic enzymes.6 In addition, some proteins contribute to desirable foam stability, whereas others may lead to undesirable haze formation. Moreover, storage proteins such as hordeins can also modify endosperm structure and thus malting quality.7−9 © 2014 American Chemical Society
Our previous investigations have been directed toward identifying metabolic proteins influencing the malting quality traits between malts produced from Canadian, Australian, and Chinese barley cultivars for breeding improvement.10,11 Overall, the malting quality of these cultivars was considered in terms of genetic background and growth environment, as well as the processes used to make malt from these cultivars. With respect to one barley cultivar, there is an interaction between the chosen malting process, the cultivar’s genetics, and the environment under which it was grown, all of which determine its suitability for brewing in terms of enzyme expression and macromolecule hydrolysis potential. To optimize malting potential, malt quality traits should therefore be linked to the malting process steps: sizing and cleaning, steeping, germination, and kilning.1 Sizing and cleaning help to obtain a homogeneous raw material and to eliminate dirt, broken seeds, and foreign materials. Steeping begins by soaking barley grains in water to raise the grain moisture content from approximately 12 to 42−46% for optimal imbibition of the seeds. During germination, gibberellins are synthesized and diffused into the aleurone layer to induce the hydrolytic enzymes’ synthesis.12,13 Received: Revised: Accepted: Published: 9310
February 13, 2014 August 31, 2014 September 2, 2014 September 4, 2014 dx.doi.org/10.1021/jf5030483 | J. Agric. Food Chem. 2014, 62, 9310−9316
Journal of Agricultural and Food Chemistry
Article
Table 1. Two Malting Processes (I and II) of Dan’er Barley malting parameters steeping germination
kilning
temperature (°C) ex-steeping moisture (%) time (h) temperature (°C) moisture (%) program
equipment
process B 16 45 120 ± 2 16, 17 on the last day 47 35−50 °C, 5 h; 55 °C, 1 h; 60 °C, 5 h; 65 °C, 6 h; 70 °C, 1 h; 77 °C, 1 h; 85 °C, 3 h Tower (300 t)
congress wort and that in the filtrate was precipitated with tannin19 and then measured. Proteomic Analysis with 2D-DIGE. Low-salt-soluble proteins of triplicates for each malt sample were extracted as described,10 with 5 mM Tris-HCl (pH 7.5) buffer containing 1 mM CaCl2. Protein concentrations were determined using the 2D-Quant kit (GE Healthcare, Pittsburgh, PA, USA). The extracted protein samples were labeled with N-hydroxysuccinimidyl-ester dyes Cy2, Cy3, and Cy5 (GE Healthcare)20 and separated by 2DE as described previously.11 The differentially labeled coresolved proteome maps within each gel were imaged separately with dye-specific excitation and emission wavelengths using a Typhoon TRIO scanner (GE Healthcare). The gel images were analyzed using the software Decyder 7.0 (GE Healthcare). Spot positions in gels were defined with the Differential In-Gel Analysis (DIA) module, and their fluorescent intensity values were standardized by forming the ratio of Cy3 or Cy5 over the Cy2 signal of the internal standard. The DIA data sets for all gels of one sample were then collectively analyzed using the Biological Variation Analysis (BVA) module, which allowed for the matching of protein spot patterns across gels and statistical analysis of matched spots. Statistical significance was assigned using Student’s t test based on log abundances of the standardized proteins (log standard abundance). Principal component analysis (PCA) was performed using the Extended Data Analysis (EDA) DeCyder module (GE Healthcare). Preparative gels were run in parallel with 2D-DIGE gels and stained with Collidal Coomassie Blue G250 and scanned by Image Master LabScan (GE Healthcare).10 Eight hundred micrograms of proteins extracted from each malt sample was loaded. Spots of interest assigned by DIGE analysis were located on the preparative 2D gels, then excised and subjected to in-gel trypsin digestion and an Ultraflex MALDI-TOF/TOF mass spectrometer (Bruker-Daltonics, Billerica, MA, USA) analysis as described previously.11 An in-house Mascot server (http://www.matrixscience.com) was used for database search, and scores calculated by the Mowse scoring algorithm in MASCOT (p < 0.05) were considered as positive identifications. Activity of Amylase and Limit Dextrinase in Crude Extracts. Crude enzyme was extracted from each malt for amylase and limit dextrinase activity assay. In detail, 2 g of malt was milled and then mixed with 20 mL of distilled water. Samples were vortexed for 3 min every 15 min during the 1 h crude enzyme extraction procedure, after which the extraction mixture was filtered. Amylase activity was determined as described previously.21 The reaction mixture comprised 1 mL of diluted crude enzyme and 1 mL of 1% (w/v) soluble starch solution in 0.05 M citrate buffer (pH 5.0). Reaction was carried out at 40 °C for 10 min and then quenched by adding 2 mL of DNS in the reaction tube. The control tube was added with dinitrosalicylic acid (DNS) prior to incubation at 40 °C. The reducing sugars liberated were estimated by DNS method22 at 540 nm. One unit of amylase activity was defined as the release of 1 mg of reducing sugar as maltose per minute under the defined assay conditions. For α-amylase activity detection, the crude enzyme was incubated at 70 °C to inactivate β-amylase before mixing with the substrate. The activity of β-amylase was calculated by subtracting the activity of α-amylase from total amylase activity. Limit dextrinase
The release and synthesis of hydrolytic enzymes initiate starch, protein, and cell wall polysaccharide degradation. When germination has proceeded to an optimal state, it is stopped by kilning to avoid excess sugar and amino acid consumption. The kilning uses two levels of temperature, mild settings (∼50−60 °C) to dry the seed and higher curing temperatures (∼80−85 °C) to develop the color and aroma of the malt. After the completion of the malting process, the enzyme potential of the malt is established. Dan’er is one of the most widely grown malt barley cultivars in China, especially along the Yangtze River. Its primary agronomic advantage is salt resistance for growing in Jiangsu province of China. However, the Dan’er malt quality traits are still not considered to be as good as those from the high-quality imported barley cultivars Metacalfe (Canada) or Gairdner (Australia). Besides the genetic deficiencies in the potential malt quality of Dan’er, the process used to produce malt may also limit its potential malting quality.11 Chinese maltsters typically use two malting processes for Dan’er cultivar that produce malts with distinctly different malt qualities. Therefore, in this investigation we have applied comparative proteomics with 2D-DIGE (two-dimensional fluorescence difference in gel electrophoresis) to highlight the differences between malts made from the Dan’er barley cultivar grown under identical environmental conditions and to study the effect of malting process schedule on malt quality. These insights were considered to provide clues for optimizing the malting process for Dan’er.
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process A 17 43 108 ± 3 17, 18 on the last day 46 45−55 °C, 3 h (5 °C/h); 60 °C, 4 h; 65 °C, 5 h; 68 °C, 1 h; 70 °C, 1 h; 77 °C, 1 h; 85 °C, 3 h Saladin Box (150 t)
MATERIALS AND METHODS
Commercial Malt Samples. The malt samples were collected from two commercial malt companies in China. The barley cultivar of Dan’er was grown in Jiangsu province in China in 2011. The harvested barleys were then malted at industrial scale (150/300 t) under two different processes, process I and process II (Table 1), which were frequently used by commercial malt companies during the same year. Malt samples (malt I from malting process I and malt II under malting condition II) were stored in sterilized sample sacks at −20 °C in our laboratory after receipt. Determination of Malt Quality Traits and Macromolecule Contents. The malt quality traits and macromolecule contents were determined as previously described.8 In particular, the filtration volume of initial turbid wort for 30 min was defined as separation rate. The European Brewery Convention (EBC) Congress mash was carried out for malt quality parameters determination by EBC official analytical methods.14 The contents of arabinoxylan (AX), β-glucan, starch, and polyphenol were determined according to the Douglas method,15 Congo-red method,16 iodine value,17 and Folin−Ciocalteu colorimetry,18 respectively. High-molecular-weight protein (HWP) representing the difference value between protein contents in the 9311
dx.doi.org/10.1021/jf5030483 | J. Agric. Food Chem. 2014, 62, 9310−9316
Journal of Agricultural and Food Chemistry
Article
boiling.26 There was little difference in the wort HWP and polyphenol contents between malts I and II. Proteome Comparison between the Two Malts from Different Malting Processes. Low-salt-soluble protein fractions containing most of the metabolic proteins were prepared from malts I and II simultaneously and resulted in high-quality 2D separation (Figure 1a,b). On average, for three replicate gels, approximately 430 protein spots were detected in each gel. In selecting candidates for further analysis, very faint spots and poorly resolved spots were eliminated, leaving 370 well-focused spots for quantitative analysis. Validated spots were matched and normalized against the internal standard. By quantitative analysis, 30 spots presented >1.5-fold change in protein abundance with Student’s t test p values of
