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Proteomic analysis of two malting barleys (Hordeum vulgare L.) and their impact on wort quality
Accepted Manuscript Proteomic analysis of two malting barleys (Hordeum vulgare L.) and their impact on wort quality Jessica Giselle Herrera-Gamboa, Claudia Berenice López-Alvarado, Esmeralda Pérez-Ortega, Luis Cástulo Damas-Buenrostro, Juan Carlos Cabada-Amaya, Benito Pereyra-Alférez PII:
S0733-5210(17)30874-3
DOI:
10.1016/j.jcs.2018.02.004
Reference:
YJCRS 2530
To appear in:
Journal of Cereal Science
Received Date: 31 October 2017 Revised Date:
7 February 2018
Accepted Date: 9 February 2018
Please cite this article as: Herrera-Gamboa, J.G., López-Alvarado, C.B., Pérez-Ortega, E., DamasBuenrostro, Luis.Cá., Cabada-Amaya, J.C., Pereyra-Alférez, B., Proteomic analysis of two malting barleys (Hordeum vulgare L.) and their impact on wort quality, Journal of Cereal Science (2018), doi: 10.1016/j.jcs.2018.02.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ABSTRACT Malted barley contributes directly to wort quality. Wort prepared with malt M1 had a
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higher alpha-amino nitrogen content, whereas wort prepared with malt M2 had a higher diastatic power. The proteome analysis (pI 3-10) in 2D-PAGE gels showed significant differences, with M1 generating 246±9.8 spots and M2 229±9.8 spots. M1 had more hydrolytic enzymes, in particular an esterase with a size of 43.7 kDa and a pI of 9.4,
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whereas M2 had more proteins involved in stress tolerance and carbohydrate metabolism,
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particularly a β-glucosidase with a size of 58.7 kDa and a pI of 7.7, which coincided with its higher diastatic power. The biological activity of M1 showed a band at ca. 150 kDa with high proteolytic activity and another band at 27 kDa with amylase activity. This study is the
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first report of amylase activity of small molecular size in malts.
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PROTEOMIC ANALYSIS OF TWO MALTING BARLEYS (Hordeum vulgare L.) AND
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THEIR IMPACT ON WORT QUALITY
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Jessica Giselle Herrera-Gamboa1, Claudia Berenice López-Alvarado1, Esmeralda Pérez-Ortega2,
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Luis Cástulo Damas-Buenrostro2, Juan Carlos Cabada-Amaya2, Benito Pereyra-Alférez1,*.
Instituto de Biotecnología, Facultad de Ciencias Biológicas. Universidad Autónoma de Nuevo
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León. Av. Pedro de Alba y Manuel L. Barragán S/N, Ciudad Universitaria, San Nicolás de los
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Garza, Nuevo León, México, CP. 66450
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Alfonso Reyes Norte Col, Bella Vista, 2202 Monterrey, Nuevo León, México
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Laboratorio de Investigación y Desarrollo, Cervecería Cuauhtémoc Moctezuma S. A de C. V.
*Corresponding Author: Dr. Benito Pereyra-Alférez, Instituto de Biotecnología, Facultad de
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Ciencias Biológicas. Universidad Autónoma de Nuevo León, México;
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Phone, 52(1)81 80859893; Email: [email protected], [email protected]
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1. INTRODUCTION
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Barley (Hordeum vulgare) is the fourth cereal most cultivated in Mexico with two grown cycles:
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Spring‐summer cycle (rainfed lands) and Fall‐winter cycle (irrigated lands) (Peñalba, 2011). Five
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varieties meet the quality criteria for brewing industry: Armida, Adabella, Esmeralda, Alina and
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Esperanza and correspond for the 70% of the total year production in the country (Peñalba,
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2011). The use of one or another variety depends on the desired characteristics of the final beer.
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Malting is a process during which the barley grain is subjected to humid conditions to activate
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the germination process and then dried. This process induces the enzymes required to transform
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the grain stock (starch and proteins) into the components required for fermentation.
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The qualities of the malt and the final beer are defined by the characteristics of the barley grains
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and the malting parameters. The barley malt chemical components, germination and viability are
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mainly determined by the grain characteristics, which are affected by the malting conditions and
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growing environments (Guo et al., 2016). Each malted barley variety generates unique flavor and
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body, which is exploited to produce different types of beer. Understanding the biochemical
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characteristics of the malt to predict the characteristics of the final beer is one of the major goals
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of the brewing industry.
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Many studies have focused on the metabolic proteins present in the malt that influence its
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quality. The studied plant species have included Arabidopsis thaliana, which is used as a study
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model, barley (Hordeum vulgare), corn (Zea mays) and rice (Oryza sativa) (Li et al. 2014). Two-
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dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the most popular proteome
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analysis method. When this method is combined with mass spectrometry identification
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procedures and enzymatic activity studies using zymography, comparisons can be performed
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between different malts to find proteins with potential as quality markers.
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Some studies, such as the study of Zhao et al. (2013), have related metabolic proteins separated
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in a pH range of 4-7 with the characteristics of two types of malts with different quality levels for
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the brewing industry. Other examples of comparative analyses using proteomic techniques
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include the study of Guo et al. (2016) who compared the proteomes of two barley varieties used
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for the food and brewing industries with an aim toward identifying the protein content in each
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variety, and the study by Jin et al. (2013), in which two malts with different degrees of
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filterability were compared.
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Various studies on the hydrolytic systems of malts have been conducted using electrophoretic
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separations with copolymerized substrates, which are better known as zymograms. These studies
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have found that distinct malt varieties can contain different band profiles with types of
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proteolytic activity at different pH values (Wrobel and Jones, 1992).
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Due to the importance of studying variations in the proteome and its relationship to the quality of
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the malt and the beer, the differences in the proteomic profiles of two varieties of malted barleys
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commonly used in Mexico brewers were studied to obtain information at the proteomic level
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concerning the differences between the varieties and to relate the proteins found in the malts with
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wort quality parameters.
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2. EXPERIMENTAL
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2.1 Biological samples: The malted barley grains (Hordeum vulgare) samples were provided by
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the Cuauhtémoc Moctezuma Brewery, Mexico. The malts were called M1 and M2 and were
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stored at -20°C prior to use. Malts were produced using two different Mexican barley varieties
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by a malting company using the same conditions.
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2.2 Protein extraction: The malts (10 g) were ground into a fine powder (< 50 µm). The
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samples were homogenized in 1 mL of acetate buffer (50 mM, pH 5.0) and extracted by vortex
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for 30 min, resting in an ice bath every 5 min. Then, they were centrifuged at 10000 g, 30 min at
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4 °C. The supernatants were dialyzed overnight at 4 °C against ten volumes of the same buffer
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(Wrobel and Jones, 1992). The soluble protein concentration was determined by the Bradford
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method. Three independent biological extractions were done to each malt.
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2.3 2D-PAGE: Prior to the separation in 2D-PAGE, 150-µg protein samples of each malt were
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cleaned with the 2D Clean-Up kit (GE Healthcare, Little Chalfont, UK). The protein mixture was
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resuspended in 125 µl of DeStreak Rehydration solution (GE Healthcare, Little Chalfont, UK)
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with 0.5 % IPG buffer (ampholyte-containing buffer) (GE Healthcare, Little Chalfont, UK). The
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samples were passively absorbed during the rehydration stage for 17 h in ReadyStrip™ IPG 7 cm
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pH 3-10 (Bio-Rad, Hercules, California, USA) strips. Isoelectric focusing was performed using
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the Protean IEF Cell System (Bio-Rad, Hercules, California, USA) for a total of 14 kVh (250 V
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for 20 min, 4000 V for 2 h and 4000 V for up to 10,000 Vh). Afterward, the strips were
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consecutively equilibrated in two equilibration buffers (375 mM Tris-HCl, pH 8.8, 6 M urea, 20
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% glycerol and 2 % SDS) supplemented with dithiothreitol (DTT) (2 %) and then iodoacetamide
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(2.5 %). The equilibrated strips were run in 12 % polyacrylamide gels (SDS-PAGE) in the
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presence of buffer (0.25 M Tris base, 2 M glycine and 1 % SDS) at 10 mA/gel for 30 min and at
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20 mA/gel for 1.5 h. The molecular weights of the proteins were determined through comparison
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of the separation between 10 and 250 kDa of the Precision Plus Protein Kaleidoscope Prestained
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Protein Standard marker (Bio-Rad, Hercules, California, USA). The gels were stained with
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Coomassie Brilliant Blue R-250 dye (0.1 % Coomassie R-250 Blue, 8 % acetic acid, 40 %
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methanol and 52 % water). At least two independent biological replicate gels were run to each
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malt.
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2.4 2D-PAGE image process and data collection: The 2D-PAGE gels were digitized using a
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GS-800 (Bio-Rad, Hercules, California, USA) densitometer and analyzed using the PDQuest
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Advanced 8.0.1 (Bio-Rad, Hercules, California, USA) software. The spot detection, background
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subtraction, image merging, master gel generation, and matching of the gels with the master
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were performed automatically during the analysis.
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2.5 Statistical analysis: Differentially expressed spots were identified; spots with a p-value
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lower than 0.05 (Student’s t test) were considered significant, and the better quality spots (above
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50 according to the software evaluation) were cut from the gel using the EXQuest Spot Cutter
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(Bio-Rad, Hercules, California, USA). All gels were processed independently in triplicate.
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2.6 Protein identification by matrix-assisted laser desorption/ionization tandem time of
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flight (MALDI-TOF/TOF): The spots were cut and sent to the Applied Biomics Company
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(Hayward, CA, USA) for protein identification. The picked spots were in-gel digested in-gel,
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desalted and spotted into wells of a MALDI plate. Mass spectra of the peptides were obtained
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using an Applied Biosystems Proteomics Analyzer. Ten to twenty of the most abundant peptides
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in each sample were further subjected to fragmentation and tandem mass spectrometry analysis.
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Both peptide mass and the associated fragmentation spectra were submitted to a GPS Explorer
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workstation equipped with a MASCOT search engine (Matrix Science, Boston, MA) to search
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the database of National Center for Biotechnology Information non-redundant (NCBInr) or
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Swiss Protein database. Search were performed without constraining molecular weight or
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isoelectric point, with variable carbamidomethylation of cysteine and oxidation of methionine
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residues, and with one missed cleavage allowed in the search parameters. Candidates with either
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protein score confidence interval (C.I.%) or Ion C.I.% greater than 95 were considered
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significant.
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2.7 Amylase and protease zymograms: The enzyme activity (zymograms) was essentially
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evaluated as reported by Wilkesman and Schroder, (2007). Briefly, total protein samples (50 µg)
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were treated under native and denaturing conditions (1 % SDS) and separated in 12 %
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polyacrylamide gels. The proteolytic activity was visualized using 0.2 % [w/v] copolymerized
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gelatin (DIBICO, Cuautitlán Izcalli, México.), and the amylolytic activity was assessed with 0.2
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% [w/v] copolymerized potato starch (Sigma, San Luis, Missouri, USA). The gels were run at 20
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mA/gel and 4°C. After the separation, the gel was washed with double-distilled water and
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incubated overnight at 40°C in 25 mM Tris and 5 mM MgCl2 at pH 5.0, 7.0 and 9.0 to assess the
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proteolytic activity and in 0.05 M sodium acetate at pH 5.0 and 6.0 to assess the amylase
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activity. The proteolytic activity was revealed with Coomassie Brilliant Blue R-250, whereas the
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amylolytic activity was revealed with Lugol's solution. The zymograms were performed in
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triplicate.
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2.8 General protease activity: Proteolytic activity was assayed by using azocasein (Sigma) as
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reported by Secades and Guijarro, (1999). Briefly, 120 µl of protein dilutions (180µg of total
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protein) was mixed with 480 µl of 1% azocasein solution (25 mM Tris, 5 mM MgCl2 at pH 7.5).
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The mixture was incubated at 30°C for 30 min. The reaction was stopped by the addition of 600
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µl of 10% trichloroacetic acid and incubated 30 min on ice. Samples were centrifuged at 15000
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g, 4 °C for 10 min. Next, 800 µl of the supernatant was neutralized by adding 200 µl of 1.8 N
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NaOH. Absorbance at 420 nm was measured. One unit of enzyme activity was defined as the
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amount which yielded an increase in A420 of 0.01 in 30 min at 30°C. Biological triplicates were
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done.
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2.9 Wort preparation and evaluation: The worts were prepared and evaluated by the Brewing
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Company in their labs according to the official analytical methods of the American Society of
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Brewing Chemists (Methods of Analysis ASBC, 1996). Samples were taken of plant wort in
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aseptic conditions.
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2.9.1 Moisture content of malt: Moisture content is determined on a 5 g of fine-grind malt
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sample by oven drying at 104°C for 3 hours following the Malt-3 method. Samples are weighted
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before and after drying and the percent of moisture content in the malt is calculated as follow: %
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moisture= (loss in weight/ weight of original sample) x 100.
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2.9.2 Color: The color was determinate following the Beer 10 method. Wort (100 ml final
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volume) was filtered through filter paper and mixed with 5 g of analytical grade diatomaceous
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earth. Let stand for 5 min and filter again to completely clarify. Fill spectrophotometer cuvette
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(1/2 in.) and determine absorbance at 430. The color is calculated by the formula: Color= 10
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(Absorbance in ½ cuvette at 430 nm)
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2.9.3 Total protein: This was measured by the classical Kjeldahl procedure described in Barley 7
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method, in which the total nitrogen in barley is distilled off as ammonia after acid digestion. If
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the results are expressed as “% protein,” the conventional factor for barley of 6.25 is used to
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convert nitrogen to protein.
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2.9.4 Wort soluble protein: Wort-soluble protein is determined spectrophotometrically using
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Wort-17 method based in the Kjeldahl method.
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2.9.5 pH: The hydrogen ion concentration is measure using a pH meter at room temperature.
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2.9.6 Diastatic power: Diastatic power is determined following the Malt-6A method by using the
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official ferricyanide reducing sugar method in a malt infusion with standard starch substrate
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under controlled conditions of time, temperature, pH, enzyme-substrate relations.
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2.9.7 Friability: The Malt 12 method determines friability and unmodified malt values, which
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relate to overall malt modification. Friability analysis involves crushing 50 g of malt sample in a
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friability instrument by 8 min run and then weighing the portion remaining on its screen. Percent
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unmodified malt is then determined by further sieving. Calculations: Friability= 100- (2 x A)
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2.9.8 Viscosity: The viscosity of the worts was measured according to method Wort 13. This
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method calculates absolute or dynamic viscosity (in centipoise units) using a viscometer
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Ostwald, with a water range of 50 - 150 s and according to the Hoeppler principle by measuring
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the rolling time of a ball through an inclined glass capillary filled with 5 ml of water or wort at
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20°C. Calculations: Viscosity= Flow time of wort x Wort density x 1.002
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2.9.9 Free amino nitrogen (FAN): Ninhydrin colorimetric method was used to determine the
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amount of FAN in worts. (Wort 12 method). Worts (1 ml) were diluted to 100 ml with distilled
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water and 2 ml of dilutions were transferred to test tubes in triplicate. Then, 1 ml of ninhydrin
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color reagent (2.8 mM Na2HPO4, 0.3 mM Ninhydrin, 4.4 mM KH2PO4, pH 6.6-6.8) was added
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to each sample and heated for exactly 16 min in boiling-water bath. Cooled for 20 min in a 20˚ C
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water bath. Add 5ml of dilution solution (9.4 mM KIO3, 38.4 % ethanol) to each sample. Mix
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thoroughly and measure absorbance at 570 nm against distilled water. Use a 0.14 mM Glycine
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solution as standard stock solution. Calculations FAN (mg/ml) = (net absorbance of test solution/
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net absorbance of standard) x 2 x dilution.
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3. RESULTS AND DISCUSSION
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3.1 MALT QUALITY
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Brewing depends mainly on the biochemical characteristics of the wort, which are directly
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related to the type of malted barley used in the preparation. In this work, the proteome of two
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brewing malts and their relation to the wort quality are reported. Both malts are cultivated in
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different seasons of the year (winter and summer) and both are commonly used in the brewing
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industry in Mexico. Their use in the process produce different flavor profiles in beer
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(Cuauhtémoc Moctezuma Brewery). The worts were produced with malts M1 (winter) and M2
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(summer), which had similar moisture content, color, total protein, soluble protein and pH
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characteristics (Table 1). Interesting differences were observed in several parameters, especially
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i) friability, ii) viscosity, iii) the Kolbach Index (KI), iv) the filtration time and v). The latter
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parameter strongly affects the process performance and the beer flavor (Table 1) (O’Rourke,
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2002)
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3.2 PROTEIN PROFILE
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Using the 2D-PAGE electrophoresis technique, an overall average of 246±9.8 spots were
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detected for malt M1 and 229±9.8 spots for malt M2 (Figure 1). Generally, the protein profiles of
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both malts were very similar, with most spots within a pI range of 4-7 and a wide molecular
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weight range from 10 to 250 kDa.
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All spots were analyzed to find changes in the protein concentration (quantitative) and to identify
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characteristic spots in each malt (differential). The results showed that 8 spots had quantitative
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differences, indicating that their concentrations significantly (p <0.05) changed approximately 2-
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fold between the malt varieties according to Student’s t-test (Supplemental Table 1). It was
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found that 17 spots were qualitatively different between the malts, of which 6 were present in
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only malt M1 and 11 in only malt M2.
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3.3
PROTEINS
IDENTIFIED
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MATRIX-ASSISTED
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DESORPTION/IONIZATION TANDEM TIME OF FLIGHT (MALDI-TOF/TOF)
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Spots were cut from the gels and 12 spots were successfully identified. The highest protein
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scoring hit with a protein score confidence interval over 95% from the database search for each
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spot was accepted as positive identification. All proteins showed good agreement between the
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theoretical and experimental molecular weight values, with slight discrepancies due to the post-
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translational modifications that occurred during the malting process (Table 2).
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3.3.1 Hydrolytic enzymes
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The conversion of starches into sugars during malting is mainly catalyzed by hydrolytic enzymes
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(Li et al. 2014), including dextrinases, amylases and glucosidases (Schmitt et al., 2013). The
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combined action of these enzymes is known as the “diastatic power” and has great influence on
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the fermentation efficiency and the beer quality (Hu et al. 2014). Among these enzymes, a beta-
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amylase (Spot 5711) present only in malt M1 and a beta-glucosidase (Spot 7703) expressed at a
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2-fold higher level in M2 were identified. The wort prepared with malt M2 had a diastatic power
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value 1.16 times higher than the value of malt M1, which could be due to the increment in beta-
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glucosidase expression. The overexpression of this enzyme might be related to this parameter.
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Spot 6301, which was present only in M2, is a hypothetical protein similar to the plant and
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bacterial peptidase family pfam04455; these proteins are believed to be part of the plant defense
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mechanism against pathogens (Marchler-Bauer et al. 2015). No information about the relation of
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this peptidase with quality parameters in malt was found.
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3.3.2 Stress tolerance
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Glutathione S-transferases are multifunctional enzymes that play important roles during the
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herbicide detoxification and stress tolerance responses and involve approximately 2% of the
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soluble proteins present in the grain (Rezaei et al. 2013). This enzyme was detected in greater
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amounts in M2 (Spot 6041) since this summer culture was subjected to the environmental
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conditions to a greater extent. Another identified protein related to environmental stress tolerance
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was the ABA-induced protein (Spot 5307); studies have shown that this protein is induced in
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response to environmental stress factors and abscisic acid concentrations. Higher expression of
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the gene has been observed in plants exposed to cold temperatures, and its presence generates
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cold resistance in plants (Kurkela and Franck, 1990). And important undesirable function of this
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protein is the suppression of α-amylase and protease genes during seed development (Gómez-
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Cardenas et al., 1999). Its presence in only M1 (Table 2) could be an explication of the lower
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diastatic power level of this malt.
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3.3.3 Defense proteins
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Chitinase-type proteins were detected in both malts (Spot 5507 in M1 and Spot 7402 in M2).
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These proteins are expressed as part of the defense system against the presence of plant
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pathogens. These enzymes catalyze the degradation of chitin present in the cells of pathogenic
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fungi; however, their accumulation has been related to the formation of temperature-induced
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turbidity in bottled white wines (Vincenzi et al. 2014). Thus, their concentrations could be
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indicative of poor quality for beer.
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3.3.4 Predicted proteins
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Within this group of identified proteins, Spot 8610 was found in malt M1. The analysis of 9
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peptide sequences revealed a high similarity with enzymes of the esterase and lipase families.
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The specificity of this family is not well defined. However, it has been shown that they catalyze
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the hydrolysis of low molecular weight esters, which are primarily flavoring agents in beer
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(Liaquat and Owusu, 2000). Spot 6604, which was present only in malt M2, is a predicted
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protein with a domain of the sugar kinase/HSP70/actin superfamily (Marchler-Bauer et al. 2015);
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HSP70 (heat shock protein 70). This family has been reported as an allergen present in barley
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and corn (Chiung et al. 2000). These proteins are expressed as an adaptive response to an
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increase in the environmental temperature (Kruse et al. 1993). Spot 2303, which exhibited higher
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expression in M2, is a predicted protein with a double-psi β barrel domain that is found in pollen
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allergens, which are structurally and functionally related to the expansins reported in corn that
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promote the growth and relaxation of the plant cell wall and participate in other processes, such
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as seed germination (Yennawar et al. 2006).
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3.4 AMYLASE AND PROTEASE ACTIVITY
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The main objective of barley malting is to convert the grain stocks, starch and protein into
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carbohydrates and amino acids assimilable by the yeast to obtain good performance during
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fermentation (Schmitt and Marinac, 2008). During this process, hydrolysis subproducts are
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generated and will define the characteristics of the wort, the behavior of the yeast during
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fermentation and, finally, the sensory characteristics of the beer. For this reason, elucidating the
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characteristics of each malt is important because the malts exhibit different characteristics for
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different varieties. For this work, we analyzed the proteolytic and amylolytic profile activities
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through zymograms using copolymerized substrates (Wrobel and Jones, 1992). In previous
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studies performed in our laboratory, we determined that the optimum proteolytic activity under
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native conditions was in the pH range from 5.0-9.0. For amylolytic activity under denaturing
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conditions, we determined an optimum pH between 5.0 and 6.0 (data not shown). After
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incubating the zymograms for 20 h at 40°C, we observed a marked difference in the levels of
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protease activity between the malts (Figure 2). Strong activity bands were detected between 60
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and 250 kDa, and a weak band was detected at approximately 40 kDa only in malt M2, which
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coincided with a low-molecular weight alkaline protease previously reported in bean seeds
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(Padmakar et al. 2005). This band was observed more clearly at pH 9.0. Although both malts
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present the same activity bands, differences were observed in the intensity levels of these
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activities. Moreover, malt M2 presented more intense activity than malt M1. Malt M1 presented
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higher activity in the 150-kDa band, and malt M2 presented higher activity in the 110-kDa band.
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Similar activity bands were reported by Grudkowska et al., 2013 in wheat leaves. They reported
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a 150kDa band with metalloproteinases activity inhibited by EDTA and a double band of
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molecular weight 115–118 kDa with aspartic proteinase activity inhibited by pepstatin A
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The general proteolytic activity was measured by the hydrolysis of azocasein (Figure 3) (Secades
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and Guijarro, 1999). The results showed that malt M1 has a significant higher level of proteolytic
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activity, 10.7 times higher that activity of malt M2 per milligram of total protein. These results
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confirmed our theory that the band of 150 kDa in malt M1 plays and important role in the
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general proteolityc activity. No differences were observed in the proteolytic activity level (Figure
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2) regarding its variation with pH, which is important since proteolytic activity is maintained at
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the same levels when the pH in the medium varies from 5.0 up to 9.0. Similar results were
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reported by Wrobel and Jones, (1993), who observed metalloprotease and serine protease activity
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bands present in barley seeds after 4 days of germination with high molecular weight ranges 84-
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220 kDa that maintained their activity up to a pH of 8.5.
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The α-amylase and β-amylase enzymes catalyze the hydrolysis of the starch present in the malt
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into free fermentable sugars and contribute to the “diastatic power” of the malt, which is an
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important malt quality factor for the brewing process (Zhang et at. 2004). Using zymograms, we
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evaluated the amylase profiles through the degradation of copolymerized starch in
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polyacrylamide gels at pH 5.0 and 6.0 (Figure 4). Both malts showed an activity region that
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ranged from 50 to 70 kDa at pH 5.0, whereas at pH 6.0, the activity region widened from 35 to
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100 kDa in both malts. It was observed a band with activity of approximately 27 kDa at pH 6.0;
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with more intensity in malt M1 and weak intensity in malt M2. A more intense and defined band
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with the same characteristics as the previous band has been detected in another brewing malt that
292
we are analyzing at present (data not shown). Amylases reported in cereal have an approximate
293
molecular weight of 45-67.2 kDa (Uno-Okamura et al. 2004). However, Uno-Okamura et al.,
294
(2004) reported an apoplastic beta-amylase of 25 kDa in Avena sativa seedlings, which was the
295
only report of a low-molecular weight amylase in plants. They described a possible physiological
296
function as molecular chaperone and to support the activity of other enzymes, such as β-
297
glucanases which are important for the brewing process, as the presence of β-glucans in the wort
298
can determine important characteristics during the process and the quality of the beer, especially
299
in relation to the viscosity (Jin et al. 2004). The more active presence of this low-molecular
300
weight amylase could be an indicator of the malt quality.
301
The parameters evaluated from the produced worts included the Kolbach index, which is an
302
indicator of the degree of protein degradation in the malt and is related to several characteristics
303
of the beer, such as the nutritional value, head stability and haze (Jin et al. 2013). The wort
304
produced with malt M1 generated a Kolbach index of 47.5 %, which was 1.2 times higher than
305
the index produced with malt M2 (KI 40.2 %). The KI is a direct function of the hydrolytic
306
activity and has been related to other important parameters of the wort that show differences in
307
our study, such as the filtration time, which was 1.7 times shorter for malt M1 than for malt M2,
308
and the free assimilable nitrogen (FAN) content. Using zymograms, it was observed that the
309
most significant difference in the hydrolytic activity between the malts was the activity level of a
310
150-kDa band in malt M1 and a 110-kDa band in malt M2. These bands are similar in molecular
311
weight to those observed by Grudkowska et al., (2013), a metalloproteinase (150-kDa) and an
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aspartic proteinase (110-kDa). If the 150-kDa band in malt M1 is indeed a metalloproteinase, this
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would be a very important quality parameter. The metalloproteinases in malt are the second more
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important for the brewing process, after cisteynilproteinases, because they play an important job
315
in the hydrolysis of storage proteins, mainly related with KI and FAN content (Grudkowska et
316
al., 2013). At present, we are working to determine the kind of proteases present in both malts.
317
3.5 DISCUSION AND CONCLUSIONS
318
During the brewing process, the malting of the barley mainly determines the assimilable protein
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and carbohydrate contents during fermentation. In this study, we found significant differences
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between two brewing malts (M1 and M2). The result shows, malt M1 has better characteristics
321
for use in the brewing process. The main advantages of M1 are a higher Kolbach index, a higher
322
FAN content, a shorter filtration time, the presence of an esterase-type enzyme (Spot 8610)
323
related to the presence of beneficial aromatic compounds in beer, a significantly higher general
324
proteolityc activity, a 150-kDa proteolytic band with higher activity and a 27-kDa amylase
325
activity band that has not been previously reported in malt. Although, we found an ABA-induced
326
protein (Spot 5307), related with an induction of hidrolytic-enzymes inhibitors (Huang et al.,
327
2017), the level of diastatic power in wort was normal for lager brewing malts (O’Rourke, 2002).
328
The latter could be compensated by the high presence of β-amylase (Spot 5711) that is not
329
influenced by the presence of ABA-induced protein (Huang et al., 2017). Chitinase-family
330
proteins (spot 5507 and 7402), present in M1 and M2, were related with the appearance of beer
331
turbidity an undesirable characteristic in beer. The wort prepared with malt M2 exhibited better
332
diastatic power but lower levels of the other quality parameters. The expression of β-glucosidase
333
genes during the malting process was positively correlated with the diastatic power (Lapitan et
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al., 2009). Based on this, our theory is that the higher level could be related to a larger amount of
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β-glucosidase. Additionally, the presence of proteins related to defense and stress tolerance that
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acted as allergens (Spots 6604 and 2303) was not a good indicator of the quality of this malt.
337
In conclusion, malt M1 produces a higher number of hydrolytic-type enzymes, which are
338
beneficial for use in the brewing process, whereas malt M2 produces higher amounts of enzymes
339
involved in carbohydrate metabolism, which, although they are advantageous for the process,
340
malt M2 also produce proteins related to defense and stress tolerance that can affect the
341
characteristics of the wort or the beer.
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The results show big differences between two types of apparently closely related malts. These
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parameters could be used in: i) genetically improving seeds, ii) establishing quality criteria for
344
selection of malts for the brewing process, iii) improving the handling of the worts and
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processes, iv) associating proteins with certain characteristics of the beer and v) selecting malt
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mixtures according to the yeast performance.
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ACKNOWLEDGMENT
FUNDING SOURCE
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We thank the National Council of Science and Technology (Consejo Nacional de Ciencia y
351
Tecnología) (CONACYT) for financial support through project PEI 231412. JG. Herrera-
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Gamboa and CB. López-Alvarado received CONACYT scholarships.
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ACKNOWLEDGMENTS
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We thank Rocío Ortiz López (CIDICS-UANL) and Víctor Aguirre (Facultad de Agronomía-
355
UANL) for their support in conducting the 2D-PAGE technique.
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AUTHOR CONTRIBUTIONS
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JGHG and CBLA designed and performed the experimental work, BPA directed the project.
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JGHG wrote the paper with the supervision of BPA. All authors provided critical feedback and
359
helped shape the project and paper. EPO, LCDB and JCCA gave the approval to the final version
360
of the paper.
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CONFLICT OF INTEREST
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All authors declare no competing financial interest.
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ABBREVIATIONS USED
365
M1, Malt 1; M2, Malt 2; 2D-PAGE, Two- dimensional polyacrylamide gel electrophoresis;
366
kVh, kiloVolts per hour; HCl, Hydrochloric acid; SDS, Sodium dodecyl sulfate; DTT,
367
Dithiothreitol; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; mA,
368
miliAmpere; kDa, kiloDalton; µg, micrograms; MgCl2, magnesium chloride; FAN, Free
369
assimilable nitrogen; KI, Kolbach index.
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0416.1992.tb01132.x
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quality discrimination. J. Agric. Food Chem., 61, 402-409. doi:10.1021/jf3034418
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FIGURE CAPTIONS Figure 1. Two-dimensional protein profiles of malts M1 and M2. Yellow spots indicate detected
466
spots and red spots indicate differential spots (T-test 95%)
467
Figure 2. Effect of pH on the proteolytic activity of malts M1 and M2 in zymograms with 0.2 %
468
gelatin
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Figure 3. General proteolytic activity per amount of total protein. Activity was measured by
470
hydrolysis of azocasein
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Figure 4. Amylase activity evaluated at different pH values using zymograms with 0.2 % starch.
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The arrow indicates an amylase activity band of 27 kDa.
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TABLES CAPTIONS
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Table 1. Parameters of the worts produced with the different malts
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Table 2. Protein identification by MALDI-TOF/TOF
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SUPPLEMENTARY MATERIAL
Supplemental Table 1. Quantitation table report of differential spots
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Table 1. Parameters of the worts produced with the different malts Malts Parameter M1 M2 Moisture (%) 4.6 4.7 Color (SRM) 2.0 2.4 Total protein (%) 12.0 12.7 Soluble protein (%) 5.7 5.1 pH 5.83 5.82 Kolbach Index (%) 47.5 40.2 Diastatic power (Base S.) 143 166 Friability (%) 70.1 51.5 Viscosity (cps) 1.58 1.61 Filtration time (min) 68 118 Alpha-amino nitrogen (ppm) 223 188
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Table 2. Protein identification by MALDI-TOF/TOF
Malt
Molecular weight Theo./Obs.
Hydrolytic enzymes a M1 62.8/59.6 5711 7703 6502 6508
pI
6.4
Identity
Accession No.
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ID Spot
b
M2
58.7/57.4
7.7
Beta-amylase; 1,4-alpha-D-glucan maltohydrolase; BetaAm1; FLAG precursor Beta-glucosidase A [Hordeum vulgare]
gi|75107132
a
M2
38.4/33.2
6.9
Glyceraldehyde-3-phosphate dehydrogenase 2, cytosolic
gi|120668
a
M2
38.7/33.2
7.3
Glyceraldehyde-3-phosphate dehydrogenase 2, cytosolic
gi|120668
gi|193073259
25.4/25
7.0
Glutathione S-transferase [Hordeum vulgare]
gi|6683765
17.7/18.9
6.6
18.9 KDa ABA-induced protein [Hordeum vulgare]
gi|1052747
Defense proteins a M1 5507
32.1/26.2
7.1
25.5/25.9
8.0
Chain A, crystalline structure of the Gh-19 Chitinase family of rye seeds Chain A, crystalline structure of an endochitinase of Hordeum vulgare L seeds at 1.8 Â resolution.
43.7/38.8
9.4
Predicted protein [Hordeum vulgare]
gi|326516774
23.0/24.2
7.0
Hypothetical protein [Hordeum vulgare]
gi|2266666
7.3
Predicted protein [Hordeum vulgare]
gi|326523037
M2
Predicted proteins a M1 8610 a M2 6301 6604 2303
M2
43.9/42.2
b
M2
18.5/18.9
5.5
Predicted protein [Hordeum vulgare]
b
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spots with qualitative differences and spots with quantitative differences.
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7402
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Stress tolerance b M2 6401 a M1 5307
gi|400977413 gi|157834680
gi|326515048
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Highlights: • Comparison of proteome of two brewing malts was done by 2D-PAGE and Zymograms
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• Quality parameters in worts were related with proteins and enzymatic activity • Difference in hydrolytic proteins were observed using zymograms
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• A small molecular size amylase was detected
S0733-5210(17)30874-3
DOI:
10.1016/j.jcs.2018.02.004
Reference:
YJCRS 2530
To appear in:
Journal of Cereal Science
Received Date: 31 October 2017 Revised Date:
7 February 2018
Accepted Date: 9 February 2018
Please cite this article as: Herrera-Gamboa, J.G., López-Alvarado, C.B., Pérez-Ortega, E., DamasBuenrostro, Luis.Cá., Cabada-Amaya, J.C., Pereyra-Alférez, B., Proteomic analysis of two malting barleys (Hordeum vulgare L.) and their impact on wort quality, Journal of Cereal Science (2018), doi: 10.1016/j.jcs.2018.02.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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ABSTRACT Malted barley contributes directly to wort quality. Wort prepared with malt M1 had a
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higher alpha-amino nitrogen content, whereas wort prepared with malt M2 had a higher diastatic power. The proteome analysis (pI 3-10) in 2D-PAGE gels showed significant differences, with M1 generating 246±9.8 spots and M2 229±9.8 spots. M1 had more hydrolytic enzymes, in particular an esterase with a size of 43.7 kDa and a pI of 9.4,
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whereas M2 had more proteins involved in stress tolerance and carbohydrate metabolism,
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particularly a β-glucosidase with a size of 58.7 kDa and a pI of 7.7, which coincided with its higher diastatic power. The biological activity of M1 showed a band at ca. 150 kDa with high proteolytic activity and another band at 27 kDa with amylase activity. This study is the
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first report of amylase activity of small molecular size in malts.
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PROTEOMIC ANALYSIS OF TWO MALTING BARLEYS (Hordeum vulgare L.) AND
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THEIR IMPACT ON WORT QUALITY
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Jessica Giselle Herrera-Gamboa1, Claudia Berenice López-Alvarado1, Esmeralda Pérez-Ortega2,
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Luis Cástulo Damas-Buenrostro2, Juan Carlos Cabada-Amaya2, Benito Pereyra-Alférez1,*.
Instituto de Biotecnología, Facultad de Ciencias Biológicas. Universidad Autónoma de Nuevo
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León. Av. Pedro de Alba y Manuel L. Barragán S/N, Ciudad Universitaria, San Nicolás de los
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Garza, Nuevo León, México, CP. 66450
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Alfonso Reyes Norte Col, Bella Vista, 2202 Monterrey, Nuevo León, México
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Laboratorio de Investigación y Desarrollo, Cervecería Cuauhtémoc Moctezuma S. A de C. V.
*Corresponding Author: Dr. Benito Pereyra-Alférez, Instituto de Biotecnología, Facultad de
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Ciencias Biológicas. Universidad Autónoma de Nuevo León, México;
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Phone, 52(1)81 80859893; Email: [email protected], [email protected]
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1. INTRODUCTION
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Barley (Hordeum vulgare) is the fourth cereal most cultivated in Mexico with two grown cycles:
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Spring‐summer cycle (rainfed lands) and Fall‐winter cycle (irrigated lands) (Peñalba, 2011). Five
19
varieties meet the quality criteria for brewing industry: Armida, Adabella, Esmeralda, Alina and
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Esperanza and correspond for the 70% of the total year production in the country (Peñalba,
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2011). The use of one or another variety depends on the desired characteristics of the final beer.
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Malting is a process during which the barley grain is subjected to humid conditions to activate
23
the germination process and then dried. This process induces the enzymes required to transform
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the grain stock (starch and proteins) into the components required for fermentation.
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The qualities of the malt and the final beer are defined by the characteristics of the barley grains
26
and the malting parameters. The barley malt chemical components, germination and viability are
27
mainly determined by the grain characteristics, which are affected by the malting conditions and
28
growing environments (Guo et al., 2016). Each malted barley variety generates unique flavor and
29
body, which is exploited to produce different types of beer. Understanding the biochemical
30
characteristics of the malt to predict the characteristics of the final beer is one of the major goals
31
of the brewing industry.
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Many studies have focused on the metabolic proteins present in the malt that influence its
33
quality. The studied plant species have included Arabidopsis thaliana, which is used as a study
34
model, barley (Hordeum vulgare), corn (Zea mays) and rice (Oryza sativa) (Li et al. 2014). Two-
35
dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the most popular proteome
36
analysis method. When this method is combined with mass spectrometry identification
37
procedures and enzymatic activity studies using zymography, comparisons can be performed
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between different malts to find proteins with potential as quality markers.
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Some studies, such as the study of Zhao et al. (2013), have related metabolic proteins separated
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in a pH range of 4-7 with the characteristics of two types of malts with different quality levels for
41
the brewing industry. Other examples of comparative analyses using proteomic techniques
42
include the study of Guo et al. (2016) who compared the proteomes of two barley varieties used
43
for the food and brewing industries with an aim toward identifying the protein content in each
44
variety, and the study by Jin et al. (2013), in which two malts with different degrees of
45
filterability were compared.
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Various studies on the hydrolytic systems of malts have been conducted using electrophoretic
47
separations with copolymerized substrates, which are better known as zymograms. These studies
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have found that distinct malt varieties can contain different band profiles with types of
49
proteolytic activity at different pH values (Wrobel and Jones, 1992).
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Due to the importance of studying variations in the proteome and its relationship to the quality of
51
the malt and the beer, the differences in the proteomic profiles of two varieties of malted barleys
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commonly used in Mexico brewers were studied to obtain information at the proteomic level
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concerning the differences between the varieties and to relate the proteins found in the malts with
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wort quality parameters.
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2. EXPERIMENTAL
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2.1 Biological samples: The malted barley grains (Hordeum vulgare) samples were provided by
58
the Cuauhtémoc Moctezuma Brewery, Mexico. The malts were called M1 and M2 and were
59
stored at -20°C prior to use. Malts were produced using two different Mexican barley varieties
60
by a malting company using the same conditions.
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2.2 Protein extraction: The malts (10 g) were ground into a fine powder (< 50 µm). The
62
samples were homogenized in 1 mL of acetate buffer (50 mM, pH 5.0) and extracted by vortex
63
for 30 min, resting in an ice bath every 5 min. Then, they were centrifuged at 10000 g, 30 min at
64
4 °C. The supernatants were dialyzed overnight at 4 °C against ten volumes of the same buffer
65
(Wrobel and Jones, 1992). The soluble protein concentration was determined by the Bradford
66
method. Three independent biological extractions were done to each malt.
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2.3 2D-PAGE: Prior to the separation in 2D-PAGE, 150-µg protein samples of each malt were
68
cleaned with the 2D Clean-Up kit (GE Healthcare, Little Chalfont, UK). The protein mixture was
69
resuspended in 125 µl of DeStreak Rehydration solution (GE Healthcare, Little Chalfont, UK)
70
with 0.5 % IPG buffer (ampholyte-containing buffer) (GE Healthcare, Little Chalfont, UK). The
71
samples were passively absorbed during the rehydration stage for 17 h in ReadyStrip™ IPG 7 cm
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pH 3-10 (Bio-Rad, Hercules, California, USA) strips. Isoelectric focusing was performed using
73
the Protean IEF Cell System (Bio-Rad, Hercules, California, USA) for a total of 14 kVh (250 V
74
for 20 min, 4000 V for 2 h and 4000 V for up to 10,000 Vh). Afterward, the strips were
75
consecutively equilibrated in two equilibration buffers (375 mM Tris-HCl, pH 8.8, 6 M urea, 20
76
% glycerol and 2 % SDS) supplemented with dithiothreitol (DTT) (2 %) and then iodoacetamide
77
(2.5 %). The equilibrated strips were run in 12 % polyacrylamide gels (SDS-PAGE) in the
78
presence of buffer (0.25 M Tris base, 2 M glycine and 1 % SDS) at 10 mA/gel for 30 min and at
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20 mA/gel for 1.5 h. The molecular weights of the proteins were determined through comparison
80
of the separation between 10 and 250 kDa of the Precision Plus Protein Kaleidoscope Prestained
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Protein Standard marker (Bio-Rad, Hercules, California, USA). The gels were stained with
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Coomassie Brilliant Blue R-250 dye (0.1 % Coomassie R-250 Blue, 8 % acetic acid, 40 %
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methanol and 52 % water). At least two independent biological replicate gels were run to each
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malt.
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2.4 2D-PAGE image process and data collection: The 2D-PAGE gels were digitized using a
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GS-800 (Bio-Rad, Hercules, California, USA) densitometer and analyzed using the PDQuest
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Advanced 8.0.1 (Bio-Rad, Hercules, California, USA) software. The spot detection, background
88
subtraction, image merging, master gel generation, and matching of the gels with the master
89
were performed automatically during the analysis.
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2.5 Statistical analysis: Differentially expressed spots were identified; spots with a p-value
91
lower than 0.05 (Student’s t test) were considered significant, and the better quality spots (above
92
50 according to the software evaluation) were cut from the gel using the EXQuest Spot Cutter
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(Bio-Rad, Hercules, California, USA). All gels were processed independently in triplicate.
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2.6 Protein identification by matrix-assisted laser desorption/ionization tandem time of
95
flight (MALDI-TOF/TOF): The spots were cut and sent to the Applied Biomics Company
96
(Hayward, CA, USA) for protein identification. The picked spots were in-gel digested in-gel,
97
desalted and spotted into wells of a MALDI plate. Mass spectra of the peptides were obtained
98
using an Applied Biosystems Proteomics Analyzer. Ten to twenty of the most abundant peptides
99
in each sample were further subjected to fragmentation and tandem mass spectrometry analysis.
100
Both peptide mass and the associated fragmentation spectra were submitted to a GPS Explorer
101
workstation equipped with a MASCOT search engine (Matrix Science, Boston, MA) to search
102
the database of National Center for Biotechnology Information non-redundant (NCBInr) or
103
Swiss Protein database. Search were performed without constraining molecular weight or
104
isoelectric point, with variable carbamidomethylation of cysteine and oxidation of methionine
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residues, and with one missed cleavage allowed in the search parameters. Candidates with either
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protein score confidence interval (C.I.%) or Ion C.I.% greater than 95 were considered
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significant.
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2.7 Amylase and protease zymograms: The enzyme activity (zymograms) was essentially
109
evaluated as reported by Wilkesman and Schroder, (2007). Briefly, total protein samples (50 µg)
110
were treated under native and denaturing conditions (1 % SDS) and separated in 12 %
111
polyacrylamide gels. The proteolytic activity was visualized using 0.2 % [w/v] copolymerized
112
gelatin (DIBICO, Cuautitlán Izcalli, México.), and the amylolytic activity was assessed with 0.2
113
% [w/v] copolymerized potato starch (Sigma, San Luis, Missouri, USA). The gels were run at 20
114
mA/gel and 4°C. After the separation, the gel was washed with double-distilled water and
115
incubated overnight at 40°C in 25 mM Tris and 5 mM MgCl2 at pH 5.0, 7.0 and 9.0 to assess the
116
proteolytic activity and in 0.05 M sodium acetate at pH 5.0 and 6.0 to assess the amylase
117
activity. The proteolytic activity was revealed with Coomassie Brilliant Blue R-250, whereas the
118
amylolytic activity was revealed with Lugol's solution. The zymograms were performed in
119
triplicate.
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2.8 General protease activity: Proteolytic activity was assayed by using azocasein (Sigma) as
121
reported by Secades and Guijarro, (1999). Briefly, 120 µl of protein dilutions (180µg of total
122
protein) was mixed with 480 µl of 1% azocasein solution (25 mM Tris, 5 mM MgCl2 at pH 7.5).
123
The mixture was incubated at 30°C for 30 min. The reaction was stopped by the addition of 600
124
µl of 10% trichloroacetic acid and incubated 30 min on ice. Samples were centrifuged at 15000
125
g, 4 °C for 10 min. Next, 800 µl of the supernatant was neutralized by adding 200 µl of 1.8 N
126
NaOH. Absorbance at 420 nm was measured. One unit of enzyme activity was defined as the
127
amount which yielded an increase in A420 of 0.01 in 30 min at 30°C. Biological triplicates were
128
done.
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2.9 Wort preparation and evaluation: The worts were prepared and evaluated by the Brewing
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Company in their labs according to the official analytical methods of the American Society of
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Brewing Chemists (Methods of Analysis ASBC, 1996). Samples were taken of plant wort in
132
aseptic conditions.
133
2.9.1 Moisture content of malt: Moisture content is determined on a 5 g of fine-grind malt
134
sample by oven drying at 104°C for 3 hours following the Malt-3 method. Samples are weighted
135
before and after drying and the percent of moisture content in the malt is calculated as follow: %
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moisture= (loss in weight/ weight of original sample) x 100.
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2.9.2 Color: The color was determinate following the Beer 10 method. Wort (100 ml final
138
volume) was filtered through filter paper and mixed with 5 g of analytical grade diatomaceous
139
earth. Let stand for 5 min and filter again to completely clarify. Fill spectrophotometer cuvette
140
(1/2 in.) and determine absorbance at 430. The color is calculated by the formula: Color= 10
141
(Absorbance in ½ cuvette at 430 nm)
142
2.9.3 Total protein: This was measured by the classical Kjeldahl procedure described in Barley 7
143
method, in which the total nitrogen in barley is distilled off as ammonia after acid digestion. If
144
the results are expressed as “% protein,” the conventional factor for barley of 6.25 is used to
145
convert nitrogen to protein.
146
2.9.4 Wort soluble protein: Wort-soluble protein is determined spectrophotometrically using
147
Wort-17 method based in the Kjeldahl method.
148
2.9.5 pH: The hydrogen ion concentration is measure using a pH meter at room temperature.
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2.9.6 Diastatic power: Diastatic power is determined following the Malt-6A method by using the
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official ferricyanide reducing sugar method in a malt infusion with standard starch substrate
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under controlled conditions of time, temperature, pH, enzyme-substrate relations.
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2.9.7 Friability: The Malt 12 method determines friability and unmodified malt values, which
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relate to overall malt modification. Friability analysis involves crushing 50 g of malt sample in a
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friability instrument by 8 min run and then weighing the portion remaining on its screen. Percent
155
unmodified malt is then determined by further sieving. Calculations: Friability= 100- (2 x A)
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2.9.8 Viscosity: The viscosity of the worts was measured according to method Wort 13. This
157
method calculates absolute or dynamic viscosity (in centipoise units) using a viscometer
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Ostwald, with a water range of 50 - 150 s and according to the Hoeppler principle by measuring
159
the rolling time of a ball through an inclined glass capillary filled with 5 ml of water or wort at
160
20°C. Calculations: Viscosity= Flow time of wort x Wort density x 1.002
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2.9.9 Free amino nitrogen (FAN): Ninhydrin colorimetric method was used to determine the
162
amount of FAN in worts. (Wort 12 method). Worts (1 ml) were diluted to 100 ml with distilled
163
water and 2 ml of dilutions were transferred to test tubes in triplicate. Then, 1 ml of ninhydrin
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color reagent (2.8 mM Na2HPO4, 0.3 mM Ninhydrin, 4.4 mM KH2PO4, pH 6.6-6.8) was added
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to each sample and heated for exactly 16 min in boiling-water bath. Cooled for 20 min in a 20˚ C
166
water bath. Add 5ml of dilution solution (9.4 mM KIO3, 38.4 % ethanol) to each sample. Mix
167
thoroughly and measure absorbance at 570 nm against distilled water. Use a 0.14 mM Glycine
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solution as standard stock solution. Calculations FAN (mg/ml) = (net absorbance of test solution/
169
net absorbance of standard) x 2 x dilution.
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3. RESULTS AND DISCUSSION
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3.1 MALT QUALITY
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Brewing depends mainly on the biochemical characteristics of the wort, which are directly
174
related to the type of malted barley used in the preparation. In this work, the proteome of two
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brewing malts and their relation to the wort quality are reported. Both malts are cultivated in
176
different seasons of the year (winter and summer) and both are commonly used in the brewing
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industry in Mexico. Their use in the process produce different flavor profiles in beer
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(Cuauhtémoc Moctezuma Brewery). The worts were produced with malts M1 (winter) and M2
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(summer), which had similar moisture content, color, total protein, soluble protein and pH
180
characteristics (Table 1). Interesting differences were observed in several parameters, especially
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i) friability, ii) viscosity, iii) the Kolbach Index (KI), iv) the filtration time and v). The latter
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parameter strongly affects the process performance and the beer flavor (Table 1) (O’Rourke,
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2002)
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3.2 PROTEIN PROFILE
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Using the 2D-PAGE electrophoresis technique, an overall average of 246±9.8 spots were
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detected for malt M1 and 229±9.8 spots for malt M2 (Figure 1). Generally, the protein profiles of
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both malts were very similar, with most spots within a pI range of 4-7 and a wide molecular
188
weight range from 10 to 250 kDa.
189
All spots were analyzed to find changes in the protein concentration (quantitative) and to identify
190
characteristic spots in each malt (differential). The results showed that 8 spots had quantitative
191
differences, indicating that their concentrations significantly (p <0.05) changed approximately 2-
192
fold between the malt varieties according to Student’s t-test (Supplemental Table 1). It was
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found that 17 spots were qualitatively different between the malts, of which 6 were present in
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only malt M1 and 11 in only malt M2.
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3.3
PROTEINS
IDENTIFIED
BY
MATRIX-ASSISTED
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DESORPTION/IONIZATION TANDEM TIME OF FLIGHT (MALDI-TOF/TOF)
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Spots were cut from the gels and 12 spots were successfully identified. The highest protein
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scoring hit with a protein score confidence interval over 95% from the database search for each
200
spot was accepted as positive identification. All proteins showed good agreement between the
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theoretical and experimental molecular weight values, with slight discrepancies due to the post-
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translational modifications that occurred during the malting process (Table 2).
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3.3.1 Hydrolytic enzymes
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The conversion of starches into sugars during malting is mainly catalyzed by hydrolytic enzymes
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(Li et al. 2014), including dextrinases, amylases and glucosidases (Schmitt et al., 2013). The
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combined action of these enzymes is known as the “diastatic power” and has great influence on
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the fermentation efficiency and the beer quality (Hu et al. 2014). Among these enzymes, a beta-
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amylase (Spot 5711) present only in malt M1 and a beta-glucosidase (Spot 7703) expressed at a
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2-fold higher level in M2 were identified. The wort prepared with malt M2 had a diastatic power
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value 1.16 times higher than the value of malt M1, which could be due to the increment in beta-
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glucosidase expression. The overexpression of this enzyme might be related to this parameter.
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Spot 6301, which was present only in M2, is a hypothetical protein similar to the plant and
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bacterial peptidase family pfam04455; these proteins are believed to be part of the plant defense
214
mechanism against pathogens (Marchler-Bauer et al. 2015). No information about the relation of
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this peptidase with quality parameters in malt was found.
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3.3.2 Stress tolerance
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Glutathione S-transferases are multifunctional enzymes that play important roles during the
218
herbicide detoxification and stress tolerance responses and involve approximately 2% of the
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soluble proteins present in the grain (Rezaei et al. 2013). This enzyme was detected in greater
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amounts in M2 (Spot 6041) since this summer culture was subjected to the environmental
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conditions to a greater extent. Another identified protein related to environmental stress tolerance
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was the ABA-induced protein (Spot 5307); studies have shown that this protein is induced in
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response to environmental stress factors and abscisic acid concentrations. Higher expression of
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the gene has been observed in plants exposed to cold temperatures, and its presence generates
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cold resistance in plants (Kurkela and Franck, 1990). And important undesirable function of this
226
protein is the suppression of α-amylase and protease genes during seed development (Gómez-
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Cardenas et al., 1999). Its presence in only M1 (Table 2) could be an explication of the lower
228
diastatic power level of this malt.
229
3.3.3 Defense proteins
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Chitinase-type proteins were detected in both malts (Spot 5507 in M1 and Spot 7402 in M2).
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These proteins are expressed as part of the defense system against the presence of plant
232
pathogens. These enzymes catalyze the degradation of chitin present in the cells of pathogenic
233
fungi; however, their accumulation has been related to the formation of temperature-induced
234
turbidity in bottled white wines (Vincenzi et al. 2014). Thus, their concentrations could be
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indicative of poor quality for beer.
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3.3.4 Predicted proteins
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Within this group of identified proteins, Spot 8610 was found in malt M1. The analysis of 9
238
peptide sequences revealed a high similarity with enzymes of the esterase and lipase families.
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The specificity of this family is not well defined. However, it has been shown that they catalyze
240
the hydrolysis of low molecular weight esters, which are primarily flavoring agents in beer
241
(Liaquat and Owusu, 2000). Spot 6604, which was present only in malt M2, is a predicted
242
protein with a domain of the sugar kinase/HSP70/actin superfamily (Marchler-Bauer et al. 2015);
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HSP70 (heat shock protein 70). This family has been reported as an allergen present in barley
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and corn (Chiung et al. 2000). These proteins are expressed as an adaptive response to an
245
increase in the environmental temperature (Kruse et al. 1993). Spot 2303, which exhibited higher
246
expression in M2, is a predicted protein with a double-psi β barrel domain that is found in pollen
247
allergens, which are structurally and functionally related to the expansins reported in corn that
248
promote the growth and relaxation of the plant cell wall and participate in other processes, such
249
as seed germination (Yennawar et al. 2006).
250
3.4 AMYLASE AND PROTEASE ACTIVITY
251
The main objective of barley malting is to convert the grain stocks, starch and protein into
252
carbohydrates and amino acids assimilable by the yeast to obtain good performance during
253
fermentation (Schmitt and Marinac, 2008). During this process, hydrolysis subproducts are
254
generated and will define the characteristics of the wort, the behavior of the yeast during
255
fermentation and, finally, the sensory characteristics of the beer. For this reason, elucidating the
256
characteristics of each malt is important because the malts exhibit different characteristics for
257
different varieties. For this work, we analyzed the proteolytic and amylolytic profile activities
258
through zymograms using copolymerized substrates (Wrobel and Jones, 1992). In previous
259
studies performed in our laboratory, we determined that the optimum proteolytic activity under
260
native conditions was in the pH range from 5.0-9.0. For amylolytic activity under denaturing
261
conditions, we determined an optimum pH between 5.0 and 6.0 (data not shown). After
262
incubating the zymograms for 20 h at 40°C, we observed a marked difference in the levels of
263
protease activity between the malts (Figure 2). Strong activity bands were detected between 60
264
and 250 kDa, and a weak band was detected at approximately 40 kDa only in malt M2, which
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coincided with a low-molecular weight alkaline protease previously reported in bean seeds
266
(Padmakar et al. 2005). This band was observed more clearly at pH 9.0. Although both malts
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present the same activity bands, differences were observed in the intensity levels of these
268
activities. Moreover, malt M2 presented more intense activity than malt M1. Malt M1 presented
269
higher activity in the 150-kDa band, and malt M2 presented higher activity in the 110-kDa band.
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Similar activity bands were reported by Grudkowska et al., 2013 in wheat leaves. They reported
271
a 150kDa band with metalloproteinases activity inhibited by EDTA and a double band of
272
molecular weight 115–118 kDa with aspartic proteinase activity inhibited by pepstatin A
273
The general proteolytic activity was measured by the hydrolysis of azocasein (Figure 3) (Secades
274
and Guijarro, 1999). The results showed that malt M1 has a significant higher level of proteolytic
275
activity, 10.7 times higher that activity of malt M2 per milligram of total protein. These results
276
confirmed our theory that the band of 150 kDa in malt M1 plays and important role in the
277
general proteolityc activity. No differences were observed in the proteolytic activity level (Figure
278
2) regarding its variation with pH, which is important since proteolytic activity is maintained at
279
the same levels when the pH in the medium varies from 5.0 up to 9.0. Similar results were
280
reported by Wrobel and Jones, (1993), who observed metalloprotease and serine protease activity
281
bands present in barley seeds after 4 days of germination with high molecular weight ranges 84-
282
220 kDa that maintained their activity up to a pH of 8.5.
283
The α-amylase and β-amylase enzymes catalyze the hydrolysis of the starch present in the malt
284
into free fermentable sugars and contribute to the “diastatic power” of the malt, which is an
285
important malt quality factor for the brewing process (Zhang et at. 2004). Using zymograms, we
286
evaluated the amylase profiles through the degradation of copolymerized starch in
287
polyacrylamide gels at pH 5.0 and 6.0 (Figure 4). Both malts showed an activity region that
288
ranged from 50 to 70 kDa at pH 5.0, whereas at pH 6.0, the activity region widened from 35 to
289
100 kDa in both malts. It was observed a band with activity of approximately 27 kDa at pH 6.0;
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with more intensity in malt M1 and weak intensity in malt M2. A more intense and defined band
291
with the same characteristics as the previous band has been detected in another brewing malt that
292
we are analyzing at present (data not shown). Amylases reported in cereal have an approximate
293
molecular weight of 45-67.2 kDa (Uno-Okamura et al. 2004). However, Uno-Okamura et al.,
294
(2004) reported an apoplastic beta-amylase of 25 kDa in Avena sativa seedlings, which was the
295
only report of a low-molecular weight amylase in plants. They described a possible physiological
296
function as molecular chaperone and to support the activity of other enzymes, such as β-
297
glucanases which are important for the brewing process, as the presence of β-glucans in the wort
298
can determine important characteristics during the process and the quality of the beer, especially
299
in relation to the viscosity (Jin et al. 2004). The more active presence of this low-molecular
300
weight amylase could be an indicator of the malt quality.
301
The parameters evaluated from the produced worts included the Kolbach index, which is an
302
indicator of the degree of protein degradation in the malt and is related to several characteristics
303
of the beer, such as the nutritional value, head stability and haze (Jin et al. 2013). The wort
304
produced with malt M1 generated a Kolbach index of 47.5 %, which was 1.2 times higher than
305
the index produced with malt M2 (KI 40.2 %). The KI is a direct function of the hydrolytic
306
activity and has been related to other important parameters of the wort that show differences in
307
our study, such as the filtration time, which was 1.7 times shorter for malt M1 than for malt M2,
308
and the free assimilable nitrogen (FAN) content. Using zymograms, it was observed that the
309
most significant difference in the hydrolytic activity between the malts was the activity level of a
310
150-kDa band in malt M1 and a 110-kDa band in malt M2. These bands are similar in molecular
311
weight to those observed by Grudkowska et al., (2013), a metalloproteinase (150-kDa) and an
312
aspartic proteinase (110-kDa). If the 150-kDa band in malt M1 is indeed a metalloproteinase, this
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would be a very important quality parameter. The metalloproteinases in malt are the second more
314
important for the brewing process, after cisteynilproteinases, because they play an important job
315
in the hydrolysis of storage proteins, mainly related with KI and FAN content (Grudkowska et
316
al., 2013). At present, we are working to determine the kind of proteases present in both malts.
317
3.5 DISCUSION AND CONCLUSIONS
318
During the brewing process, the malting of the barley mainly determines the assimilable protein
319
and carbohydrate contents during fermentation. In this study, we found significant differences
320
between two brewing malts (M1 and M2). The result shows, malt M1 has better characteristics
321
for use in the brewing process. The main advantages of M1 are a higher Kolbach index, a higher
322
FAN content, a shorter filtration time, the presence of an esterase-type enzyme (Spot 8610)
323
related to the presence of beneficial aromatic compounds in beer, a significantly higher general
324
proteolityc activity, a 150-kDa proteolytic band with higher activity and a 27-kDa amylase
325
activity band that has not been previously reported in malt. Although, we found an ABA-induced
326
protein (Spot 5307), related with an induction of hidrolytic-enzymes inhibitors (Huang et al.,
327
2017), the level of diastatic power in wort was normal for lager brewing malts (O’Rourke, 2002).
328
The latter could be compensated by the high presence of β-amylase (Spot 5711) that is not
329
influenced by the presence of ABA-induced protein (Huang et al., 2017). Chitinase-family
330
proteins (spot 5507 and 7402), present in M1 and M2, were related with the appearance of beer
331
turbidity an undesirable characteristic in beer. The wort prepared with malt M2 exhibited better
332
diastatic power but lower levels of the other quality parameters. The expression of β-glucosidase
333
genes during the malting process was positively correlated with the diastatic power (Lapitan et
334
al., 2009). Based on this, our theory is that the higher level could be related to a larger amount of
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β-glucosidase. Additionally, the presence of proteins related to defense and stress tolerance that
336
acted as allergens (Spots 6604 and 2303) was not a good indicator of the quality of this malt.
337
In conclusion, malt M1 produces a higher number of hydrolytic-type enzymes, which are
338
beneficial for use in the brewing process, whereas malt M2 produces higher amounts of enzymes
339
involved in carbohydrate metabolism, which, although they are advantageous for the process,
340
malt M2 also produce proteins related to defense and stress tolerance that can affect the
341
characteristics of the wort or the beer.
342
The results show big differences between two types of apparently closely related malts. These
343
parameters could be used in: i) genetically improving seeds, ii) establishing quality criteria for
344
selection of malts for the brewing process, iii) improving the handling of the worts and
345
processes, iv) associating proteins with certain characteristics of the beer and v) selecting malt
346
mixtures according to the yeast performance.
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ACKNOWLEDGMENT
FUNDING SOURCE
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We thank the National Council of Science and Technology (Consejo Nacional de Ciencia y
351
Tecnología) (CONACYT) for financial support through project PEI 231412. JG. Herrera-
352
Gamboa and CB. López-Alvarado received CONACYT scholarships.
353
ACKNOWLEDGMENTS
354
We thank Rocío Ortiz López (CIDICS-UANL) and Víctor Aguirre (Facultad de Agronomía-
355
UANL) for their support in conducting the 2D-PAGE technique.
356
AUTHOR CONTRIBUTIONS
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JGHG and CBLA designed and performed the experimental work, BPA directed the project.
358
JGHG wrote the paper with the supervision of BPA. All authors provided critical feedback and
359
helped shape the project and paper. EPO, LCDB and JCCA gave the approval to the final version
360
of the paper.
361
CONFLICT OF INTEREST
362
All authors declare no competing financial interest.
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ABBREVIATIONS USED
365
M1, Malt 1; M2, Malt 2; 2D-PAGE, Two- dimensional polyacrylamide gel electrophoresis;
366
kVh, kiloVolts per hour; HCl, Hydrochloric acid; SDS, Sodium dodecyl sulfate; DTT,
367
Dithiothreitol; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; mA,
368
miliAmpere; kDa, kiloDalton; µg, micrograms; MgCl2, magnesium chloride; FAN, Free
369
assimilable nitrogen; KI, Kolbach index.
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REFERENCES
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FIGURE CAPTIONS Figure 1. Two-dimensional protein profiles of malts M1 and M2. Yellow spots indicate detected
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spots and red spots indicate differential spots (T-test 95%)
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Figure 2. Effect of pH on the proteolytic activity of malts M1 and M2 in zymograms with 0.2 %
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gelatin
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Figure 3. General proteolytic activity per amount of total protein. Activity was measured by
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hydrolysis of azocasein
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Figure 4. Amylase activity evaluated at different pH values using zymograms with 0.2 % starch.
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The arrow indicates an amylase activity band of 27 kDa.
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Table 1. Parameters of the worts produced with the different malts
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Table 2. Protein identification by MALDI-TOF/TOF
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Supplemental Table 1. Quantitation table report of differential spots
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Table 1. Parameters of the worts produced with the different malts Malts Parameter M1 M2 Moisture (%) 4.6 4.7 Color (SRM) 2.0 2.4 Total protein (%) 12.0 12.7 Soluble protein (%) 5.7 5.1 pH 5.83 5.82 Kolbach Index (%) 47.5 40.2 Diastatic power (Base S.) 143 166 Friability (%) 70.1 51.5 Viscosity (cps) 1.58 1.61 Filtration time (min) 68 118 Alpha-amino nitrogen (ppm) 223 188
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Table 2. Protein identification by MALDI-TOF/TOF
Malt
Molecular weight Theo./Obs.
Hydrolytic enzymes a M1 62.8/59.6 5711 7703 6502 6508
pI
6.4
Identity
Accession No.
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ID Spot
b
M2
58.7/57.4
7.7
Beta-amylase; 1,4-alpha-D-glucan maltohydrolase; BetaAm1; FLAG precursor Beta-glucosidase A [Hordeum vulgare]
gi|75107132
a
M2
38.4/33.2
6.9
Glyceraldehyde-3-phosphate dehydrogenase 2, cytosolic
gi|120668
a
M2
38.7/33.2
7.3
Glyceraldehyde-3-phosphate dehydrogenase 2, cytosolic
gi|120668
gi|193073259
25.4/25
7.0
Glutathione S-transferase [Hordeum vulgare]
gi|6683765
17.7/18.9
6.6
18.9 KDa ABA-induced protein [Hordeum vulgare]
gi|1052747
Defense proteins a M1 5507
32.1/26.2
7.1
25.5/25.9
8.0
Chain A, crystalline structure of the Gh-19 Chitinase family of rye seeds Chain A, crystalline structure of an endochitinase of Hordeum vulgare L seeds at 1.8 Â resolution.
43.7/38.8
9.4
Predicted protein [Hordeum vulgare]
gi|326516774
23.0/24.2
7.0
Hypothetical protein [Hordeum vulgare]
gi|2266666
7.3
Predicted protein [Hordeum vulgare]
gi|326523037
M2
Predicted proteins a M1 8610 a M2 6301 6604 2303
M2
43.9/42.2
b
M2
18.5/18.9
5.5
Predicted protein [Hordeum vulgare]
b
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spots with qualitative differences and spots with quantitative differences.
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Stress tolerance b M2 6401 a M1 5307
gi|400977413 gi|157834680
gi|326515048
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Highlights: • Comparison of proteome of two brewing malts was done by 2D-PAGE and Zymograms
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• Quality parameters in worts were related with proteins and enzymatic activity • Difference in hydrolytic proteins were observed using zymograms
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• A small molecular size amylase was detected