Effect of growth conditions and genotype on barley yield and Î²-glucan

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Food and Beverage Chemistry/Biochemistry

Effect of growth conditions and genotype on barley yield and #-glucan content of kernels and malt Ivan Tomasi, Valeria Sileoni, Ombretta Marconi, Umberto Bonciarelli, Marcello Guiducci, Stefano Maranghi, and Giuseppe Perretti J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00891 • Publication Date (Web): 14 May 2019 Downloaded from http://pubs.acs.org on May 14, 2019

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Journal of Agricultural and Food Chemistry

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Effect of growth conditions and genotype on barley yield and -glucan content of kernels and

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malt

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Ivan Tomasi1, Valeria Sileoni1,2, Ombretta Marconi1,2*, Umberto Bonciarelli1, Marcello Guiducci1,

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Stefano Maranghi2, Giuseppe Perretti1,2 1 University

of Perugia, Department of Agricultural, Food and Environmental Science, Borgo XX

Giugno, 06121, Perugia, Italy 2

University of Perugia, Italian Brewing Research Centre, via San Costanzo s.n.c., 06126, Perugia,

Italy

* Corresponding author e-mail: [email protected] Tel: +39 075 585 7941 - Fax: +39 075 585 7946 Department of Agricultural, Food and Environmental Science - University of Perugia, via San Costanzo s.n.c. 06126 Perugia, Italy

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Abstract

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The study was conducted to evaluate the effect of growth conditions and genotype on the barley

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yield and β-glucan content of the grain and malt. Total and water-extractable (W-E) β-glucans and

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their molecular and structural properties were analyzed in nine 2-row barley varieties and

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corresponding malts. The total β-glucan content of barley is not influenced by year or by the

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cultivar, while the grain yield and W-E β-glucan content are significantly influenced by the year.

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Barley W-E β-glucans have a molecular weight between 1.0×105 and 4.0×105 Da and a random coil

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conformation. β-Glucan levels in malt are significantly lower than in barley, and neither the total

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nor the W-E β-glucans are influenced by environmental factors or genetic aspects. W-E β-glucans

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are mainly composed of fractions with Mw below 1.0×105 Da. In conclusion the molecular

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characterization of β-glucans, could represent a powerful tool to understand their role in the

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brewing process.

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KEYWORDS: β-glucan, β-glucan molecular properties, barley genotype, barley growth conditions,

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barley yield, malting.

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Introduction

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Barley (Hordeum vulgare L.) belongs to the Poaceae family and grows across many latitudes and

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longitudes with high productivity1.

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The main uses of barley are as animal fodder, raw material for the production of beer and certain

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distilled beverages and an ingredient in health food formulations due to its high content of

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biologically active compounds such as dietary fiber, especially β-glucans, and phenolic

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compounds2. Before use in the brewery, the barley must first be converted into malt by the malting

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process, which consists of several wetting and drying treatments of barley grain and involves

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complex biochemical processes. It is well documented that the malting quality of barley is

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evaluated by physiological, physical and chemical examination. However, quality parameters are

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strongly dependent on geo-location, environmental factors, varietal aspects, type (covered or naked

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type, two- or six-row type), spring or winter variety, fertilization rate, and more3-7. The malting

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quality of barley is affected not only by major chemical components in grains, such as protein and

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starch but also by many minor constituents, in particular β-glucans and arabinoxylan. In fact,

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inadequate grain malting has negative effects on the overall malt quality and is responsible for

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incomplete cell wall degradation and malt modification, giving low extract yields3. β-Glucans and

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arabinoxylans represent approximately 85% of the total nonstarch polysaccharides in barley and are

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located mainly in the endosperm cell wall and in the aleurone layer, respectively,8-11 and are

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involved in the cytolytic modification of malt. β-Glucans are composed of linear chains of glucose

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residues polymerized through β-1–3 and β-1-4 linkages. β-1-4 linkages occur in groups of two to

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four, while β-1-3 linkages occur singly. Therefore, the structure is dominated by β-1-3-linked

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cellotriosyl and cellotetraosyl units. The rest of the structure consists of longer blocks of 4-15 1-4-

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linked β-D-glucopyranosyl units12-14. The presence of β-1-3 linkages reduces the tendency to pack

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into stable, regular molecular aggregates and consequently influences the β-glucan solubility. The

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total content of β-glucans in barley normally ranges from 2 to 8%, depending on both genetic and

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environmental factors, and approximately 70% of barley β-glucans are in soluble form, although 3 ACS Paragon Plus Environment


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some parameters, such as temperature, solvent type, extraction time and enzymatic treatment, can

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influence the yield and structural characteristics of soluble β-glucans15-18.

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The decomposition of hemicelluloses is carried out during the malting process due to the

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stimulation of endogenous enzymes. The enzymatic activity increases during steeping and reaches

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its maximum during germination. During this process, the β-glucan content significantly decreases

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because specific linkages are broken, leading to the formation of lower molecular weight fractions

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with different molecular properties and solubilities19-22. During mashing, the endo-β-1-4-glucanases,

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active between 35 °C and 50 °C, can affect β-glucan content, while the β-glucan-solubilizing

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enzyme, active between 55 °C and 70 °C can solubilize them. However, the β-glucan content in

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malt and wort does not depend on the initial content of β-glucans in barley, but it is strongly

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influenced by the enzyme activity during malting and mashing.. β-glucans are often associated with

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problems of poor performance in lautering and filtration of the beer due to their ability to increase

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the viscosity of the solutions and form gels, hazes, and precipitates23. β-Glucan gel formation

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depends on numerous factors, such as shear forces, conditions and temperatures in storage cellars,

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which are characteristic factors of each individual manufacturing facility. Very often, neither the

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concentration nor the viscosity can be easily correlated with these phenomena24. Unable to establish

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a certain correlation between total β-glucan content in malt and the subsequent filterability of wort

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or beer, the focus has shifted towards the analysis of the different molecular weight fractions and

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their properties.

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Generally, in barley, the Mw values roughly range from 1.0 × 104 to 1.0 × 106 Da, and higher values

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are found using mild extraction conditions25,

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demonstrated that barley β-glucans undergo several changes in molecular weight distribution and

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structure, and the molar mass of the most abundant fraction decreased from approximately 2.6 ×105

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Da for barley to approximately 1.0 ×105 Da for malts.

26.

In a previous work, the same authors22


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The present study focused on the evaluation of the effect of genotype and environmental conditions

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on barley yield and quality parameters for malting, including total and water-extractable (W-E) β-

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glucans and their molecular properties. The β-glucan molecular characterization was performed by

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high-performance size-exclusion chromatography after extraction from the flour, and the data were

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correlated to the quality attributes of barley and malt. The main goal of this study was to examine

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the malting performance of nine barley cultivars from two different years grown under contrasting

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climatic conditions from the point of view of β-glucan molecular modification with the aim of

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investigating new parameters to evaluate the malting quality of kernels and the brewing

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performance of the malt.

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Materials and methods

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Experimental site, crop management and treatments

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Several 2-row barley varieties (namely, 7 spring varieties: Bambina, Belgravia, Concerto, Prague,

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Propino and Quench and 2 winter varieties: Scala and Violetta) were compared in two field

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experiments carried out in 2012/13 and 2014/15 at the experimental station (FIELDLAB) of the

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Department of Agricultural, Food and Environmental Sciences of the University of Perugia, Italy.

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The experiments were set up in a randomized block design with 3 replicates. The field lab is located

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on the Tiber River alluvial plain at 42.956°North, 12.376°East, at 163 m a.s.l. The soil is a typical

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Fluventic Haplustept clay-loam (20% sand, 46% silt and 34% clay, 1.4 Mg m-3 bulk density) that is

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subalkaline (pH= 7.8), poor in organic matter (12 g om kg-1, C/N ratio= 11) and extractable

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phosphorus (29.9 mg P2O5 kg-1, Olsen method) and rich in exchangeable potassium (258 mg K2O

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kg-1, int. method).

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The location is characterized by a mean annual rainfall of 831 mm (1921–2015), with November as

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the wettest month (106 mm on average) and July as the driest month (37 mm on average). The

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mean air temperature is 13.2 °C (1951–2015), ranging from 23.2 °C in July to 4.0 °C in February.

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In each year, the previous crop had been soft wheat, and the soil was fertilized just after wheat

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harvest with 75 kg P2O5 ha-1 as superphosphate. 5 ACS Paragon Plus Environment


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Barley was sown on 13 December 2012 and 12 December 2014 in single rows 0.15 m apart using a

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plot driller, with a sowing density of 400 kernels m-2. A single plot consisted of 10 barley rows that

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were each 7 m long.

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The crop received 120 kg N ha-1 as ammonium nitrate, split between tillering (i.e., 90 kg ha-1 on 14

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February 2013 and 2015) and the beginning of shooting (i.e., 30 kg ha-1 on 28 March 2013 and

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2015). The crop was maintained weed free by spraying herbicide postemergence. Barley was

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harvested on 17 July 2013 and 29 June 2015 with a plot combine.

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Weather conditions

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Weather data were collected by an automatic meteorological station inside the FIELDLAB. Figure

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1 shows the weather conditions during the barley crop cycle in 2012/13 and 2014/15 and ten-day

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mean air temperatures (full lines) compared to ten-day averages from 1951-2015 (dotted lines). Full

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bars indicate 10-day cumulated rainfall compared to the 1921-2015 average. Harrows indicate

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sowing (S) and harvest (H) dates.

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Weather conditions were contrasting in the two experimental years: the year 2013 was unusually

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rainy, as total rainfall from October to June was almost twice as much the average (1143 mm vs 661

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mm), and rains were quite frequent during the entire crop cycle. As a consequence, soils maintained

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low oxygenation for long periods, and deep drainage was intense; thus, barley plants experienced

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adverse soil conditions during the long part of their life cycle. In contrast, in the year 2014/15, total

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rainfall was near the average (i.e., 653 mm), and rains were well distributed; thus, soil conditions

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were almost optimal for barley plants during the entire crop cycle. Both years were warmer than

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usual, especially the second one, in which mean air temperatures were at least 2.0 °C higher than

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the 1951-2015 average during almost the entire crop cycle.

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Barley grain analyses

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Quality attributes important for malting barley (such as grain moisture, protein content, germinative

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energy, thousand-grain weight and sieving) were determined using Analytica EBC methods27. All

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analyses were performed in duplicate. 6 ACS Paragon Plus Environment

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Total β-glucan content in barley was measured according to the enzymatic method proposed by

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McClear and Glennie-Holmes28 in 1985 using a commercial assay kit (Megazyme International

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Ireland, Bray, Ireland). W-E β-glucans were fractionated by high performance size-exclusion

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chromatography (HPSEC) and characterized by a triple detector (TDA), following the method

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proposed by Marconi et al. in 201422. The used system was composed of a Knauer 1050 solvent

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delivery system, an HTA 300 L autosampler with a 100 μL mounted loop, a TSK PWXL guard

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column and two serially connected columns (TSKgel G5000PW, TSKgel G4000PW, Tosoh

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Corporation, Tokyo, Japan), kept at 40 °C. The eluent was 0.1 M NaNO3 containing 0.05% NaN3

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injected at a flow rate of 1 mL/min. The TDA was a Viscotek 270 model (Malvern Instruments Ltd.,

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Malvern, United Kingdom) (low and right angle light scattering, and viscometer) and a serially-

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connected Viscotek VE3850 refractive index detector kept at 35 °C. Raw data were analyzed using

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Viscotek OmniSEC software. The method was tested using 2.29 × 105 Da molecular weight β-

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glucan standard (Megazyme International Ireland, Bray, Ireland), which chromatogram is shown in

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figure 2. HPSEC-TDA analysis allows to evaluate molecular weight distributions (average molar

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mass Mn, given by the sum of the absolute masses of the various moles of molecules divided by the

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number of moles, and the weighted molar mass Mw, given by the sum of the molar masses of the

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various molecules divided by the weight), intrinsic viscosity [η], hydrodynamic radius (Rh) and

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Mark–Houwink parameters. The Mark-Houwink equation expresses the relationship between the

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intrinsic viscosity (η) and the molar mass (M): [η] = KMα, which is plotted in a double logarithmic

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scale against the Mark-Houwink constant; logK and exponent α are obtained from the intercept and

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slope, respectively. An important property of exponent a is that it provides information about the

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polymer conformation. Generally, for flexible polymer molecules in thermodynamically good

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solvents, the exponent a is approximately 0.65–0.7529. Higher values of a, e.g., equal or above the

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unity, can be found for less flexible rod-like macromolecules, such as some polysaccharides or

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polyelectrolytes. All analyses were performed in triplicate.

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Micromalting process and malt analyses 7 ACS Paragon Plus Environment


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Malting was conducted in a micromalting pilot plant provided by the Italian Brewing Research

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Centre (CERB, University of Perugia, Casalina, Italy). The plant included 4 independent

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steeping/germination tanks as well as 4 independent drying tanks, and each tank had the capacity to

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hold 4 drums filled with 0.5 kg of sample. The barley was steeped twice, once at 18 °C for 5 h

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followed by an air rest at 17 °C for 16 h and then again at 16 °C for 4 h followed by an air rest at 16

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°C for 24 h. Germination was conducted at 15 °C for 72 h. The kilning program was 15 h at 55 °C,

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4.5 h at 72 °C and finally 3.5 h at 82 °C.

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Standard quality attributes (moisture, extract, pH, viscosity, total protein, soluble protein, Kolbach

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index (KI), free amino nitrogen (FAN), friability, fermentability and diastatic power) were

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determined following the Analytica EBC methods27. All analyses were performed in duplicate.

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Total β-glucans and W-E β-glucans in malt were measured as specified for barley. All analyses

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were performed in triplicate.

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Statistical model and analyses

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The statistical analyses were performed by using Statgraphics Centurion XVI version 16.1.11

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(StatPoint Technologies, Inc., Warranton, VA). The significant variations between the different

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samples were discriminated using a one-way analysis of variance (ANOVA, p 1.2)31. The Mw was significantly

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influenced by the year, with a trend opposite to the W-E β-glucan content, meaning that the higher

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are the weighed molar masses of the soluble β-glucans, the lower their content. The average value

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of 0.65 of the exponent α of the Mark-Houwink equation indicates a random coil conformation. The 10 ACS Paragon Plus Environment

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intrinsic viscosity ranged from 2.89 to 3.99 dL/g in accordance with other researchers25. The results

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showed that the barley W-E β-glucans are mainly composed of polymers with medium Mw,

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between 1.0 × 105 and 4.0 × 105 Da, which represent 66% of the total on average.

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To better explain the relationship between the considered parameters and the influence of year and

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cultivar on barley quality, the correlation table (Table 4) and the PCA biplot (Figure 3) are shown.

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First, the year seems to have a greater influence than the cultivar on the considered parameters

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because samples harvested in 2015 are mainly in the top-right part of the plot, while the samples

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harvested in 2013 are mainly in the bottom-left part of the plot; samples of the same variety do not

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always show similar trends during the two years. The first principal component explains

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approximately 35% of the variance and discriminates the samples on the basis of the relative

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moisture, grain yield, grain dimension and protein content. In fact, samples on the right part of the

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plot show the highest values of relative moisture, grain yield, grain dimension and protein content,

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in contrast to samples on the left side of the plot. In fact, it can be seen in the correlation table that

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the high fractions of sieving (>2.5 mm) are positively and very significantly related to the grain

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yield, TKW and protein content. The total β-glucan content is close to the lower sieving fraction in

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the PCA biplot, indicating that smaller kernels show higher β-glucan contents. In fact, the total β-

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glucan content is negatively correlated with the TKW in a very significant way. Moreover,

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according to other researchers, total β-glucan content is negatively related to the protein content in a

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highly significant way16. In fact, the higher crude protein content in barley is accompanied by lower

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contents of starch and dietary fiber32. The total β-glucan content is not related to the W-E β-glucan

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content and its molecular properties or to the standard quality parameters. The W-E β-glucan

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content and its molecular properties discriminate the samples along the second principal

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component, which explains 16% of the variance. Samples in the bottom part of the plot show the

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highest W-E β-glucan content and the lowest weighed molar masses, and the opposite is true for

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samples in the top part of the plot. In fact, the W-E β-glucans and the Mw are negatively correlated 11 ACS Paragon Plus Environment


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in a very significant way, highlighting that the W-E β-glucans with lower molecular weights are

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more soluble.

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Standard quality attributes of malts obtained from the barley samples, determined according to EBC

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methods, are shown in Table 5. Most of the quality attributes of the analyzed malt samples are in

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the normal range of a pale malt, confirming the good malting attitude of barley samples and the

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efficiency of the malting process. Most of the quality attributes of malt are influenced by the year

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with the exception of moisture content, indicating good malting reproducibility. The extract values

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range from 80.7 wt % (d.m.) of Violetta to 85.6 wt % (d.m.). of Zeppelin., both harvested in 2015,

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and it is significantly influenced by the cultivar, with Zeppelin, Concerto, Propino and Belgravia

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showing the highest values and Violetta the lowest. Moreover, the extract is significantly influenced

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by the year, with the samples from 2015 showing, on average, higher values than the samples from

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2013. Malts from 2015 show, on average, higher values of viscosity and protein content, and these

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parameters are significantly influenced by year. The values of these parameters should decrease

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during malting as a consequence of the enzymatic activities23. Moreover, the protein content is also

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significantly influenced by the cultivar, with Quench, Concerto, Belgravia and Zeppelin showing

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the lowest values and Violetta and Scala showing the highest values. These values partially confirm

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the extract results; in fact, considering that the extract is linked to the enzymatic activity, it can be

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expected that the cultivars that show the highest extract values also have the lowest protein content

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and vice versa3. Moreover, the stronger protease activity of malts from 2013 barleys is also

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confirmed by the higher values of KI, soluble protein and free amino nitrogen (FAN) content,

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which are significantly influenced by the year. Additionally, these malts also show higher values of

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fermentability, indicating a stronger activity of α- and β-amylases, and friability, indicating a

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general better modification; both of these parameters are significantly influenced by the year.

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Looking at the data, it can be concluded that in 2015, grains showed a higher protein content than in

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2013, leading to less modified malts. The data confirm results previously obtained by the same

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authors33, and they clearly indicate that the weather conditions have a great influence on malt 12 ACS Paragon Plus Environment

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quality. Concerning the influence of cultivar, Scala and Violetta varieties, which showed the highest

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protein content and the greatest kernel dimension, lead to malt with a high level of protein and low

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extract, indicating poor modification. This statement is also confirmed by their high values of

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viscosity and by their low values of KI and friability.

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The total and W-E β-glucan contents in malt samples are shown in Table 2. According to previous

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studies, β-glucans in malt are significantly lower than in barley grains, with values ranging from

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0.12 to 0.62 wt % (d.m.) for the total and from 0.09 to 0.70 wt % (d.m.). for W-E β-glucans. The

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factorial analysis shows that neither the total nor the W-E β-glucans are influenced by

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environmental factors or genetic aspects. Moreover, as previously suggested by the same authors22,

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no correlation was found between total β-glucans in barley grains and in final malt, indicating that

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low-level β-glucans in grains may not be sufficient to obtain a low-β-glucan malt, without a good

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potential to produce β-glucanases. This aspect is very important in the selection of the malting

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cultivar because varieties with good malting potential may be rejected on the basis of a high level of

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total β-glucans34. In fact, the residual β-glucan content in final malt is mainly affected by the

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enzymatic activity. To better understand the degradation of β-glucans from barley to malt, it is

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useful to define a “reduction index” (Δ%) expressed as (β-glucans in barley – β-glucans in malt)/β-

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glucans in barley *100. This parameter clearly provides the percentage of β-glucans that were

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degraded during malting. As expected, the reduction index values are very high, with an average of

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89% for both total and W-E β-glucans, and they are not influenced by the year or the cultivar. Malts

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from 2013 grain, which are better modified according to quality attribute results, have, on average,

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higher values of the W-E β-glucan reduction index than those from 2015. Moreover, malts obtained

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from Scala and Violetta varieties, which are poorly modified, show the lowest reduction index

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values for both total and W-E β-glucans and consequently the highest content of these compounds,

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even if this difference was not evident in the barleys. This means that β-glucanase activity, which is

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strongly related to malt modification, has a decisive influence on the β-glucan content of malt. The

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action of this enzyme also affects the molecular properties of β-glucans in the final malt, leading to 13 ACS Paragon Plus Environment


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a decrease of the Mn values, mostly in malts from 2013 barleys, in correspondence with the higher

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reduction index. On the other hand, Mw values decreased mainly in the malts from 2015 barleys,

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leading to lower values; in fact, this parameter is very significantly influenced by year.

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Nevertheless, this degradation does not change the structure of the polymer that, on average,

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maintained the random coil conformation represented by α values of approximately 0.65, as for

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barley. However, small values of α indicated a rod-like conformation caused by the shortening of

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the chains. The degradation does not change the broad distribution of the polymers (Mw/Mn >1.2).

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The intrinsic viscosity and the hydrodynamic radius are highly significantly influenced by the year.

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In fact, following the Mw trend, they show lower values in malts from 2015 barleys. The intrinsic

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viscosity shows a significant reduction during malting in both the considered years, while the

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hydrodynamic radius is considerably reduced only in malts from 2015 barleys. The degradation of

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W-E β-glucans taking place during malting is evident observing that, unlike barley, the malt W-E β-

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glucans are mainly composed of polymers with low Mw (< 1.0 × 105 Da), which represent 46.7% of

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the total on average. As a consequence, the percentage of fractions with medium Mw (between 1.0 ×

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105 Da and 4.0 × 105 Da) is considerably reduced with respect to barley, passing from 66 to 38.8%

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of the total, while the class with high Mw (>4.0 × 105 Da) does not change and represents 14.5%.

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To better understand the relationship between the considered parameters and the influence of year

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and cultivar on malt quality, the correlation table (Table 6) and the PCA biplot (Figure 4) are

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shown. Additionally, in this case, for barley, the year seems to have a greater influence than the

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cultivar on the considered parameters because malts from 2013 barleys, are better modified and are

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mainly in the top-right part of the plot, while the sample malts from 2015 barleys were less

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modified and are mainly in the bottom-left part of the plot; samples of the same variety do not

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always show similar trends in the two years. The first principal component explains 40% of the

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variance and discriminates the malt samples on the basis of their modification. In fact, on the top-

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right part of the plot, there are quality attributes related to protein degradation, such as soluble

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protein, KI and FAN, which are positively and very significantly correlated each other and with 14 ACS Paragon Plus Environment

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other indices of modification, fermentability and friability, In the left part of the plot, there are total

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protein, viscosity and pH, which are expected to decrease during malt modification. These

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parameters are very significantly and positively correlated with each other and significantly and

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negatively correlated with the other indices of modification, such as KI, FAN, friability and

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fermentability. Nevertheless, these correlations between the standard quality attributes are already

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well known.

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The interesting results are those concerning β-glucans. In fact, total β-glucans of malt are positively

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and very significantly correlated with viscosity and significantly and negatively correlated with

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friability and fermentability. These results confirm literature data35 and indicate that a high content

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of β-glucans in a malt, related to a low activity of β-glucanase, is a good indicator of poor

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modification. Moreover, total β-glucans are positively and significantly correlated with the W-E β-

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glucans, and as a consequence, these compounds are significantly negatively correlated with

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friability, fermentability and KI. These correlations are more significant than those between the

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same modification parameters and the total β-glucans, indicating that the W-E portion is more

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representative than the total content. We can suppose that W-E β-glucans are more related to β-

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glucanase activity than total β-glucan content, and thus, a low level of W-E β-glucans in malt

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indicates good β-glucanase activity. Furthermore, W-E β-glucans are highly significantly positively

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related with the viscosity of wort, and thus, lautering could be affected by W-E rather than total β-

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glucan content. Moreover, W-E β-glucans are highly significantly positively related to the total

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protein content. In fact, the degradation of macromolecules occurring during malting requires the

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combined action of a series of enzymes, including α-amylase, β-amylase, protease, β-glucanase, β-

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D-xylosidase and β-(1,4)-endoxylanase. Consequently, it is clear that low-modified malts show a

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low enzymatic activity and high content of β-glucans and protein. Moreover, as found in barley, the

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W-E β-glucans and the Mw are significantly negatively correlated.

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In conclusion, the factorial analysis showed a variation in the barley quality parameters strongly

355

dependent on both the years and the variety. On the other hand, the total β-glucan content of barley 15 ACS Paragon Plus Environment


Page 16 of 37

356

was not influenced by year or by the cultivar, and it was negatively related to the protein content

357

and the kernel size. The W-E β-glucans in barley were approximately 70% of the total, and their

358

content was significantly influenced by the year but not significantly correlated with the standard

359

quality attributes. Most of the barley W-E β-glucans (70%) were composed of a fraction with a

360

molecular weight between 1.0 × 105 and 4.0 × 105 Da, a very broad distribution of the polymers and

361

a random coil conformation.

362

Most of the quality attributes of malt were influenced by the year of cultivation. The total and W-E

363

β-glucan contents in malt were significantly lower than in barley because of the enzymatic activity,

364

which also implies a decrease in the molecular weight, the intrinsic viscosity and the hydrodynamic

365

radius, even if the polydispersity and the conformation do not change with respect to barleys. No

366

correlation was found between total β-glucans in barley and in final malt. The total β-glucan content

367

in malt was significantly related to the wort viscosity and negatively related to the main indices of

368

modification, such as fermentability and friability. Additionally, W-E β-glucans in malt were highly

369

and negatively related to the common modification parameters with greater significance, indicating

370

that the W-E portion is more representative than the total content. W-E β-glucans are mainly

371

composed of fractions with Mw below 1.0 × 105 Da, and consequently, these molecular fractions

372

can significantly influence the wort viscosity. As reported elsewhere, Congress wort showed a high

373

content of low-MW β-glucans, and thus, their characterization in malt may have a key role in the

374

prediction of lautering performance36.

375

In this context, the molecular characterization of β-glucans, instead of total content determination,

376

represents a powerful tool to understand the role of this polymer in the brewing process, and the

377

characterization of β-glucans in wort may be useful to predict future problems during filtration.

378 379 380

Abbreviations used


Page 17 of 37


381

W-E β-glucans: water-extractable β-glucans; Da: Dalton; Mw: weighted molar mass; a.s.l: above sea

382

level; C/N: carbon/nitrogen; EBC: European Brewery Convention; HPSEC: high performance size-

383

exclusion chromatography; TDA: triple detector; η: intrinsic viscosity; Rh: hydrodynamic radius; K

384

and α: Mark-Houwink equation parameters; KI: Kolbach index; FAN: free amino nitrogen; PCA:

385

principal component analysis; TKW: thousand-kernel weight; wt %: percent weight; d.m.: dry

386

matter; Mn: average molar masses; VISCO: viscosity; NTOT: total protein; NSOL: soluble protein;

387

FRI: friability; FERM: fermentability; WEBG: W-E β-glucan; TBG: Total β-glucan.

388

Acknowledgement

389

This research was supported by the Italian Ministry of Instruction, University and Research, special

390

grant PRIN 2010-2011—prot. 2010ST3AMX_004.



391

Page 18 of 37

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16. Izydorczyk, M. S.; Storsley, J.; Labossiere, D.; MacGregor, A. W.; Rossnagel, B. G.

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Variaton in total soluble β-glucan content in hulless barley: effects of thermal physical and

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enzymic treatments. J. Agric. Food Chem. 2000, 48, 982-989.

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17. Holtekjiølen, A. K.; Uhlen, A.K.; Bråthen, E.; Sahlstrøm, S.; Knutsen, S. H. Contents of

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18. Ahmad, A.; Anjum, F. M.; Zahoor, T.; Nawaz, H.; Dilshad, S. M. R. Beta Glucan: a valuable functional ingredient in foods. Crit. Rev. Food. Sci. 2012, 52, 201-212. 19. Henry, R. J., The carbohydrates of barley grains- a review. J. Inst. Brew. 1988, 94, 71-78. 19 ACS Paragon Plus Environment


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22. Marconi, O.; Tomasi, I.; Dionisio, L.; Perretti, G.; Fantozzi, P. Effects of malting on

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molecular weight distribution and content of water-extractable β-glucans in barley. Food

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23. Briggs, D. E. The biochemistry of malting. In: Malts and malting, 1st edition; Blackie Academic and Professional: London, UK, 1998; pp. 133-229.

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24. Jin, Y. L.; Speers, A.; Paulson, A. T.; Stewart, R. J. Effects of β -Glucans and

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Environmental Factors on the Viscosities of Wort and Beer. J. Inst Brew. 2004, 110, 104–

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25. Irakli, M.; Biliaderis, C. G.; Izydorczyk, M. S.; Papadoyannis, I. N. Isolation, structural

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features and rheological properties of water-extractable β-glucans from different Greek

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barley cultivars. J. Sci. Food Agric. 2004, 84, 1170–1178.

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26. Bohm, N.; Kulicke, W. M. Rheological Studies of Barley (1→3)(1→4)-β-Glucan in

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Concentrated Solution: Mechanistic and Kinetic Investigation of the Gel Formation.

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Carbohydrate Research 1999, 315, 302–311.

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27. European Brewery Convention. Analytica-EBC, 5th edition; Fachverlag Hans Carl, Nürnberg, Germany, 2007. 28. McClear, B. V.; Glennie-Holmes, M. Enzymic quantification of (1→3) (1→4) β-D-glucan in barley and malt. J. Inst. Brew. 1985, 91, 285–295.

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29. Podzimek, S. (2011). Polymers. In: Light Scattering, Size Exclusion Chromatography and

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30. Magliano, P. N.; Prystupa, P.; Gutiérrez-Boem, F. H. Protein content of grains of different size fractions in malting barley. J. Inst. Brew. 2014; 120, 347–352. 31. Ram, A. Fundamentals of polymer engineering, 1st edition; Plenum Press: New York, N. Y., 1997. 32. Biel, W.; Jacyno, E. Chemical composition and nutritive value of spring hulled barley varieties. Bulg. J. Agric. Sci. 2013, 19, 721-727.

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33. Marconi, O.; Amigo Rubio, J. M.; Sileoni, V.; Sensidoni, M.; Perretti, G.; Fantozzi, P.

475

Influence of barley variety, timing of nitrogen fertilisation and sunn pest infestation on

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malting and brewing. J. Sci. Food Agric. 2011, 91, 820–830.

477

34. Edney, M. J.; LaBerge, D. E.; Langrell, D. E. Relationships among the β-glucan contents of

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barley, malt, malt congress extract, and beer. J. Am. Soc. Brew. Chem. 1998, 56, 164-168.

479

35. Özkara, R.; Basman, A.; Köksel, H.; ÇElík, S. Effects of cultivar and environment on β -

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glucan content and malting quality of Turkish barleys. J. Inst. Brew. 1998, 104, 217-220.

481

36. Tomasi, I.; Marconi, O.; Sileoni, V.; Perretti, G. Validation of a high performance size-

482

exclusion chromatography method to determine and characterize b-glucans in beer wort

483

using a triple-detector array. Food Chem. 2017, 214, 176–182.

484



485

Figure captions

486

Figure 1: weather condition during barley crop cycle in 2012/13 and 2014/15

Page 22 of 37

487 488

Figure 2: HPSEC-TDA chromatogram of 2.29×105 Da molecular weight βglucan standard

489

(Megazyme International Ireland, Bray, Ireland).

490 491

Figure 3: Barley Principal Component Analysis (PCA)

492

PC: principal component, RM: relative moisture, TKW: Thousand Kernels Weight, GE:

493

germinative energy, S: sieving fraction, TBG: Total β-glucan. WEBG: Water-Extractable β-glucan,

494

Mn: average molar mass, Mw: weighted molar mass, alpha and log K: Mark–Houwink equation

495

parameters, IV: intrinsic viscosity, Rh: hydrodynamic radius.

496 497

Figure 4: Malt Principal Component Analysis (PCA)

498

PC: principal component, RM: relative moisture, VISCO: viscosity, NTOT: total protein, NSOL:

499

soluble protein, KI: Kolbach Index, FAN: Free Amino Nitrogen, FRI: friability, FERM:

500

fermentability, TBG: Total β-glucan. WEBG: Water-Extractable β-glucan, Mn: average molar mass,

501

Mw: weighted molar mass, alpha and log K: Mark–Houwink equation parameters, IV: intrinsic

502

viscosity, Rh: hydrodynamic radius.

503


Page 23 of 37

504


Table 1: influence of the year and cultivar on the grain yield and quality attributes of the seven spring and two winter barley varieties. Bambina

Belgravia

Concerto

Prague

Propino

Quench

Scala

Violetta

Zeppelin

p year

p cultivar

3.59×10-8***

0.4874

6.0 × 10-4***

0.3480

0.2147

2.0×10-4***

0.7377

0.3654

0.0136*

0.0354*

4.0 × 10-3**

0.0641

Grain yield (t ha-1) 2013

3.06

Aab

4.05

Abcd

3.04

Aab

2.93

Aa

3.66

Aabcd

3.37

Aabc

4.54

Ad

3.73

Aabcd

4.38

Acd

2015

8.63

Bbc

8.62

Bbc

8.44

Bb

8.80

Bbc

7.84

Ba

8.77

Bbc

8.51

Bbc

7.82

Ba

9.00

Bc

2013

11.6

Aa

11.9

Aa

11.7

Aa

11.7

Aa

11.6

Aa

11.7

Aa

11.6

Aa

11.9

Aa

11.8

Aa

2015

12.1

Ba

12.0

Ba

12.1

Ba

11.8

Ba

12

Ba

12.2

Ba

11.9

Ba

12

Ba

12.3

Ba

2013

37.5

Aab

35.3

Aa

36.0

Aab

35.3

Aa

40.1

Aab

33.3

Aa

46.2

Ac

44.0

Ac

37.5

Aab

2015

37.7

Aab

36.8

Aa

37.6

Aab

36

Aa

38.2

Aab

37.4

Aa

45.1

Ac

44.9

Ac

38.4

Aab

Moisture (%)

Thousand Kernels Weight (g)

Germinative energy 4mL (%) 100.

100.

2013 96.0

Aa

99.0

Aa

97.0

Aa

97.0

Aa

97.0

Aa

97.0

Aa

0

Aa

0

Aa

98.0

Aa

98

Aa

100

Aa

97

Aa

99

Aa

100

Aa

96

Aa

99

Aa

97

Aa

97

Aa

2013

9.4

Aa

8.9

Aa

9.1

Aa

9.5

Aa

9.5

Aa

8.9

Aa

10.2

Aa

9.9

Aa

8.9

Aa

2015

9.4

Aa

9.9

Bab

9.4

Aa

9.5

Aa

9.5

Aa

9.8

Bab

Bbc

11.6

Bc

9.7

Bab

87.5

Ac

89.3

Acd

87.9

84.0

Ab

94.6

Ae

81.6

Af

95.8

Aef

2015 Total protein (wt % d.m.)

11

Sieving > 2.5mm (%) 2013

Acd

Aa

96.9

90.1

Ad



2015

93.2

Bab

95.8

Bc

95.5

Bc

92.3

Ba

96.0

Ac

93.1

Bab

Page 24 of 37

96.4

Acd

97.4

Bd

94.0

Bb

n: two analytical replicates. Grain yield: mean of four replicates. d.m.: dry matter. Capital letters refer to comparison between years of the same varieties. while lower-case letters refer to comparison between varieties of the same year. Values followed by the same letter are not significantly different (p < 0.05). P values result from the two-way factorial ANOVA. *p < 0.05 (significant). **p < 0.01 (very significant). ***p < 0.001 (highly significant).


Page 25 of 37


506

Table 2: influence of the year and cultivar on the total and water-extractable β-glucan content (wt % d.m.) and their Δ% value, in different barley

507

varieties and correspondent malts. Bambina Belgravia

Concerto Prague

Propino

Quench

Scala

Violetta

Zeppelin p year

p cultivar

Barley Total β-glucan

W-E β-glucan

2013 3.28

Aa

3.66

Ac

3.52

Abc

3.32

Aa

3.52

Abc

3.44

Aab

3.43

Bab

3.24

Ba

3.62

Ac

2015 3.72

Bd

3.59

Acd

3.73

Ad

3.36

Ac

3.58

Acd

3.78

Bd

2.77

Ab

2.13

Aa

3.54

Acd

2013 2.48

Bb

2.42

Bab

2.22

Aab

2.32

Bab

2.37

Bab

2.08

Ba

2.08

Aa

2.83

Bc

3.11

Bd

2015 2.02

Abc

2.14

Ac

2.21

Ac

1.78

Aab

1.74

Aab

1.50

Aa

2.01

Abc

1.73

Aab

1.95

Abc

2013 0.21

Aa

0.49

Ac

0.47

Bc

0.45

Ac

0.18

Aa

0.12

Aa

0.53

Ac

0.49

Ac

0.33

Ab

2015 0.17

Aa

0.41

Ab

0.16

Aa

0.41

Ab

0.19

Aa

0.46

Bb

0.62

Ac

0.42

Ab

0.25

Aa

2013 0.10

Aa

0.26

Bd

0.12

Aa

0.19

Bbc

0.16

Aab

0.12

Aa

0.29

Ad

0.21

Ac

0.12

Aa

2015 0.11

Aab

0.09

Aa

0.15

Bb

0.13

Aab

0.17

Ab

0.14

Bb

0.70

Be

0.63

Bd

0.26

Bc

0.592

0.244

4.0 × 10-3**

0.451

0.737

0.134

0.217

0.193

0.064

0.772

0.274

0.121

Malt Total β-glucan

W-E β-glucan

2013

94

87

87

86

95

97

85

85

91

2015

95

89

96

88

95

88

78

80

93

2013

96

89

95

92

93

94

86

93

96

2015

95

96

93

93

90

91

65

64

87

Δ% (Total β-glucan)

Δ% (W-E β-glucan)

n: three analytical replicates. d.m.: dry matter, W-E: water-extractable, Δ% = (β-glucan in barley – β-glucan in malt)/ β-glucan in barley *100. Capital letters refer to comparison between years of the same varieties, while lower-case letters refer to comparison between varieties of the same year. Values followed by the same letter are not significantly different (p < 0.05). P values result



Page 26 of 37

from the two-way factorial ANOVA. *p < 0.05 (significant), **p < 0.01 (very significant), ***p < 0.001 (highly significant).


Page 27 of 37

509


Table 3: influence of the year and cultivar on the molecular weight and structural properties of W-E β-glucan of barley and malt. Barley

Malt

2013

2015

p year

p cultivar

2013

2015

p year

p cultivar

Mn (105 Da)

1.66Ba (1.09-2.27)

1.53Aa (1.14-1.96)

0.450

0.553

0.82Aa (0.49-1.03)

1.29Aa (0.46-2.86)

0.088

0.522

Mw (105 Da)

2.37Aa (1.69-3.19)

3.08Bb (1.98-4.24)

0.040*

0.647

3.03Ab (1.01-5.76)

1.50Aa (0.59-3.56)

0.011**

0.075

α

0.67Aa (0.65-0.71)

0.63Aa (0.53-0.81)

0.171

0.198

0.69Aa (0.52-0.96)

0.63Aa (0.41-0.76)

0.329

0.206

log K

-3Aa (-3.23 - -2.89)

-2.84Aa (-3.72 - -2.34)

0.185

0.155

-3.32Aa (-4.67 - -2.44)

-3.30Aa (-4.15 - -2.07)

0.971

0.261

[η] (dL/g)

3.46Ba (2.89-3.99)

3.46Ba (2.94-3.87)

0.975

0.049*

2.14Ab (1.44-3.10)

1.12Aa (0.61-1.92)

1.0 × 10-3***

0.327

24.22Aa (19.10-34.86)

23.45Ba (19.80-25.41)

0.601

0.279

20.36Ab (15.57-25.98)

14.75Aa (10.93- 9.27)

1.0 × 10-3***

0.068

2.43 Bb (2.08-3.10)

1.90 Ba (1.50-2.21)

0.004**

0.451

0.17 Aa (0.10-0.29)

0.26 Aa (0.09-0.70)

0.217

0.193

0.45 Ba (0.15 - 0.77)

0.39 Ba (0.08 - 0.65)

0.507

0.397

0.08 Aa (0.05 - 0.13)

0.12 Aa (0.10 - 0.56)

0.439

0.263

1.71 Bb (1.46 - 2.14)

1.17 Ba (0.89 - 1.31)

0.001***

0.477

0.06 Aa (0.02 - 0.12)

0.11 Aa (0.01 - 0.33)

0.222

0.541

0.27 Ba (0.09 - 0.42)

0.34 Ba (0.17 - 0.51)

0.123

0.170

0.03 Aa (0.01 - 0.06)

0.03 Aa (0.01 - 0.06)

0.903

0.674

Rh (nm)

Total WEBG (wt % d.m.) WEBG with Mw2.5mm

Mn

Mw

[η]

Rh

WEBG

TBG

0.799***

0.138

0.145

0.417

0.617**

-0.158

0.584*

-0.226

0.201

0.022

-0.083

-0.012

-0.020

0.203

0.435

-0.234

0.398

0.218

0.167

-0.425

0.118

0.345

0.801***

0.729***

-0.212

-0.187

-0.399

0.171

-0.031

-0.599**

0.137

0.458

0.061

0.139

-0.123

0.367

0.102

0.016

0.634**

-0.190

0.013

-0.169

0.027

-0.407

-0.820***

-0.280

0.227

-0.166

0.182

-0.259

-0.225

0.608**

0.522*

0.182

-0.252

0.122

0.417

0.152

-0.628**

0.179

0.283

-0.113

0.176

0.196

0.007

Mn Mw [η] Rh WEBG

0.156

513 514

RM: Relative Moisture, TKW: Thousand Kernels Weight, GE: germinative energy, S: Sieving, Mn: average molar mass, Mw: weighted molar mass, [η]: intrinsic viscosity, Rh:

515

hydrodynamic radius, WEBG: W-E β-glucan, TBG: Total β-glucan. Asterisks indicate the significance of correlation:. *p < 0.05 (significant), **p < 0.01 (very significant), ***p

516

< 0.001 (highly significant).



517

Page 30 of 37

Table 5: influence of the year and cultivar on the quality attributes of the malts from the nine different barley varieties. p Bambina

Belgravia

Concerto

Prague

Propino

Quench

Scala

Violetta

Zeppelin

p year

cultivar

Moisture (%) 2013

4.0

Aa

4.2

Aa

4.2

Aa

4.1

2015

4.0

Aa

4.0

Aa

4.2

Aa

4.2 Aa

83.1

Ab

83.3

Ab

Aa

4.2

Aa

4.2

Aa

4.3

Aa

4.3

Aa

4.3

Aa

3.9

Aa

4.0

Aa

4.4

Aa

3.9

Aa

81.5

Aa

82.1

81.3

Aa

83.4

Ab

0.1720 4.3 Aa

0.4359

Extract (wt % d.m.) 2013

82.4

Aab

81.2

Aa

83.2

Ab

Aab

0.0120 4.9 × 10-3**

2015

84.2

Bcd

83.9

Ac

84.9

Bde

83.0

Bb

84.0

Ac

83.5

Bbc

82.4

Aab

80.7

Aa

85.6

Be

2013

5.90

Aa

5.91

Aa

5.89

Aa

5.94

Aa

5.86

Aa

5.88

Aa

5.99

Aa

5.97

Aa

5.90

Aa

2015

5.93

Aa

6.01 Ab

5.98

Aab

6.08

Abc

6.04 Bbc

6.07

Bbc

6.00 Ab

6.12

Ac

6.01

Ab

2013

1.45

Aa

1.45

Aa

1.43

Aa

1.46

Aa

1.44

Aa

1.44

Aa

1.48

Aa

1.48

Aa

1.43

Aa

2015

1.43

Aa

1.48

Aa

1.45

Aa

1.49

Aa

1.44

Aa

1.49

Aa

1.62

Bb

1.57

Bb

1.46

Aa

8.63

Aab

7.31

Aa

8.25

Aab

8.63

Aab

8.25

Aab

7.69

Aa

9.56

Ab

9.38

Ab

7.88

Aa

*

pH 7.0 × 10-3***

0.2413

0.0334*

0.0856

Viscosity (mPa s at 12°P)

Total Protein (wt % d.m.) 2013

0.0125 3.7 × 10-3**

2015

8.88

Ab

9.13

Bbc

8.13

Aa

9.63

Ac

9.31

Abc

8.63

Aab

10.44

Ad

11.63

Be

8.56

Aab

*

Soluble Protein (wt %


Page 31 of 37


d.m.) 2013

4.44

Ba

4.19

Aa

3.88

Aa

4.44

Aa

4.38

Aa

3.94

Aa

4.44

Aa

4.44

Ba

4.00

Aa

2015

3.56

Aa

3.81

Aa

4.06

Aa

3.94

Aa

3.94

Aa

3.81

Aa

4.00

Aa

3.75

Aa

3.75

Aa

2013

51.3

Bb

57.2

Bc

47.0

Aa

51.6

Bb

53.0

Bb

51.1

Bb

46.5

Ba

47.2

Ba

50.8

Bb

2015

39.8

Abc

41.9

Acd

49.8

Ae

40.8

Abc

42.4

Acd

44.1

Ad

38.4

Ab

32.3

Aa

44.0

Ad

2013

196

Bb

191

Bab

175

Bab

189

Bab

191

Bab

172

Ba

181

Bab

184

Bab

187

Bab

2015

161

Ab

159

Aab

154

Aab

131

Aa

153

Aab

146

Aab

156

Aab

136

Aab

149

Aab

2013

98

Ab

95

Ab

99

Ab

99

Bb

99

Ab

99

Ab

87

Ba

96

Bb

99

Ab

2015

98

Ab

92

Ab

97

Ab

90

Ab

96

Ab

93

Ab

70

Aa

74

Aa

97

Ab

2013

84.4

Aa

84.3

Ba

84.2

Aa

84.4

Aa

84

Aa

84.7

Ba

82.2

Aa

83.8

Aa

85.2

Ba

2015

84.2

Aa

82.1

Aa

82.9

Aa

83.3

Aa

84.8

Aa

82.7

Aa

81.3

Aa

82.4

Aa

83.2

Aa

5.1 × 10-3**

0.6802

1.0 × 10-3***

0.3520

1.0 × 10-4***

0.3130

0.0228*

0.0555

7.3 × 10-3**

0.0760

Kolbach Index

FAN (mg/L)

Friability (%)

Fermentability (%)

n: two analytical replicates. d.m.: dry matter, FAN: Free Amino Nitrogen Capital letters refer to comparison between years of the same varieties, while lower-case letters refer to comparison between varieties of the same year. Values followed by the same letter are not significantly different (p < 0.05). P values result from the two-way factorial ANOVA. *p < 0.05 (significant), **p < 0.01 (very significant), ***p < 0.001 (highly significant).

518



Page 32 of 37

519

Table 6: correlation coefficients between malt quality attributes, total and W-E β-glucan content and their molecular weight and structural

520

proprieties.. pH

VISCO

NTOT

NSOL

KI

FAN

FRI

Mw

[η]

Rh

0.422

-0.167

-0.010

-0.150

WEBG

-0.388

-0.455

0.027

-0.265

pH

0.595**

0.732***

-0.392

-0.771***

-0.840***

-0.609**

-0.60**

0.164

-0.684**

-0.519*

-0.638**

0.423

0.315

0.797***

-0.107

-0.640**

-0.453

-0.960***

-0.78***

-0.232

-0.529*

-0.189

-0.503*

0.890***

0.603**

-0.166

-0.860***

-0.547*

-0.828***

-0.63**

0.021

-0.461

-0.249

-0.453

0.712***

0.395

0.624**

0.729***

0.171

0.162

-0.565*

0.255

0.441

0.278

-0.068

0.179

0.801***

0.687**

0.577*

-0.339

0.478*

0.469*

0.511*

-0.521*

-0.206

0.48*

0.511*

-0.308

0.534*

0.598**

0.601**

-0.294

-0.015

0.773***

0.180

0.567*

0.166

0.501*

-0.908***

-0.572*

0.168

0.621**

0.305

0.577*

-0.605**

-0.518*

0.081

-0.421

-0.038

-0.145

-0.356

0.408

0.725***

-0.478*

-0.612**

0.851***

-0.067

0.188

-0.408

-0.163

NSOL KI FAN FRI FERM Mn Mw [η] Rh WEBG

-0.517*

TBG

-0.385

NTOT

0.008

Mn

Extract 0.001

VISCO

0.353

FERM

-0.308

0.506*

521

VISCO: viscosity, NTOT: total protein, NSOL: soluble protein, KI: Kolbach Index, FAN: Free Amino Nitrogen, FRI: friability, FERM: fermentability, Mn: average molar mass, Mw: weighted

522

molar mass, α and logK: Mark–Houwink equation parameters, [η]: intrinsic viscosity, Rh: hydrodynamic radius, WEBG: W-E β-glucan, TBG: Total β-glucan. Asterisks indicate the significance of

523

correlation:. *p < 0.05 (significant), **p < 0.01 (very significant), ***p < 0.001 (highly significant).


Page 33 of 37

524


Figure 1

525 526



527

Page 34 of 37

Figure 2

Response (mV)

Refractive Index (RI) Right Angle Light Scattering (RALS) Low Angle Light Scattering (LALS) Viscometer (V)

528

Retention Volume (mL)

529


Page 35 of 37

530


Figure 3

531

532 533



534

Page 36 of 37

Figure 4

535


Page 37 of 37

536


For Table Of Contents Only

537


Effect of growth conditions and genotype on barley yield and Î²-glucan

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