From native malt to pure starch – Development and characterization of a purification procedure for modified starch

Accepted Manuscript From native malt to pure starch – development and characterization of a purification procedure for modified starch M. Rittenauer, L. Kolesnik, M. Gastl, T. Becker PII:

S0268-005X(15)30157-0

DOI:

10.1016/j.foodhyd.2015.11.025

Reference:

FOOHYD 3205

To appear in:

Food Hydrocolloids

Received Date: 2 June 2015 Revised Date:

15 October 2015

Accepted Date: 18 November 2015

Please cite this article as: Rittenauer, M., Kolesnik, L., Gastl, M., Becker, T, From native malt to pure starch – development and characterization of a purification procedure for modified starch, Food Hydrocolloids (2015), doi: 10.1016/j.foodhyd.2015.11.025. 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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Rittenauer, M., Kolesnik, L., Gastl, M., Becker, T.: Email (Corresponding Author): [email protected]

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Technische Universität München, Institute of Brewing and Beverage Technology, Research Group Raw Materials based Brewing and Beverage Technology, 85354 Freising, Germany, Tel.: +49 8161 71 3653

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From native malt to pure starch – development and characterization of a purification procedure for modified starch

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Abstract

Starch characteristics influence the gelatinization process, which is an important prerequisite for the saccharification required in many industrial processes. In order to determine these characteristics in barley malt, an adapted purification procedure allowing to preserve the native starch composition and simultaneously segregating the amylolytic enzymes which were formed during the germination is indispensable.

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Therefore, this research aimed to develop a method based on a combination of dry milling, micro-sieving and density gradient centrifugation. The impact on the starch characteristics was evaluated for three germinated barley varieties.

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The purified starches showed starch contents greater than 90% and proteins contents less than 0.4%. Yields ranged from 40.3 to 48.6%, depending on the variety. Considering the starch properties, the amylose/amylopectin ratio was not modified during the purification. The circularity of the granules as well as the ratio of A- and B-type granules remained constant. The particle size distribution of A-granules was not shifted, B-granules with a specific diameter of 5 to 10 µm were slightly reduced in dependency of the native granule composition. The highest impact could be observed on the amylolytic enzymes, which were completely segregated regardless of their initial value. The standard deviation of repeatability was less than 5%, except for the determination of B-type particle size distribution (7%).

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The newly developed procedure supplements existing isolation methods of unmalted grains by enabling the purification of germinated barley in a reproducible manner, without altering the native starch properties and by providing pure starch free of amylolytic activity.

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Keywords: Starch isolation, Germinated barley, Enzyme segregation, Confocal-laserscanning-microscopy (CLSM), Granule morphology

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Raw material properties have a major influence on food production processes as well as on the final product quality. In many food systems, starch characteristics and their processinduced alterations play a key role. In the brewing industry, starch composition and morphology affect the gelatinization of raw materials during the saccharification process. An incomplete gelatinization of starch minders the enzymatic susceptibility (Kongseree & Juliano, 1972; Preedy, 2009; Slack & Wainwright, 1980; Snow & O'Dea, 1981; Sullivan & Johnson, 1964) and results in lower substrate levels required for alcoholic fermentation. The impact on the final beer quality such as a change of palate fullness, the occurrence of turbidity and an increased risk of microbial contamination remain nowadays still unclear (Schuell, 2012).

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Barley malt is the most common source of starch in the brewing industry. The determination of starch characteristics and related gelatinization properties of germinated barley remains a major challenge. The combination of amylolytic enzymes in malt samples and the addition of water, necessary for starch analyzes like the Dynamic-Scanning-Calometry (DSC), lead to measurement errors resulting from starch degradation during the sample preparation and measurement phases (Derde, Gomand, Courtin, & Delcour, 2012; Leman, Bijttebier, Goesaert, Vandeputte, & Delcour, 2006; Leman, Goesaert, Vandeputte, Lagrain, & Delcour, 2005; van Steertegem, Pareyt, Brijs, & Delcour, 2013).

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In case of ungerminated grains with no or very low amylolytic activity, starch isolation and subsequent analysis helped to gain knowledge about structure, composition and morphology of starches as well as the impact on rheological, swelling and gelatinization behavior (Arendt & Zannini, 2013; Bamforth, 2003; BeMiller & Whistler, 2009; Tester, Karkalas, & Qi, 2004). The prerequisite for the validity of these results is an isolation method, which only marginally modifies the native starch composition and characteristics. In order to fulfill this requirement, an adaption and validation of the purification method for the targeted raw material is necessary in order to prevent partial loss of granule fractions (especially the smaller granules) or granule damage during the isolation (Lindeboom, Chang, & Tyler, 2004; Wu, Li, & Gilbert, 2014). Furthermore a complete segregation of amylolytic enzymes during the purification is necessary in case of germinated grains.

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A wide range of isolation methods for cereal starches were developed with their own advantages and disadvantages. Most are based on a typical scheme, which can be divided in three parts (BeMiller & Whistler, 2009; Lindeboom et al., 2004): Firstly whole or cracked grains are steeped in water (South & Morrison, 1990), acid (McDonald & Stark, 1988; Morrison, Milligan, & Azudin, 1984) or mercuric chloride (Arbuckle & Greenwood, 1958; Greenwood & Thomson, 1959) to prepare the resistant grains for the following milling or grinding process. In the next step one or a combination of the following methods is applied to remove proteins and cellular material: Sieving, conventional- or density gradient centrifugation, enzymatic degradation by exogenous enzymes or extensive washing in toluene. Finally the purified starch is washed in water and acetone and dried afterwards.

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The first isolation procedure which was depending on the described scheme was based on steeping the kernels in mercuric chloride and removing the proteins by shaking the filtered crude starch in a toluene containing saline suspension (Arbuckle & Greenwood, 1958;

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1. Introduction

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Greenwood & Thomson, 1959). The advantage of this physical denaturation and removal of protein is that no chemical or enzymatic degradation of starch is expected (Greenwood & Robertson, J. S. M., 1954). The predominant existence of large A-type granules (> 10 µm, Lindeboom et al., 2004) in the resulting starch was explained by the remaining of small Btype starch granules (< 10 µm, Lindeboom et al., 2004) in the toluene containing saline solution (Bathgate & Palmer, 1972).

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A different approach of deproteinization is described by Morrison et al. (1984). Kernels were softened by steeping in acid followed by rubbing and centrifugation. The brown proteinaceous layer was removed from the top of the white starch pellet. In further investigations McDonald and Stark (1988) demonstrated that the brown layer contains beside protein also small starch granules which are lost if the layer is removed.

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Deproteinization of starch without a loss of B-type granules became possible through density centrifugation (South & Morrison, 1990). Therefore whole kernels were degermed, cracked and steeped in water. The separation of starch from the less dense substances such as cellular material, storage proteins and starch granule surface proteins was achieved by centrifuging through 80% (w/v) cesium chloride solution (South & Morrison, 1990; Sulaiman & Morrison, 1990).

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Based on the described methods, further purification procedures were developed for different types of cereals (Bohacenko, Chmelik, & Psota, 2006; Ibañez et al., 2007; Peng, Gao, AbdelAal, Hucl, & Chibbar, 1999; Pérez, Haros, & Suarez, 2001; Tester, Yousuf, Kettlitz, & Roper, 2007; Wankhede et al., 1990). All of these established methods focus however on unmalted grains, consisting of native starch and a restricted enzymatic activity compared to germinated grains (Georg-Kraemer, Mundstock, & Cavalli-Molina, 2001).

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During the germination, starch and its surrounding matrix is significantly modified (Shaik, Carciofi, Martens, Hebelstrup, & Blennow, 2014). This biotransformation is initiated by enhanced enzymatic activity in the grains (Kuntz & Bamforth, 2007; Palmer, 1972). Proteins (Jones & Budde, 2005; Schmitt, Skadsen, & Budde, 2013), cell wall components (Hrmova et al., 1997; Wang, Zhang, Chen, & Wu, 2004) and starch are hydrolyzed differently:

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The enzymatic degradation of starch granules is based on morphological properties such as their size and structure (Dona, Pages, Gilbert, & Kuchel, 2011). Small granules are preferably hydrolyzed from the outside by surface erosion (Bamforth, 2003; Naguleswaran, Vasanthan, Hoover, & Bressler, 2013; Palmer, 1972) without occurence of pinholes or channels on the surface (Kano, Kunitake, Karakawa, Taniguchi, & Nakamura, 1981; MacGregor & Ballance, 1980). In contrast, enzymatic hydrolysis of large granules during germination is located in defined areas, leading to pitting, hole formation and appearance of radial channels (Kano et al., 1981; MacGregor & Ballance, 1980; Palmer, 1972; You & Izydorczyk, 2007).

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These biotransformations increase the susceptibility of starch to chemical (Naguleswaran et al., 2013; You & Izydorczyk, 2007) and enzymatic (Chung, Cho, Park, Kweon, & Lim, 2012; MacGregor & Ballance, 1980) modifications. Therefore, chemical or enzymatic treatments cause a decrease of the total starch content (Chu, Hasjim, Hickey, Fox, & Gilbert, 2014; M. S. Izydorczyk, 2001; You & Izydorczyk, 2007), the molecular weight and the radius of gyration and shift the amylose/amylopectin (AM/AP) ratio (Greenwood & Thomson, 1959; Kano et al., 1981). This as well as the remaining amylolytic enzymes in the grain must be considered for the development of an adapted starch purification procedure.

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The objective of the present work is to isolate premodified starch from a germinated and enzymatically grain matrix without altering its native morphological and chemical characteristics. Furthermore, the resulting starch has to be free of amylolytic enzyme activity allowing to add the required inert substrate for water based rheological, pasting and gelatinization analyses.

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Therefore an alternative method without steeping the kernels in acid or water is required in order to prevent continuing starch modification. In order to preserve the native starch granule distribution, alternatives for the removal of the protein-starch layer after centrifugation and the washing in toluene must be found. To validate the new purification procedure, the initial malt samples just as the purified starch samples are analyzed chemically, enzymatically and morphologically and the results are analyzed statistically.

2. Materials and methods

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2.1 Sample materials

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Purifications were performed on three different barley varieties (Hordeum vulgare L.) which were grown in Germany in 2013 and micro-malted afterwards. A 2-rowed spring barley (‘Marthe’, Saaten-Union GmbH, Germany) and a 2-rowed winter barley (‘Wintmalt’, KWS Lochow GmbH, Germany) were used as samples of established commercial malting barley varieties. A food-waxy barley (betaGERSTE, Dieckmann Seeds GmbH & Co. KG, Germany) was used as a variety with an altered starch composition. All samples were malted according to the standardized micro-malting procedure of the (Anger, 2006).

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2.2 Purification procedure Starch of the malts was isolated without an initial steeping step. Therefore, the friable grains were dry milled in a laboratory mill (LM 3100, Perten Instruments, Sweden). The impact of the mill regarding broken starch granules was determined microscopically by static imaging. Different amounts of malt flour (4.0 g, 5.5 g, 7.0 g) were dispersed in 50 mL distilled water (20 °C) in order to maximize the amount of purified starch for further analyses. A preliminary purification of these samples was achieved by sieving the solution through monofilament textile filters (Schwegmann Filtrationstechnik GmbH, Germany) with different mesh sizes (50, 80, 100, 200, 400 µm). The filtrate was transferred into a 50 mL conical tube (diameter of cylindrical section: 28 mm, height: 114 mm, material: polypropylene, Sarstedt AG & Co, Germany) and centrifuged (Rotina 420R, Andreas Hettich GmbH & Co.KG, Germany) at varying rotating speeds (2000 g, 3500 g, 5000 g) for 5 minutes. The liquid supernatant was removed. The starch pellet, including the brown proteinaceous layer on top, was resuspended in 5 mL distilled water to remove remaining cell wall material, proteins and enzymes. The suspension was carefully laid on 30 mL of an 80% (m/v) cesium chloride (CsCl) solution and centrifuged at 3500 g for 5 minutes. The clear liquid supernatant was discarded and the residue washed twice with distilled water and once with acetone (purity > 99%). Afterwards, the pure starch was air dried for 24 h and stored afterwards in a desiccator (Silica Gel, relative humidity < 10%, 20 °C).

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In order to account the differing initial amount of starch the yield was calculated based on the following equation:

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ACCEPTED ‫ܿݎܽݐݏ ݈ܽݐ݋ݐ‬ℎ ݅݊ ‫ ݂݀݁݅݅ݎݑ݌‬MANUSCRIPT ‫ܿݎܽݐݏ‬ℎ (݀. ܾ. ) ݉‫ݏ݌ܵ × ݏ݌ݓ × ݏ݌‬ Y= = ‫ܿݎܽݐݏ ݈ܽݐ݋ݐ‬ℎ ݅݊ ݈݉ܽ‫݀( ݎݑ݋݈݂ ݐ‬. ܾ. ) ݉݉ × ‫݉ܵ × ݉ݓ‬ 165 166 167 168 169

Where mps is the resulting weight of purified starch (g), mm the initial weight of malt flour, which remained constant at 5.5 g for all the purifications, wps is the water content of purified starch (%), wm is the water content of malt flour (%), Sps is the total starch content of purified starch (%) and Sm is the total starch content of malt flour (%).

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2.3 Chemical and enzymatic analyses

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The starch and AM/AP content of the malt flour and the purified starch samples were determined enzymatically using the K-TSTA and K-AMYL enzyme kits (Megazyme, Ireland) and the results are reported on a dry weight basis. The test kit includes relative standard deviations within the laboratory smaller than 5% for pure starches and up to 10% for cereal flours. The α-amylase (EC 3.2.1.1) and β-amylase (EC 3.2.1.2) activity were determined using the α- and β-assay kit (Ceralpha and Betamyl3 method, Megazyme Ireland). The results are expressed in Ceralpha and Betamyl-3 units respectively. Every sample was measured in duplicate.

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The crude protein content of the starches and flours was determined based on the Kjeldahl method using a Kjeltec™ 2400 Auto Analyzer Unit (FOSS GmbH, Germany). A conversion factor of 6.25 was used to convert the analyzed percentage of nitrogen into the crude protein content (Mitteleuropäische Brautechnische Analysenkommisision, 2006). Therefore, the determined value represents the sum of proteinaceous nitrogen including enzymes, proteins and their metabolites (peptides, amino-acids) as well as non-protein nitrogen originating from amines, ribonucleic acids and their derivates.

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2.4 Visualization of samples

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Sample materials, intermediate products, wastages and the purified starches were stained with dyes in order to detect impurities (1% [w/w], Light green SF yellowish, Merck KGaA, Germany) and starch granules (0.3% [w/w] Safranine O, Merck KGaA, Germany). The samples were visualized by confocal laser scanning microscopy (CLSM, Eclipse Ti, Laser 448 nm and 635 nm, 20x air objective, Nikon Instruments Europe, Netherlands).

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2.5 Size distribution of starch granules

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Size distributions were determined by static imaging (Morphologi G3, Malvern Instruments Ltd., UK). Malt flour and starch samples were dispersed by compressed air on a glassy surface using the integrated dry powder dispersion unit. Microscopic pictures were taken automatically in a defined area around the selectable starting point. These pictures were analyzed regarding morphological characteristics in real time until 15’000 particles were characterized (Morphologi Software 8.1, Malvern Instruments Ltd). For every sample three different starting points were analyzed resulting in at least 45’000 particles per sample. Compared to other analysis, static imaging provides the selective determination of particles by morphological properties like circularity and size. This enables the exclusion of agglomerates and non-starch particles, the determination of broken starch granules as well

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To yield enough material for the targeted analyses, three batches of each variety were purified simultaneously. Trials were conducted in quadruplicates.

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as the categorization of starch granules into two classes; A-type (circle equivalent diameter, CED, 10-40 µm) and B-type (CED, 2-10 µm). The initial relative quantity based frequency of particles (q0) is additionally transformed into a relative volume frequency (q3) using the following formula: ‫ݍ‬3 (‫= )ݔ‬

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212 Where x is the circle equivalent diameter.

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In order to describe and compare the distributions mathematically, mean, mode, median as well as representative diameters CEDx(10), CEDx(50) and CEDx(90) were calculated. The latter describe the diameter of a particle, where 10, 50 and 90% of the particles are smaller than this diameter.

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3. Results and Discussion

3.1 Evaluation of the purification procedure

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For a homogenous pulverization of malt kernels, samples were dry milled without steeping to prevent enzymatic modifications. In order to investigate the impact of milling on the starch granules, samples were analyzed by static imaging (Morphologi G3). Less than 1‰ of all granules showed reduced circularities (< 0.7) and were identified as broken granules. In order to achieve a preliminary purification within 10 minutes and to ensure the segregation of husk fragments and cell wall material, suspensions with concentrations between 4.0 and 7.0 g were micro-sieved through filters of pore sizes ranging from 50 to 400 µm.

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Successful filtrations with a reasonable duration (< 20 min.) could be achieved with pore sizes of 80 and 100 µm (Figure 1). For meshes with smaller pores (50 µm), the duration of filtration exceeded 60 minutes which is an inacceptable long residence times of the amylolytic enzymes. The use of meshes with bigger pores (200, 400 µm) allowed to reduce the filtration duration to below 5 minutes, the microscopy evaluation of these samples however revealed high residues of husk fragments and cell wall material in the filtrate. To standardize the procedure (allowing for comparison) only filters with the smallest possible mesh size (80 µm) were used for the following purifications.

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A rotation speed of 3500 g during the centrifugation of the filtrate ensures the formation of a stable starch pellet. During the successive density centrifugation, this rotation speed also provides the retention of proteins and cell wall material in the CsCl phase as well as the sedimentation of starch granules in combination with the formation of a stable starch pellet on the bottom of the tube. Investigations of the separated supernatant by CLSM indicate the presence of proteins, cell wall material and a limited amount of starch granules. Compared to the relative granule composition of the final starch pellet, the supernatant consists more Btype granules (+3%). This circumstance could be explained by the STOKES equation, describing the sedimentation rate (vs) depending on the radius and on the density difference of the particles and the surrounding fluid:

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2‫ ܲߩ( ∗ ݃ ∗ ܲݎ‬− ߩ‫) ܨ‬ 9ߟ‫ܨ‬

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Where rP is the circle equivalent radius, g is the centrifugation force, ρP is the density of the particles, ρF is the density of the fluid and ηF is the dynamic viscosity of the fluid.

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Derived from this equation smaller B-granules feature a minor sedimentation rate compared to A-granules. The continuous decreasing density difference caused by the density gradient of the CsCl solution leads to a further reduction of the sedimentation rate of both types of starch granules. The combination of these effects might lead to extended centrifugation times or agglomerates with proteins or cell wall material, especially in case of B-granules. This hypothesis is supported by the observations made by purification trials with different initial weights of the sample: Masses of 4 to 5.5 g of malt flour result in an almost complete sedimentation of starch granules and the formation of a stable pellet on the bottom of the centrifugation tube. An increase in sample weight to 7 g results however in an accumulation of sample material in the cesium chloride phase. This accumulation forms a blockage hindering the remaining sample material to pass through and results in a significantly reduced mass (< 50%) of the formed starch pellet and an intolerable degree of separation after the centrifugation. To maximize the obtained amount of purified starch, 5.5 g malt flour were used for the following purification. The final procedure is illustrated in Figure 2.

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3.2 Chemical and enzymatic investigations

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The aim of the purification is to isolate starch out of a native matrix in the highest possible purity. Barley contains mainly starch and protein but also minor components such as minerals and lipids. The insufficient removal of these other components impacts the quality of the isolated starch. Chemical and enzymatic analysis of the malt flours and purified starches (summarized in Table 1) were carried out to determine the outcome of the purifications. It could be observed that the pure starch yield is depending on the barley variety and substrate used for the purification. The highest yield was obtained for malt flour of winter barley (48.6% ± 0.3%), followed by spring (41.9% ± 0.8%) and waxy barley (40.3% ± 0.3%).

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The ratio of starch and crude protein is determined by the raw material. Flour of waxy malt shows the lowest amount of total starch in combination with the highest crude protein content. Malts of spring and winter barley have significantly higher starch and lower crude protein contents as it is generally desired for the production of malt (Anger, 2006; Narziss & Back, 2012; Palmer, 2000). By the purification the starch content could be increased to over 90% and the crude protein content could be decreased to less than 0.4% in all samples. These levels are comparable to the contents found in previous starch purification studies (Bohacenko et al., 2006; Greenwood & Thomson, 1962, Tester & Qi, 2004, 2004).

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The impact of the purification on the starch composition can be determined by the AM/AP ratio. Comparable results were determined in the malts of spring and winter barley and no significant alteration was observed due to the purification. Waxy barley malt shows a low amylose content (11.2%) which is decreased by 4% during the purification. However, this decrease is within the range of the specified standard deviation provided by the manufacturer.

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In contrast to previous starch purification procedures using ungerminated grains, the segregation of amylolytic enzymes, which are expressed during the germination, is a primary aim of the developed procedure. Remarkable differences regarding the initial amounts of alpha- and beta amylases are measurable in the different malt flours (Table 1). Malt of spring barley exhibited by far the highest amylolytic activity being approximately 30% higher for both, alpha and beta amylase, compared to the one of germinated winter and waxy barley. The concerning amylases were accumulated in addition to other proteins, cell wall material, and B-type granules in the brown layer of the centrifuged filtrate after micro-sieving. These impurities, excepting the majority of B-type granules were successfully segregated during the density gradient centrifugation through cesium chloride (compare Figure 1D). After this purification step, neither alpha- nor beta amylolytic activity was detected in any of the starch samples. Regarding the setup of the purification process, this fact speaks in favor to keep the impure brown layer in order to preserve the native starch granules configuration, especially the presence of B-type granules. However, agglomerations of starch granules with proteins or cell wall substances in the brown layer or during the successive density centrifugation cannot be excluded completely.

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The quantity based starch granule distributions of barley malt flours show a bimodal behavior (Figure 3) which is typical for granules of barley starches (BeMiller & Whistler, 2009). B-type granules are dominant by quantity in all cases but the ratios of A and B-type granules depend on the barley variety (Table 2). Spring and winter barley show similar compositions of starch granules. Waxy barley has a significantly higher amount of small granules (+2.8%). This observation agrees with previous results reported by several investigations (Fredriksson, Silverio, Andersson, Eliasson, & Åman, 1998; Schirmer, Höchstötter, Jekle, Arendt, & Becker, 2013; Tang, Watanabe, & Mitsunaga, 2002). During the purification the ratios of A- and B-granules of all samples were not significantly modified (Table 2).

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The quantity and the volume based (Figure 4) granule size distributions differ noticeably from each other. As the volume increases with the cube of the radius, larger A-type granules become dominant, resulting in a nearly unimodal and symmetrical volume based distribution curve. For spring and waxy barley malt the purification process did not lead to a significantly different volume based granule distribution. In case of winter barley a slight but significant decrease of B-granules is however observable. This circumstance might be explained in a variety dependent, stronger starch granule-protein adhesion in the endosperm of the barley kernel (Palmer G. H., 1991). In the studies of Brennan, Harris, Smith, and Shewry (1996), a strong starch-protein binding is linked with poor malting characteristics, especially if B-type granules are completely engulfed in protein. They concluded that the degree of protein coating seems to have a bigger influence on the malting quality than the total amount of crude protein has. In case of the purification process, protein adhesions on B-type granules might lead to a reduced particle density. According to the law of STOKES, this results in a minor sedimentation rate which might explain the loss of B-granules in case of winter barley.

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3.3 Morphological investigation

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To determine a possible impact of the purification on the morphological starch characteristics on the granule types, a separate characterization of the unimodal A- and B-type fractions is required. Therefore the bimodal particle size distribution is split into two unimodal fractions which are summarized in Table 3 (A-type) and 4 (B-type).

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The morphology of A-type granules can be compared with large granules originating from ungerminated barley (Naguleswaran et al., 2013; Tang et al., 2002), including the observation that granules of waxy barley indicate a reduced diameter.

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A standard deviation of repeatability below 5% could be achieved for all purified samples. Therefore the procedure to purify starch can be assumed as reproducible. Furthermore no significant deviations between the circularity of granules in malt flour (0.88) and purified starch (0.89) were observed. Modes and medians (CEDx50) of the particle size distributions were not significantly altered by the purification process. The mean values of spring and winter barley remained constant, the mean of waxy barley was however reduced by 5%.

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The results of static imaging indicate that size reduction of starch granules by enzymatic degradation as well as size enhancement or shape transformation due to granule swelling during the purification process were successfully and reproducibly suppressed. Pinholes on the surface of starch granules were not observed before and after the purification, neither by static imaging nor by CLSM. The amount of broken starch granules remained at a constant low level (< 1‰).

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In case of B-type granules (Table 4), the standard deviation of repeatability was with 7% slightly higher compared to the one of the A-granule fraction (5%). The significantly higher degree of circularity of 0.95 on average compared to the A-type granules indicates an almost spherical shape. The purification had no significant impact on the circularity of B-type granules of spring- and waxy barley but on the circularity of winter barley. However, the increase in circularity (+2%) has to be considered in the context of a very low standard deviation of repeatability, which is lower than 1%. Therefore the results are in a reasonable range.

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B-type granules of spring and winter barley showed reduced diameters (decrease of mean and mode by 13% in average) after the purification. As it can be seen in Table 4 and Figure 5, the amount of B-type granules in the range of 2–5 µm increased in combination with a decrease of granules ranging from 5–10 µm. Because of the dependency of these relative results and the fact that no small B-type granules were added during the purification, the loss of B-type granules with a diameter from 5–10 µm might be a possible explanation. Aggregation of granules, break up due to shear forces or enzymatic hydrolyze might be responsible for this minor shift.

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In contrast, no significant impact on the morphology of purified waxy starch could be observed. This divergent behavior might result from the compositional differences of the raw material illustrated in Table 4 and Figure 5. Flours of spring and winter barley demonstrate comparable granule distributions where approximately 50% of all particles lie in the range of 5 to 10 µm. However, in waxy flour only 25% of the granules lie in this specific range. Furthermore it needs to be considered that the reduced total starch content in waxy flour results in a lower total number of starch granules in the sample.

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An explanation for the loss of those particles could be the already mentioned partial restraining during the density centrifugation. Proteins, cell wall material and especially B-type

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granules accumulate during the centrifugation process in the CsCl phase hindering following starch granules to pass by and thus agglomerating. If a critical concentration of granules is reached this might lead to the reduced diameters of B-type granules.

4. Conclusion This study aimed at developing and evaluating a reproducible starch isolation procedure for modified starch from germinated barley. A combination of milling, micro-sieving, density centrifugation and acetone treatment was determined to be most suitable ensuring reproducible results, the preservation of native starch characteristics and a complete segregation of amylolytic enzymes.

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The granule morphology of the raw materials and the respective purified starches show bimodal granule distributions by number as well as nearly unimodal granule distributions by volume. The dominance of A- or B-type granules is therefore depending on the considered type of distribution. Within these general conditions, the final granule composition is determined by the native starch composition of the respective barley variety. Commercial malting barleys show consistent results regarding chemical starch composition and granule morphology. Waxy barley malt however, shows a reduced amount of total starch, a typical high level of amylopectin and a lower percentage of B-type granules ranging from 5–10 µm. The results suggest a correlation of the initial amount of granules ranging from 5–10 µm and the loss of these B-type granules during the purification. Therefore the mean diameter of Btype granules of spring and winter barley was reduced during the purification but no impact on the morphological characteristics of waxy barley within the B-type granule fraction was observed.

396 397 398 399 400 401 402

For a further improvement of the procedure, a screening of the raw material in advance might help to determine the amount of critical B-type granules and adapt the sample weight or adjust the parameters of density centrifugation. An increase of the available cross section during the density centrifugation could prevent agglomerations and residues in the CsCl phase. However, it has to be mentioned that the described reduction did not inevitably lead to a significant change of the general A- / B-type ratio of the purified starch samples and thus can be tolerated in most cases.

403 404 405 406 407 408 409

Summarized the developed method provides a reproducible manner to isolate starch from germinated barley varieties. By preserving the native granule composition and morphology the obtained starch can be used for further specific investigations. The segregation of amylolytic enzymes avoids adulterations of measurement results. Furthermore the segregated enzymes might be regained and used as an authentic source of malt enzymes for prospective trials. However, the impact of the purification (centrifugation, ionic strength of CsCl solution) on the different classes of enzymes must be determined previously.

410 411

The described purification procedure supplements existing isolation methods of unmalted grains and can be adapted to other germinated cereals in the future.

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5. Acknowledgements The authors thank the Fraunhofer Institute for Process engineering and Packaging (IVV, Germany, Freising) for the possibility of static imaging and the kind and helpful support.

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Anger, H. M. (2006). Brautechnische Analysenmethoden - Band 1: Methodensammlung der Mitteleuropäischen Brautechnischen Analysenkommisision. Freising: Selbstverlag der MEBAK. Arbuckle, A. W., & Greenwood, C. T. (1958). 535. Physicochemical studies on starches. Part XIII. The fractionation of oat and wheat starches. Journal of the Chemical Society (Resumed), 2626. Arendt, E. K., & Zannini, E. (2013). Barley. In Cereal Grains for the Food and Beverage Industries (pp. 155-201e). Elsevier. Bamforth, C. Dr. (2003). Barley and Malt Starch in Brewing: A General Review. MBAA Technical Quarterly, 40(2), 88–97. Bathgate, G. N., & Palmer, G. H. (1972). A Reassessment of the Chemical Structure of Barley and Wheat Starch Granules. Starch - Stärke, 24(10), 336– 341. BeMiller, J. N., & Whistler, R. L. (2009). Starch: Chemistry and technology (3rd ed.). London: Academic. Bohacenko, I., Chmelik, J., & Psota, V. (2006). Determination of the Contents of A- and B-Starches in Barley using Low Angle Laser Light Scattering. Czech Journal of Food Science, 24(1), 11–18. Brennan, C. S., Harris, N., Smith, D., & Shewry, P. R. (1996). Structural differences in the mature endosperms of good and poor malting barley cultivars. Journal of Cereal Science, 24(2), 171–177. Chu, S., Hasjim, J., Hickey, L. T., Fox, G., & Gilbert, R. G. (2014). Structural Changes of Starch Molecules in Barley Grains During Germination. Cereal Chemistry Journal, 91(5), 431–437. Chung, H.-J., Cho, D.-W., Park, J.-D., Kweon, D.-K., & Lim, S.-T. (2012). In vitro starch digestibility and pasting properties of germinated brown rice after hydrothermal treatments. Journal of Cereal Science, 56(2), 451–456. Derde, L. J., Gomand, S. V., Courtin, C. M., & Delcour, J. A. (2012). Characterisation of three starch degrading enzymes: thermostable βamylase, maltotetraogenic and maltogenic α-amylases. Food chemistry, 135(2), 713–721. Dona, A. C., Pages, G., Gilbert, R. G., & Kuchel, P. W. (2011). Starch granule characterization by kinetic analysis of their stages during enzymic hydrolysis: 1H nuclear magnetic resonance studies. Carbohydrate Polymers, 83(4), 1775–1786. Fredriksson, H., Silverio, J., Andersson, R., Eliasson, A.-C., & Åman, P. (1998). The influence of amylose and amylopectin characteristics on gelatinization and retrogradation properties of different starches. Carbohydrate Polymers, 35(3–4), 119–134. Georg-Kraemer, J. E., Mundstock, E. C., & Cavalli-Molina, S. (2001). Developmental Expression of Amylases During Barley Malting. Journal of Cereal Science, 33(3), 279–288. Greenwood, C. T., & Robertson, J. S. M. (1954). Physicochemical studies on starches. Part I. The characterization of the starch present in the seeds of

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6. References

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TABLE CAPTIONS Table 1: Chemical and enzymatic characterization of malt flours and corresponding purified starches, ± sd., (n = 4).

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Table 2: Quantity and volume ratios of starch granules of the different barley malt flours and resulting purified starches; ± sd., (n = 4).

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Table 3: Morphological characteristics of unimodal A-granule fractions of malt flours and resulting purified starches (quantity based); ± sd., (n = 4).

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Table 4: Morphological characteristics of unimodal B-granule fractions of malt flours and resulting purified starches (quantity based); ± sd., (n = 4).

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FIGURE CAPTIONS

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Figure 1: Confocal-laser-scanning-micrographs: Starch granules and cell wall material appear green, proteins red; scale bar: 200 µm; malt flour (A), residue on a 80 µm micro-sieve (B), crude starch (C), supernatant after density gradient centrifugation (D), purified malt starch (E)

Figure 2: Standardized procedure applied to purify barley malt starch

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Figure 3: Relative frequency of starch granules by quantity of different barley malt flours (smoothed over 9 points, n = 4): Spring barley; Winter barley; Waxy barley;

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Figure 4: Relative frequency of starch granules by volume of different barley malt flours (smoothed over 9 points, n = 4) Spring barley; Winter barley; Waxy barley;

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Figure 5: Cumulative particle size distributions by quantity of B-type granules of different malt flours and respective purified starches (smoothed over 9 points, n = 4) Spring Barley Malt; Spring Barley Starch; Waxy Barley Malt; Waxy Barley Starch Results of winter barley malt and starch are not shown for reasons of clarity but are comparable to those of spring barley malt and starch (compare Table 4)

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Starch Content (% w/w db)

Protein Content (% w/w db)

Amylose (%)

Amylopectin (%)

α - Amylase β - Amylase (U/g) (U/g)

Spring Barley Malt Flour

57.3 ± 0.4

9.9 ± 0.1

26.9 ± 1.2

73.1 ± 1.2

308 ± 9

990 ± 2

Purified Starch

90.5 ± 1.2

0.3 ± 0.1

24.6 ± 1.8

75.4 ± 1.8

n.d.

n.d.

Malt Flour

58.3 ± 1.2

8.6 ± 0.1

27.9 ± 0.1

72.1 ± 0.1

189 ± 9

752 ± 4

Purified Starch

90.7 ± 1.3

0.4 ± 0.1

27.6 ± 0.1

72.4 ± 0.1

n.d.

n.d.

48.9 ± 0.8

11.0 ± 0.1 0.3 ± 0.1

11.2 ± 0.7

204 ± 3

725 ± 4

Waxy Barley Malt Flour

a

b

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b

91.9 ± 2.3 92.8 ± 1.8 Purified Starch 7.2 ± 1.8 n.d. Means are significantly different but within the specified standard deviation provided by the manufacturer (paired t-test, p < 0.05; two-tailed).

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n.d.

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Quantity Ratio (%)

Volume Ratio (%)

A-Type

B-Type

A-Type

B-Type

Malt Flour

23.3 ± 1.2

76.7 ± 1.2

93.7 ± 1,9

6.3 ± 1.9

Purified Starch

25.8 ± 1.5

74.2 ± 1.5

94.9 ± 0,9

5.1 ± 0.9

Malt Flour

24.0 ± 1.8

76.0 ± 1.8

92.2 ± 1,3

a

7.8 ± 1.3

Purified Starch

24.3 ± 1.1

75.7 ± 1.1

94.6 ± 0,6

b

5.4 ± 0.6

Malt Flour

20.9 ± 1.1

79.1 ± 1.1

91.8 ± 0.8

8.2 ± 0.8

Purified Starch

21.0 ± 1.6

79.0 ± 1.6

90.0 ± 1.0

10.0 ± 1.0

Spring Barley

Winter Barley

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Means in the same row with different letters are significantly different (paired t-test, p < 0.05; two-tailed).

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CED Mean CED Mode CEDx(10) CEDx(50) CEDx(90) Circularity (µm) (µm) (µm) (µm) (µm) (-)

Spring Barley a

16.7 ± 0.9 24.8 ± 0.6 0.88 ± 0.01

b

17.7 ± 0.5 24.5 ± 0.5 0.90 ± 0.02

a

16.7 ± 0.2 24.3 ± 0.3 0.87 ± 0.02

b

16.9 ± 0.4 24.3 ± 0.5 0.88 ± 0.01

17.6 ± 0.6

18.6 ± 0.2 10.9 ± 0.2

Purified Starch 18.2 ± 0.4

18.9 ± 0.5 12.0 ± 0.3

Malt Flour

Malt Flour

17.5 ± 0.2

18.3 ± 0.6 11.1 ± 0.2

Purified Starch 17.7 ± 0.4

18.2 ± 0.5 11.5 ± 0.2

Waxy Barley Malt Flour

a

16.0 ± 0.1

14.6 ± 0.5

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0.90 ± 0.01

b

0.90 ± 0.01

10.9 ± 0.1 15.0 ± 0.1 21.9 ± 0.3

b

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Purified Starch 15.2 ± 0.2 14.0 ± 0.6 10.7 ± 0.1 14.4 ± 0.2 20.3 ± 0.4 Means in the same row with different letters are significantly different (paired t-test, p < 0.05; two-tailed)

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CED Mean CED Mode CEDx(10) CEDx(50) CEDx(90) Circularity (µm) (µm) (µm) (µm) (µm) (-)

Spring Barley Malt Flour Purified Starch

a

a

5.2 ± 0.3

5.1 ± 0.2

b

4.6 ± 0.2

2.9 ± 0.2

a

5.0 ± 0.3

b

2.6 ± 0.1

4.3 ± 0.2

a

2.9 ± 0.2

a

4.8 ± 0.2

4.4 ± 0.2

a

0.95 ± 0.01

b

0.96 ± 0.01

a

0.94 ± 0.01

a b

7.7 ± 0.3

b

7.0 ± 0.2

a

7.7 ± 0.2

Winter Barley a

5.1 ± 0.2

Purified Starch

4.4 ± 0.1

b

4.3 ± 0.2

b

2.5 ± 0.1

b

4.1 ± 0.1

b

6.6 ± 0.2

b

0.96 ± 0.01

4.2 ± 0.1

3.7± 0.1 3.8 ± 0.1

2.2 ± 0.1

3.8 ± 0.1

6.8 ± 0.1

0.95 ± 0.01

4.2 ± 0.1 2.3 ± 0.1 3.7 ± 0.1 6.9 ± 0.2 Purified Starch Means in the same row with different letters are significantly different (paired t-test, p < 0.05; two-tailed)

0.95 ± 0.01

4.8 ± 0.3

Waxy Barley

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ACCEPTED MANUSCRIPT Whole barley malt kernels

↓ Crushing of grain by lab mill Dispersing 5.5 g of homogeneous malt flour in 45 mL distilled water

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↓ Malt flour-water suspension (Fig. 1A)

↓

Micro-sieving through filter with 80 µm pores

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Centrifugation of the filtrate (3500 g; 5 min.) Discarding of supernatant (Fig. 1B)

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↓

Crude starch (Fig. 1C)

↓

Dispersing of crude starch in 5 mL distilled water Centrifugation of crude starch suspension through 30 mL of 80% CsCl (3500 g; 5 min.)

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Discarding of supernatant (Fig. 1D)

Washing of the residue (twice) with distilled water (Centrifugation: 3500 g; 5 min., twice) Acetone washing and air drying (Centrifugation: 3500 g; 5 min.)

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Pure Starch (Fig. 1E)

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HIGHLIGHTS An adapted starch purification procedure for germinated barley was developed

•

3 barley varieties were germinated, purified and analyzed

•

Starch content was > 90%, protein content < 0,4% after the purification

•

Starch composition was not altered significantly

•

Amylolytic enzymes were completely segregated during the purification

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•