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Biochemical heterogeneity of malt is caused by both biological variationand differences in processing: I. Individual grain analyses of biochemicalparameters in differently steeped barley (Hordeum vulgare L.) malts

Maik Kleinwchter a, Christian Mller b, Frank-Jrgen Methner b, Dirk Selmar a,a Institut fr Panzenbiologie, Technische Universitt Braunschweig, Mendelssohnstrae 4, D-38106 Braunschweig, Germanyb Institut fr Biotechnologie, Technische Universitt Berlin, Seestrae 13, D-13353 Berlin, Germany

a r t i c l e i n f o

Article history:Received 30 April 2013Received in revised form 28 August 2013Accepted 16 September 2013Available online 25 September 2013

Keywords:Hordeum vulgare L.MaltingBiochemical homogeneitySteep aeration

a b s t r a c t

Using individual grain analyses, the degree of inherent biological variation in germinating barley seedshas been established. Even under homogenous laboratory conditions, the activities of the germination-related enzymes a-amylase, b-amylase and b-glucanase varied by a factor of two to three. The compar-ison with single grain analyses of different industrially produced malts (steeping systems withoutaeration, with air suction and pressurised aeration) revealed that the heterogeneity of these malts nearlytripled. This increase may be due to the gradients in O2 and CO2 that arise in large industrial steepingvessels. The most homogenous malting in the industrial systems was achieved without any aerationduring steeping. Therefore, to improve homogeneity, the common practise of steep aeration should beomitted. Germination progression was quite different within the three exhaustively aerated attempts,which indicated that gaseous composition was not the only factor affecting germination progression.

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1. Introduction

Malting is the process of converting cereal seeds into malt foruse in brewing or distilling. In the rst stage of this process, thecereal seeds1, e.g., barley (Hordeum vulgare L.), are germinated. Ger-mination is induced by the imbibition of the barley seeds in steepingvessels for 12 d. This so-called steeping process generally includes23 immersion phases which are split up with dry rest periods. Aftersteeping, the germinating grains are incubated for a further 34 d ingermination boxes for sprouting. Seedling development is then ter-minated by kiln-drying. The aim of the germination phase of maltingis to induce the synthesis of germination-related enzymes, i.e., cellwall and protein-degrading enzymes (e.g., b-glucanases and differ-ent proteases), as well as starch mobilising enzymes (e.g., a- andb-amylases). Owing to the concerted activities of these enzymes,the starch stored in amyloplasts can be hydrolysed, leading to the re-

lease of low molecular carbohydrates, such as glucose, maltose, mal-totriose, etc. These provide nutrition to the developing embryo.Furthermore, a suitable degree of cellular modication is requiredfor the subsequent brewing process, since the brewing performanceof the malt (e.g., the lterability of the mash) strongly depends onthe degree of breakdown in cellular structures. In the third stageof malting, kilning, the seedlings are dried and slightly roasted in akiln-oven at temperatures ranging from 50 C to 90 C. This rapidlyaborts all metabolic processes by water deprivation and heat effectsand, due to the high temperatures, Maillard reactions take place,which lead to the formation of several of the typical colour and a-vour compounds found in malt (for detailed information on malting,refer to Kunze, 2010; Narziss, 1999).

Many studies have demonstrated that barley seed germinationand early seedling development are strongly inuenced by exter-nal factors, such as the temperature, the availability of O2 andthe prevailing CO2 concentration (Kleinwchter, Meyer, & Selmar,2012; Narziss, 1999). It is well-known that a certain concentrationof O2 is required for regular barley seed germination and seedlingdevelopment: under low O2 partial pressure, the germination pro-gression is retarded, while it is completely inhibited in the absenceof O2 (e.g., Kleinwchter et al., 2012; Narziss, 1999). After beingincubated for 78 d under anoxia, barley seeds even die (Perata,Loreti, Guglielminetti, & Alpi, 1998). With respect to the underlyingmechanisms, Hanson and Jacobson (1984) reported that the tran-scription of germination-related enzymes (i.e., a-amylase) didnot occur under anoxia. For this reason, barley embryos are

0308-8146/$ - see front matter 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.foodchem.2013.09.090

Corresponding author. Tel.: +49 (0) 531 391 5881; fax: +49 (0) 531 391 8180.E-mail addresses: [email protected] (M. Kleinwchter), c.mueller@

tu-berlin.de (C. Mller), [email protected] (F.-J. Methner),[email protected] (D. Selmar).

1 Sensu stricto barley grains represent fruits, i.e., caryopses. Due to the specicfusion of seed coat and pericarp, it is not possible to distinguish between fruit andseed. With respect to most aspects, grains indeed should be denoted as fruits.However, in all works that mainly deal with the characteristic physiological attributesof seeds, e.g., germination, the term seed should be used, since the basic metabolicreactions are related to seed physiology and not to fruit physiology. Accordingly, inthis treatise, barley grains are denoted as seeds.

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supposed to die due to carbohydrate starvation. However, furtherdetails on the inhibition of barley seed germination under anoxiousconditions have not been elucidated until now. Interestingly, sim-ilar inhibitory effects on barley seed germination have recentlybeen shown for CO2 despite the presence of ambient O2 concentra-tions (Kleinwchter et al., 2012). Yet, the mechanisms behind thisphenomenon are unclear.

In line with these ndings, it is commonplace in industrial malt-ing to aerate extensively during steeping and subsequent germina-tion in order to avoid delays in germination caused by O2deciency or enhanced CO2 concentrations (Albers, Drost, & Pes-man, 1983; French & McRuer, 1990; Gibbons, 1983). Nonetheless,the practise of steep aeration is considered controversial by someauthors; Wilhelmson et al. (2006) showed that aeration at thebeginning of the steeping process had only a limited effect on bar-ley seed germination and malt quality. Therefore, aeration seemsnot to be necessary at the beginning of steeping. Other researchersgenerally question the common practise of steep aeration, since anexcessive supply of O2 could also strongly promote the growth ofthe embryos, which would lead to enhanced substance losses dur-ing malting (Cantrell, 1987; Enari, Linnahalme, & Linko, 1961; Kelly& Briggs, 1992).

It is also known that besides the overall levels of biochemicalparameters (e.g., enzyme activities and b-glucan contents), theirdistribution, i.e., the homogeneity in the batch, also plays animportant role in the brewing performance of the malt (Palmer,2000), but the impact of steep aeration on the biochemical homo-geneity of malt has not been analysed until now. In the last decade,de S & Palmer published three related articles (de S & Palmer,2004, 2006; Palmer, 2000) which exhaustively dealt with thedetermination of biochemical homogeneity (i.e., that of b-glucan-ase activity, b-glucan contents and nitrogen content), as well aswith the impact of biochemical homogeneity on brewing perfor-mance (i.e., the lterability of the mash). Unfortunately, the stan-dard (recommended) methods for malt analysis are not capableof detecting inhomogeneities, since these methods are based onthe analysis of pooled, homogenised samples, from which onlyaverage values can be obtained. Thus, there is a need to performindividual grain analysis and to arrange and analyse the data in fre-quency distributions instead of displaying them as average values(Palmer, 2000). Furthermore, the authors found that the homoge-neity of the protein and the b-glucan breakdown had a direct im-pact on the brewing performance of the malt. In turn, de S andPalmer (2004) came to the conclusion that inhomogeneities inendosperm modication, where a small, but signicant, numberof grains showed limited breakdown of cell wall (b-glucan) mate-rials, could cause severe brewhouse problems with respect to thelterability of the mash.

This study addresses how the processing, in particular steepaeration, impacts on the biochemical homogeneity of malt. Ini-tially, the corresponding enzyme assays for standard malt analysishad to be scaled-down so that they were able to quantify the en-zyme activities in individual barley grains (3050 mg). In orderto monitor the progression and homogeneity of the germinationprocesses, the activities of a- and b-amylase and b-glucanase weredetermined. Additionally, c-aminobutyric acid (GABA) contentswere analysed as an indicator for plant stress metabolism (Kle-inwchter et al., 2012).

In a second step, small-scale laboratory germinations to ensureuniform germination conditions were performed in order to estab-lish a reference for assessing the biochemical homogeneity of themalts produced under different processing conditions. Subse-quently, samples from three different steeping systems (withoutaeration, air suction and pressurised aeration) were analysed byindividual grain analysis. In order to assess homogeneity, the datawere arranged in graphs that display the distribution of the values

in combination with the average value and the coefcient of vari-ation (measure for the degree of homogeneity).

2. Materials and methods

2.1. Germination under laboratory conditions (continuous aeration)

The barley seeds (Hordeum vulgare L., var. Braemar) used for thisexperiment derived from a single batch of malting barley (CargillDeutschland GmbH, Salzgitter, Germany). The barley was steepedin a steeping vessel and then germinated for several days in specialgermination boxes to mimic the industrial malting process in asimplied manner. For this, exactly 150 g of the barley seeds weresoaked for 24 h in 1 L of tap water in an Erlenmeyer ask. Pressur-ised air was introduced through a gas frit (30 L/h). To ensure anevenly distribution of oxygen, the steeping good was consistentlymixed using a magnetic stirrer. After steeping, the moisture con-tent of the barley seeds was about 42%. Then the seeds were spreadout on a slightly elevated sieve placed on a plastic socket in a clos-able plastic container (26 10 9 cm). The plastic socket allowedthe seedlings to be uniformly exposed to a stream of air (30 L/h)throughout the entire germination period (72 h). The air intro-duced into the germination systemwas saturated with water usingan impinger. The layer thickness of the germination good was


Saladin boxes and one circular box). In the germination boxes, thebarley seedlings were intensively aerated with humidied andtempered air. The beds of barley were thoroughly stirred threetimes a day by vertical screws. The layer thicknesses of the germi-nation beds were 0.9 m (Saladin boxes) and 1.20 m (circular box).The temperature in the different germination boxes varied be-tween 15 C and 19 C. Samples were taken after 96 h total germi-nation time (including the steeping period) at three randomlychosen points in the barley beds using a sampling lance. After athorough mixing, the plant material was directly shock-frozen inliquid N2. Single, randomly chosen grains from these samples werethen homogenized using a Retsch MM 200 ball mill and stored at20 C before enzyme activity and GABA analysis were performed.

2.3. Determination of a- and b-amylase and b-glucanase activities

In order to determine a- and b-amylase and b-glucanase activ-ities, standard assays from Megazyme (Ireland) were used (Ceral-pha, Betamyl-3, b-amylase, and malt & Bacterial b-glucanase &cellulase, respectively). The substrate, p-nitrophenyl maltoheptao-side, was used to determine a-amylase activity. Its hydrolysisyields p-nitrophenyl maltosaccharide, which is further cleaved inthe presence of excess a-glucosidase to yield glucose and p-nitro-phenol, the concentration of which is measured photometrically(400 nm). The betamyl-3 method, used for the determination ofb-amylase activity, employs p-nitrophenyl-b-maltotrioside as thesubstrate yielding p-nitrophenyl-b-glucoside. This is hydrolysedby b-glucosidase and gives rise to glucose and p-nitrophenol,which is quantied photometrically as in the a-amylase assay.Analysis of b-glucanase activity is based on the cleavage of aninsoluble azo-barley glucan substrate. When the dyed substrateis hydrolysed by glucanases, dyed fragments are liberated, whichare then separated from the insoluble substrate by centrifugation.The absorbance (590 nm) of the supernatant corresponds to theglucanase activity. The standard procedures recommended by themanufacturer were used, but the sample weight and extractionbuffer volumes were reduced proportionally. In order to determinethe a- and b-amylase activities, the homogenised plant materialsof single barley grains (3050 mg, 15 per time point) were ex-tracted with 500 lL of extraction buffer, corresponding to a reduc-tion of 1/10 in the suggested values. In the case of b-glucanase,single homogenised barley grains were extracted with 800 lL ofextraction buffer, which also corresponded to a reduction of 1/10.Corresponding comparisons of the mean enzyme activities gainedfrom 15 individual grain analyses with those from the analysesof pooled samples proved the applicability of the reduction of 1/10 for both assays.

2.4. Determination of GABA contents

GABA was quantied by HPLC according to Bytof, Knopp, Schi-eberle, Teutsch, and Selmar (2005). Before extraction, 20 mg ofthe homogenised barley grains was spiked with norvaline(40 nmol) as internal standard. For extraction, 10 mL of sulfosali-cylic acid (4% w/v) was added to the freeze dried powder and thenthe extraction mixture was sonicated for 30 min and incubatedovernight at 4 C. After mixing, a 2 mL aliquot of the extractionsolution was centrifuged and ltered. The amino acids were deri-vatised with o-phthaldialdehyde (OPA) before HPLC analysis usinga Spark Holland Midas autosampler. The amino acid derivativeswere separated on a C18 column (Nucleosil 100, 5 lm, MachereyNagel, 250 4.0 mm) using a binary gradient (A, 5% MeOH, 5%ACN, 2% THF, 88% 50 mM sodium acetate buffer, pH 6.2; B, 40%MeOH, 40% ACN, 20% sodium acetate buffer) at a ow rate of1.3 mL/min. The derivatives were detected using an RF-551 Shima-dzu uorescence detector (kex = 334 nm; kem = 425 nm) and

quantied by external calibration. Duplicate analyses were carriedout on each sample.

2.5. Data analysis

Each series of analyses consisted of 15 single analyses of 15individual barley grains. In order to display the biochemical homo-geneity of the malts, the data were arranged in frequency distribu-tions (establishment of a reference) or in bar charts (comparisonbetween different steeping systems). In the bar charts, the meanvalue of the 15 single analyses, the maximal relative differencesfrom the mean value, and the coefcient of variation (relative stan-dard deviation for the data set) are displayed. The GABA contentswere not statistically analysed to clearly distinguish between thisstress related low molecular metabolite and the enzymes, whichare used as markers for germination processes.

3. Results and discussion

3.1. Small-scale germination assays: estimation of the inherentbiological variation

Before the variability caused by the different industrial process-ing methods could be estimated, the inherent biological variationhad to be determined. Thus, small-scale germination assays, ensur-ing identical conditions for each seed, were conducted. The princi-pal nding of this experiment was the large individual extent towhich the analysed biochemical parameters varied (a-amylase,b-amylase and b-glucanase activity as well as GABA contents). Thiscan be seen by the wide spread of the values in the distributioncharts after 96 h of germination (Fig. 1). However, these variationswere not seen in the ungerminated barley seeds. The variabilityincreased over time during seedling development, which is shownby the time-dependent broadening of the distribution pattern.Fluctuations by a factor of two to three seem to be standard (e.g.,a-amylase activity after 96 h of germination; Fig. 1A). A detailedevaluation revealed that the parameters followed differentprogression patterns.

The a-amylase and b-glucanase activity (Fig. 1A and C) was ab-sent or very low in the ungerminated barley seeds (0 and 24 h), afact that is well-known from the analysis of pooled samples(Narziss, 1999). With respect to the variations in these two param-eters, the ungerminated seed material was nearly homogenous.After being incubated for more than 24 h, the distribution patternsof a-amylase and b-glucanase activity shifted to the right, whichreected an increase in enzyme activity. Intriguingly, the individ-ual levels of enzyme activity in the older seedlings varied morethan at the beginning of germination, which is shown by the muchbroader width of the distribution patterns. In particular, after 96 h,the a-amylase activity (Fig. 1A) varied from approximately 1 to3 nkat/mg DW, corresponding to a factor of three. Over the courseof germination, biosynthetic activities are induced; the corre-sponding enzymes are synthesised and their activities increasesteadily. The speed of the increase in a-amylase and b-glucanaseactivity was different within individual seedlings because of differ-ences in their individual germination-associated biosyntheticactivity. However, since particular attention was paid to ensuringuniform germination conditions (e.g., temperature control, lowlayer thickness, uniform ow of air through the bed etc.), differ-ences in the metabolic reactions caused by varying external condi-tions can be excluded. The differences must therefore be due to theinherent biological variation.

Differing patterns for the changing enzyme activity were foundfor b-amylase activity (Fig. 1B). The ungerminated seeds (0 h) al-ready revealed distinct enzyme activities, which varied from 0.15

M. Kleinwchter et al. / Food Chemistry 147 (2014) 2533 27


to 0.4 nkat/mg DW, corresponding to a factor of two to three. Thus,the variation range for b-amylase activity in the original, ungermi-nated barley seeds was quite similar to that found for a-amylaseand b-glucanase activity after the seed had been germinated for96 h. At the beginning of the germination period (24 h), the meanb-amylase activities increased slightly, which was shown by a min-or shift in the distribution pattern to the right to higher values.Subsequently, the b-amylase activities remained on a similar levelfor the rest of the incubation period (4896 h). The variation rangeremained relatively constant (by a factor of two to three). The nd-ing that b-amylase, in contrast to a-amylase and b-glucanase, is al-ready present in mature barley seeds, is in accordance with theresults of previous studies (Narziss, 1999; Sopanen & Laurire,1989). The slight increase in b-amylase activity at the beginningof germination (24 h) is not related to any additional de novo syn-thesis (Sopanen & Laurire, 1989), but due to the fact that some ofthis enzyme occurs in a bound, inactive form, which is instantly re-leased to its free, active form by proteases during germination.

The GABA distribution charts (Fig. 1D) show that the accumula-tion pattern of this typical stress metabolite was completely differ-ent from the distribution patterns of the enzymes. Most of thebarley seeds consistently had relatively low GABA concentrations,ranging from 2 to 10 mg/100 g DW over the course of the entireincubation period. However, with the progression of germinationand growth processes (7296 h), some grains accumulated extraor-dinarily high GABA concentrations (e.g., 24 and 44 mg/100 g DW;96 h). The general germination-related increase in GABA concentra-tion previously reported (Inatomi & Slaughter, 1971; Kleinwchteret al., 2012), is not due to a slight uniform increase in the GABA con-tents in all seeds of a malt batch, but is caused by a low, but signif-icant, number of deviators. One explanation of this unexpected

nding is that some grains may suffer from severe stress, whereasothers are not affected. In this context, it has to be noted that GABAis accumulated in response to various stresses (e.g., high tempera-tures, water deciency, physical injuries, biotic stresses etc.; for re-view see Bown & Shelp, 1997; Satya Narayan & Nair, 1990).Accordingly, there might be numerous particular causes, e.g., slightinjuries or spatial infections, which are responsible for the individ-ual differences in GABA accumulation.

The data on the enzyme activity mentioned above were com-pared with the results of the individual grain analyses for b-glucan-ase activity reported by de S and Palmer (2004), which are theonly individual grain analyses published so far. This comparisonrevealed that the variations by factors of two to three establishedin this study have to be considered as very low. The variations inb-glucanase activity reported by de S and Palmer were muchhigher (between a factor of six to eight). One suggestion for thismay be that the micro-malting system used in their studies gaverise to much higher heterogeneities, with respect to the germina-tion conditions, compared to the small-scale apparatus used in thisstudy. Alternatively, it could be due to the fact that the barley usedby these authors was relatively inhomogeneous and had higherinherent biological heterogeneity, which may be caused by unevenripening of the grains in the eld.

Since the differences in the germination state, and thus in theactivities of germination-related enzymes, are most pronouncedduring the later stages of the malting process, the evaluation ofhomogeneity of the malts derived from different processing ap-proaches, should be performed using malts from the late phaseof germination (i.e., 96 h). Accordingly, this strategy was followedin order to evaluate and to visualise the heterogeneity of malts de-rived from the different industrial-scale processing systems.

A B

C D

freq

uenc

y of

a m

easu

red

valu

e

0.5 1 1.5 2 2.5 -amylase activity [nkat/mg DW] (0.5 nkat/mg-intervals)

15

15

15

15

15

0 h

24 h

48 h

72 h

96 h

-amylase

0

0

0

0

0

0

-amylase activity [nkat/mg DW] (0.05 nkat/mg-intervals) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

freq

uenc

y of

a m

easu

red

valu

e 0 h

24 h

48 h

72 h

96 h

-amylase 15

15

15

15

150

0

0

0

0 0

10 20 30 40GABA concentration [mg/100 g DW] (2 mg/100 g-intervals)

0 h

24 h

48 h

72 h

96 h

GABA 15

15

15

15

150

0

0

0

0 0

freq

uenc

y of

a m

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-glucanase activity [nkat/mg DW] (0.5 nkat/g-intervals) 2 4 6 8 10 12 14 16 18

freq

uenc

y of

a m

easu

red

valu

e 0 h

24 h

48 h

72 h

96 h

-glucanase 15

15

15

15

150

0

0

0

0 0

Fig. 1. Distribution of biochemical parameters in barley seeds during germination and early seedling growth. Special emphasis was put on ensuring uniform germinationconditions (e.g., temperature control, low layer thickness and uniform ow of air). Germination processes were monitored in terms of (A) a-amylase, (B) b-amylase and (C) b-glucanase activity. The level of stress metabolism (D) was determined using the concentration of c-aminobutyric acid (GABA).

28 M. Kleinwchter et al. / Food Chemistry 147 (2014) 2533


3.2. Industrial-scale germinations: impact of steep aeration on thebiochemical homogeneity of malt

Malt samples produced via three different industrial-scalesteeping systems (without aeration, air suction during dry rests,and temporary pressurised aeration) were investigated by singlegrain analyses. The germination conditions were similar in all threeprocesses. The seeds were subjected to different conditions by thethree industrial approaches only in the rst 3040 h (for details seeSection 2.2.). The water content of the seeds after steeping wasabout 4143% and this was more or less identical for all samples.As mentioned above, the enzyme activities for establishing the het-erogeneities were determined after 96 h. In the corresponding g-ures, the small-scale laboratory experiment data (germinationunder almost homogeneous conditions) were added as a standardand a reference (Figs. 25) for the data from the three differentindustrial malting processes.

With respect to a-amylase activity (Fig. 2), the most obviousnding was that the malt germinated under dened, uniform lab-oratory conditions (continuous aeration) was much more homoge-neous than all the industrially processed malts. This is best shownby a standard deviation (CV) of only 19.4% for the laboratory ap-proach compared to 33.9% for the malt produced without aeration;65.9% for air suction and 45.6% for pressurised aeration (Fig. 2). Thesame situation is shown by the individual grain analyses for b-amylase activity (Fig. 3). Again, the malt produced under denedlaboratory conditions had the lowest heterogeneity (CV = 26.1%)followed by the industrial-scale process without steep aeration(CV = 31.1%). Just like a-amylase, the b-amylase activity for thetwo industrial-scale processes, where steep aeration was applied,was considerably more heterogeneous (CV = 48.1% and 70.8%).The data on b-glucanase activity conrmed the previous ndings(Fig. 4) that the highest biochemical homogeneity was estimated

for the malt produced under dened laboratory conditions(CV = 17.7%), whereas the malts derived from the industrial pro-cesses were less homogeneous, as shown by the CV-values, whichmore than doubled. For further clarication, the CV-values for allthree enzymes (a- and b-amylase and b-glucanase) were averagedfor each processing method and the corresponding mean valuesare displayed in Table 1. The malt germinated under dened anduniform lab conditions had only small heterogeneities, shown bya mean coefcient of variation of just 21.1%. In contrast, the meanCV of all the industrially produced malts was more than double(47.1%). Thus, the tremendous increase in heterogeneity must bedue to heterogeneities in the malting conditions at the industrialfacilities. Accordingly, it could be deduced that the most heteroge-neous conditions are to be found in the steeping vessels with airsuction, followed by those with pressure aeration. The mosthomogenous conditions seem to be found in industrial malting ap-proaches where aeration during steeping is omitted.

The main differences between steeping systems with and with-out aeration should be due to the concentrations of O2 and CO2. Inthis context, it has to be considered that the germinating seedsconsume O2 and liberate CO2. Whereas in unaerated approaches,related equilibrium concentrations will result throughout thebatch, directed aeration will create massive differences. Substantialgradients in the concentration of O2 and CO2 will be establisheddue to the large size of the industrial steeping vessels (Alberset al., 1983). Moreover, the aeration may also affect the tempera-ture of the malt bed (Narziss, 1999). The spatial heterogeneitiesin gaseous composition and temperature create strongly inhomo-geneous germination conditions, which will directly impact onthe progression of metabolic processes. As a consequence, the bio-chemical homogeneity of the complete malt batch could be im-paired. In line with these arguments, in the lab scale trial, theheterogeneities were much lower because the bed layer was only

156.5%

72.8%

CV = 65.9%

= 0.7

-am

ylas

e ac

tivity

[ nk

at /

mg

DW

]

individual seedlings 0

1

2

3

4

CV = 33.9% = 2.2

-am

ylas

e ac

tivity

[ nk

at /

mg

DW

]


1

2

3

4

98.3%

82.8%

CV = 45.6%

-am

ylas

e ac

tivity

[ nk

at /

mg

DW

]


1

2

3

4

= 1.2

-am

ylas

e ac

tivity

[ nk

at /

mg

DW

]


1

2

3

4

= 2.1

CV = 19.4%

A B

C D

without steep aeration air suction

temporary steep aeration laboratory

33.2%

92.5%

30.6%

42.2%

Fig. 2. The a-amylase activities of 15 individual barley seedlings of differently processed green malts after 96 h of germination: (A) without steep aeration, (B) with airsuction during dry rests and (C) with temporary pressurised steep aeration. As a reference, the data obtained by germination under dened laboratory conditions have beenadded (D; see also Fig. 1A, 96 h). In addition, the average enzyme activity () of the 15 individual grain analyses is given. The heterogeneity of the malt is displayed by thecoefcient of variation (CV; relative standard deviation for the corresponding data set) and the maximum relative differences from the mean value.



about 12 cm, which provided identical gaseous concentrationsand temperatures for all seeds. Thus, with regards to malt homoge-neity, the common practise of steep aeration should be omitted.

Yet, to substantiate these ndings further studies are required, inwhich, for instance, the causes of biochemical heterogeneitiesshould be elucidated.

A B

C D

0

0.2

0.4

0.6

0.8

-am

ylas

e ac

tivity

[ nk

at /

mg

DW

]

individual seedlings

CV = 70.8%

= 0.3

179.8%

75.3%

0

0.2

0.4

0.6

0.8

-am

ylas

e ac

tivity

[ nk

at /

mg

DW

]


CV = 26.1%

= 0.4 38.7%

58.2%

0

0.2

0.4

0.6

0.8

-am

ylas

e ac

tivity

[ nk

at /

mg

DW

]


CV = 48.1%

= 0.3 80.7%

97.0%

0

0.2

0.4

0.6

0.8

-am

ylas

e ac

tivity

[ nk

at /

mg

DW

]


CV = 31.1% = 0.4

77.7%

31.4%



Fig. 3. The b-amylase activities of 15 individual barley seedlings of differently processed green malts after 96 h of germination: (A) without steep aeration, (B) with air suctionduring dry rests and (C) with temporary pressurised steep aeration. As a reference, the data obtained by germination under dened laboratory conditions have been added (D;see also Fig. 1B, 96 h). In addition, the average enzyme activity () of the 15 individual grain analyses is also given. The heterogeneity of the malt is displayed by the coefcientof variation (CV; relative standard deviation for the corresponding data set) and the maximum relative differences from the mean value.

0

4

8

12

16

-gl

ucan

ase

activ

ity [

nkat

/ g

DW

]


= 9.1

CV = 48.0%

0

4

8

12

16

-gl

ucan

ase

activ

ity [

nkat

/ g

DW

]


= 12.4 CV = 17.7%

-gl

ucan

ase

activ

ity [

nkat

/ g

DW

]

0

4

8

12

16


= 9.4

CV = 39.0%

0

4

8

12

16

-gl

ucan

ase

activ

ity [

nkat

/ g

DW

]


= 11.0

CV = 41.9%

A B

C D



67.6%

91.6%

50.5%

96.8%

40.6%

77.3%

35.0%

27.4%

Fig. 4. The b-glucanase activities of 15 individual barley seedlings of differently processed green malts after 96 h of germination: (A) without steep aeration, (B) with airsuction during dry rests and (C) with temporary pressurised steep aeration. As a reference, the data obtained by germination under dened laboratory conditions have beenadded (D; see also Fig. 1C, 96 h). In addition, the average enzyme activity () of the 15 individual grain analyses is also given. The heterogeneity of the malt is displayed by thecoefcient of variation (CV; relative standard deviation for the corresponding data set) and the maximum relative differences from the mean value.



3.3. Industrial-scale germinations: impact of steep aeration ongermination progression

Apart from the estimation and evaluation of homogeneity, theextent of the corresponding enzyme activities is also relevant formalt quality. Accordingly, germination progression is of specialinterest. Steep aeration is recommended for rapid germinationand thus for cellular modication and mobilization of starch. How-ever, high germination rates are undesirable because of the in-crease in malting losses, and some researchers suggest that steepaeration should be omitted (Kelly & Briggs, 1992). In contrast,other researchers have reported delays in germination due to O2deciency or enhanced CO2 concentrations, when steep aerationwas omitted (Albers et al., 1983; French & McRuer, 1990; Gibbons,1983). Since the activities of a-amylase and b-glucanase rise dra-matically as germination time increases, germination progressioncan be evaluated by analyzing the activities of these enzymes.The results from this study showed that the mean a-amylase activ-ity of the aerated lab-scale approach and that of the industrialsteeping process without aeration were quite similar (2.1 and2.2 nkat/mg DW, respectively; Fig. 2). The induction and progres-sion of metabolic activity in the two batches of seeds were thesame, even though the O2 supply and the CO2 concentrationsshould be quite different. Surprisingly, the mean activities of thetwo aerated industrial-scale processes were drastically lower (1.2and 0.7 nkat/mg DW, respectively; Fig. 2), which suggested that

germination had been strongly retarded. Similar effects were alsoobserved for the mean b-glucanase activities, although the differ-ences observed were less pronounced. Again, the aerated lab ap-proach and the industrial malting without aeration had verysimilar mean activities (12.4 and 11.0 nkat/g DW, respectively;Fig. 4), which were higher than the two aerated industrial maltings(9.4 and 9.1 nkat/g DW, respectively). When changes in enzymeactivities are used to document germination progression, it hasto be considered that ungerminated seeds reveal already signi-cant b-glucanase activity, whereas a-amylase is totally absent inungerminated seeds (Fig. 1). When comparing the mean b-amylaseactivity, all the values were very similar. This was expected as largeamounts of this enzyme are present in the ungerminated seeds andonly minor increases of its activity are likely to occur. Conse-quently, there are only minor differences in b-amylase activity,even when germination progression differs strongly. A comparisonof the mean values conrms the suggestions made for a-amylaseand b-glucanase with regards to the differences in germinationrates. The mean values for b-amylase in the aerated lab approachand industrial malting without aeration were identical (0.4 nkat/mg DW; Fig. 3) and were slightly higher than the two aeratedindustrial approaches (0.3 nkat/mg DW).

These ndings clearly indicate that germination progressiondiffers strongly, depending on the steeping conditions. As outlinedin the introduction, some researchers argue that the common prac-tise of aeration in industrial steeping is not mandatory to achieve

0

40

80

120

GAB

A c

onte

nt[ m

g / 1

00 g

DW

]


= 36.2

0

40

80

120

GAB

A c

onte

nt[ m

g / 1

00 g

DW

]


= 7.7 0

40

80

120

GAB

A c

onte

nt[ m

g / 1

00 g

DW

]


= 36.3

0

40

80

120

GAB

A c

onte

nt[ m

g / 1

00 g

DW

]


= 20.0

A B

C D



Fig. 5. The c-aminobutyric acid contents (GABA) of 15 individual barley seedlings of differently processed green malts after 96 h of germination: (A) without steep aeration,(B) with air suction during dry rests and (C) with temporary pressurised steep aeration. As a reference, the data obtained by germination under dened laboratory conditionshave been added (D; see also Fig. 1D, 96 h). In addition, the average GABA content () of the 15 individual grain analyses is also given.

Table 1Mean coefcients of variation ( CV) for all the a- and b-amylase and b-glucanase activities (n = 45) and mean GABA concentrations of the differently processed malts. The GABAvalues were recalculated after the exclusion of statistical outliers (variance more than double the mean value).

Lab-scale Industrial

Steep aeration Without aeration Air suction Forced air Mean

CV (%) 21.1 35.6 61.6 44.2 47.1 GABA (mg/100 g DW) 3.5 20.0 23.8 21.2 21.7



optimal germination progression (Wilhelmson et al., 2006),although such an approach is generally applied in the daily maltingroutine. According to these researchers, the omission of aerationhad no negative effect, unless the time of O2 deciency does notexceed the initial phase of the process. Consequently, there shouldbe no difference in germination progression in both the aeratedand non-aerated approaches. In this context, the nding that thegermination rate in the two aerated industrial-scale approacheswas drastically lower than that in the non-aerated approach wassurprising. The identication of possible reasons for this becomeseven more complicated if one considers that the fastest germina-tion occurred with the continuously aerated lab-scale approach,where high O2 and low CO2 concentrations were present, and inthe non-aerated industrial approach, where the O2 levels werelow and the CO2 concentration high. These contrasting conditionsfor the two processes may indicate that gaseous composition isnot solely responsible for the observed differences in the germina-tion progression. For clarication, a further experimental lab-scaleapproach was conducted, where during steeping (24 h wet steep)the seeds had been either aerated, as outlined in Section 2.1., werenot aerated, or were purged with nitrogen. The subsequent germi-nation conditions were identical in all cases and the seeds wereaerated. The activity of a-amylase and b-glucanase did not differsignicantly between the three approaches and non-aerationturned out to be slightly benecial (Figs. S1 and S2, supplementaldata). In contrast, coleoptile growth was fastest using the steepaerated approach (Fig. S3, supplemental data), a fact that is inaccordance with the well known acceleration of germination re-lated growth processes by aeration (e.g., Albers et al., 1983; French& McRuer, 1990). Clearly, the growth processes and biosynthesis ofgermination related enzymes were not progressing in parallel,which indicates that the regulation of these two processes maybe different and is not impacted by exogenous factors in the sameway.

The question arises, which factors, apart from gaseous composi-tion, may directly or indirectly affect the various germination pro-cesses. As purging with air causes a vaporization-relatedtemperature decline, differing temperature in the differentiallyaerated assays might also contribute to the observed differencesin germination progression. Such effects can easily be substanti-ated using lab scale approaches. However, in industrial steepingsystems, these differences in temperature do not become apparentdue to the massive production of heat by the metabolic activities ofthe germinating seeds.

3.4. Industrial-scale germinations: impact of steep aeration on stressmetabolism

The plant stress metabolite GABA (Fig. 5) generally shows acompletely different distribution pattern to the enzyme activities.As already described in the context of Fig. 1D (lab germination;Section 3.1.), the bulk of the barley seeds accumulated relativelylow amounts of GABA (2030 mg/100 g DW; Fig. 5AD), but someoutliers had extraordinary high GABA contents (e.g., >120 mg/100 g DW; Fig. 5B and C). Thus, some of the barley grains seemedto suffer from severe stress, whereas others were not affected.However, the particular reasons for these tremendous GABA accu-mulations are not clear (e.g., injury or partial infection; see alsoSection 3.1.). Yet, when the estimated outliers, i.e., those grainscontaining more than twice the mean GABA content, were ex-cluded from the calculation, the mean values for all the industri-ally produced malts were quite similar: 20.0, 23.8 and 21.1 mg/100 g DW, respectively. However, the corresponding mean valueof 21.7 mg/100 g DW was about six times higher than that ofthe grains processed under laboratory conditions (3.5 mg/100 gDW), which suggested a markedly higher general stress level in

industrially produced malts. Yet, as the supply of O2 strongly dif-fered due to the aeration regime applied, anoxia-related GABAaccumulation (e.g., Chung, Jang, Cho, & Lim, 2009; Kleinwchteret al., 2012) could be excluded as a possible cause. Moreover,due to the relatively similar temperatures during steeping andthe equal water content of the steeped grains, temperature aswell as water effects should also be ineligible as stress inducers.Thus, the causes of the signicant differences in the stress levelsbetween industrially steeped barley seeds and those generatedunder laboratory conditions remain unclear. Apart from the fac-tors already mentioned, the major difference between industriallyprocessed malts and those processed under laboratory conditionsare the mechanical strains due to the higher layer thickness inindustrial steeping and germination systems. However, it is notknown how such mechanical strains could induce these corre-sponding stress reactions. GABA accumulation is a complex issue,and has been reported to be triggered by a number of quite dif-ferent stress reactions (Inatomi & Slaughter, 1971; for reviewsee Bown & Shelp, 1997; Satya Narayan & Nair, 1990). Accord-ingly, further studies are required, in order to draw unambiguousconclusions on the feasibility of using individual grain analyses ofthe GABA contents as a marker for process-related biochemicalinhomogeneities.

4. Conclusion

In conclusion, our ndings clearly indicate that steep aeration isnot a basic requirement for obtaining appropriate germination rateand sufcient levels of the relevant enzyme activities, and that itrather should be omitted in order to prevent the establishmentof biochemical inhomogeneities during malting in industrialfacilities.

Moreover, it is intriguing that the differences in the steepingprocedure still are manifested at the end of the germination per-iod, although the conditions for all samples throughout the fol-lowing three days had been identical. This is in accordance withthe old saying of German maltsters Das Malz wird in der Weichegemacht, which means The malt is made whilst steeping.

This article represents the rst part of a two-part study dealingwith the impact of steep aeration on the homogeneity of barleymalt. As such, it involved the preparatory work for the establish-ment of individual grain analysis for the assessment of biochemicalhomogeneity and comparative analysis of differently steeped bar-ley malts. The second part of this two-part study, entitled Impactof aeration differences on the manifestation of biochemical param-eters, will concentrate on the causes of biochemical heterogene-ities in differently steeped malts.

Acknowledgements

This research project was supported by the German Ministry ofEconomics and Technology (via AiF) and the FEI (Forschungskreisder Ernhrungsindustrie e.V., Bonn, Germany). Project AiF-16299N. We thank Winrich von Bierbrauer zu Brennstein (Oettin-ger Brauerei, Braunschweig, Germany), Batrice Conde-Petit, UrsKeller and Eliana Zamprogna (all Buhler Group, Uzwil, Switzerland)for their engagement and support of this research project. Theircontributions and ideas are greatly acknowledged.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.foodchem.2013.09.090.



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