norja lasko lipolytic and oxidative changes of barley

J. Inst. Brew., September-October, 1993, Vol. 99, pp. 395-403

LIPOLYTIC AND OXIDATIVE CHANGES OF BARLEY LIPIDS DURING MALTING

AND MASHING

By A. Kaukovirta-Norm and S. Laakso

Helsinki University of Technology, Department of Chemical Engineering, Laboratory of Biochemistry

and Microbiology, SF-02150 Espoo, Finland

AND

P. Reinikainen and J. Olkku

Oy Lahden Polttimo Ab, P.O. Box 14, SF-I52U Lahti 21, Finland

Submitted 17 December 1992

Lipolytic and oxidative changes of barley lipids were studied during malting and mashing. The amount

of lipid decreased by 23% during malting and changes in the composition of lipid classes were minor.

On the other hand, during mashing the amount of free fatty acids (FFA) increased which indicated,

that lipid hydrolysis had occurred. The same phenomenon was seen when malt flour was soaked in

water at 23°C. The triglyceride (TG) and polar lipid (PL) contents were reduced and the proportion of

FFA in total lipids was increased. Following similar soaking of barley flour, TG and PL were reduced

but the accumulation of FFA and especially linoleic acid (LA) was slight. The results were consistent

with the data on lipoxygenase activity (LOX) during malting. During steeping LOX decreased and was

15-20% of the activity of raw barley at the beginning of germination. The activity remained low during

germination but rose sharply in the middle of kilning only to decrease again to a very low level at the

end of kilning (5%). This in combination with the fact that the proportion of FFA remained high in the

soaked malt samples suggests that the oxidation by LOX is negligible in the malt samples. However,

the data suggest that mashing of barley, but not that of malt, includes the potential for the formation

of highly polar lipid oxidation products.

Key Words: Barley, malting, lipids, lipase-lipoxygenase

Introduction

In the initial stages of malting, water activates the barley

embryo and the hydrolysis of biopolymers begins with the

rise of the activities of hydrolytic enzymes. However, lipid

metabolism of germinating barley differs from that of other

macromolecules. Lipase activity is already present in barley"

and the activity is known to increase by over fourfold during

malting1. Lipoxygenase activity has also been reported to

increase during germination of barley4-10. Lipases and phos-

pholipases act in concert with lipoxygenase: the polyunsatur-

ated fatty acids released from triglycerides and phospholipids

are preferentially oxidized by lipoxygenase. However, only

minor differences exists between barley and malt lipids1.

During mashing up to 30% of malt lipids may be oxidized2

even though only 2-4% of initial lipoxygenase activity has

been reported to be present in malt after kilning4-12. It is

surprising that such a low lipoxygenase activity would be

responsible for the oxidation of such a high amount of lipid.

A more detailed understanding of this anomaly would be

valuable since the lipoxygenase activity of malt is closely

related to the nonenal potential of wort5. We approached

the problem by parallel determinations of changes in lipid

composition and lipoxygenase activity during the malting

process and under conditions simulating mashing.

Materials and Methods

Barley

The malting barleys chosen for the lipoxygenase assays

were the Finnish varieties Ingrid, Kilta, Kustaa, Kymppi,

Pirkka, Pokko, and Porno harvested in both 1990 and 1991,

and the other varieties Etu (1989), Elo (1990), Plaisant

(1990), Ariel (1989), Formula (1989), Golf (1989), Grit

(1989), Harrington (1987), Triumph (year not known), and

Pipkin (1990) (year of harvesting after the variety name).

The malting and mashing experiments and the water soaking

tests were done with the variety Kymppi (1990).

Other materials

The standards for thin-layer chromatography (TLC) and

gas-liquid chromatography (GLC) analyses and the substrate

for the lipoxygenase assay were purchased from Sigma and

were as follows: dipentadecanyolphosphaditylcholine

(P-7285), heptadecanoic acid (H-3500), triheptadecanoin

(T-2151), dipentadecanoin (D-8508), heptadecanoic acid

methyl ester (H-4515), and linoleic acid (L-1376). Silica gel

plates (5721) were purchased from Merck. HPLC grade sol

vents were used for all fatty acid analyses. All other chemicals

were of reagent grade or of a higher grade.

Malting

The variety Kymppi was malted to give lager malts using

a procedure which included a 44-h steeping with varying wet

and dry periods, a 6-day germination and a 21-h kilning.

The green malt samples were freeze-dried and stored at room

temperature.

Mashing

The malt was ground to coarse flour with a coffee mill.

A 80-g sample was suspended in 400 ml distilled water and

micromashed in a BRF CM2 automatic mash bath (ADE

Engineering) with the following time and temperature pro

cedure: 30 min at 40°C, 30 min at 52°C, 40 min at 63°C,

30 min at 70°C, and 10 min at 80°C. The temperature was

raised one centigrade degrees/min. The wort was centrifu-

gated (3800 rpm) for 10 min and filtered under pressure

through Seitz K 900 plate. The samples were freeze-dried

and stored at room temperature. Mashing in the laboratory

was carried out in a water bath with an identical temperature

program as in the micromashing using a BRF CM2 bath.

This document is provided compliments of the Institute of Brewing and Distilling www.ibd.org.uk Copyright - Journal of the Institute of Brewing

396 BARLEY LIPIDS [J. Inst. Brew.

The wort and spent grains were separated by ccntrifugation

(3800 rpm) for 10 min, and freeze-dried.

Water soaking

For soaking, extraction and lipoxygenase assays the barley

and malt samples were ground to a fine powder with a

Schnitzer (model KE) natural stone flour mill. A O.S-g flour

sample of either barley or malt was soaked for 15 h in a 50-

ml shaking flask (200 rpm, 23°C) in 2.5 ml of distilled water.

Prior to lipid extraction of the unsoaked control samples,

2.5 ml of distilled water was added after the chloroform-

methanol (2:1, v/v) addition.

Lipid extraction

The samples were extracted by shaking for 6 h (250 rpm,

28°C) in 19 volumes of chloroform-methanol (2:1, v/v)

according to the method of Folch et al.1. The mixtures were

centrifugated for 10 min (6000 rpm) to remove insoluble

material. The extraction was repeated for 2 h with the same

amount of chloroform-methanol, and the extracts were com

bined and evaporated to dryness in a rotary evaporator.

Lipids were dissolved in 10 ml of chloroform-methanol

(100:1, v/v), divided into 200-1000 u.1 portions in test tubes,

evaporated to dryness under N2, and stored at -20°C under

N2 until analysis. The samples were used to determine the

absolute and relative amounts of fatty acids in total lipids

and major Upid classes.

Separation of major lipid classes by TLC

The extracts were redissolved in 200 u.1 of chloroform-

methanol (100/1, v/v) and supplemented with 50 u.g each of

the polar lipid (PL), diglyceride (DG), free fatty acid (FFA)

and triglyceride (TG) standards, and applied on silica plates.

The chromatograms were developed in petroleum ether-

diethyl ether-acetic acid (80:30:1, v/v). Lipid classes were

visualized by spraying with 0.01% Rhodamine 6G and

detected under UV light, scraped off, and used for fatty acid

determination.

Preparation and analysis of fatty acid methyl esters

Fatty acids were saponified and converted to methyl esters

as described by Suutari et al. (1990). The methyl esters were

analyzed using GLC and major fatty acids were identified

by comparing their retention times with those of known

standards. The total extractable fatty acids were determined

by adding 30 fig of internal standard, heptadecanoic acid

methyl ester to each sample prior to saponification and

methylation.

Gas chromatography

A Hewlett-Packard Model 5890A gas chromatograph

equipped with a flame ionization detector, a capillary inlet

system, a HP-FFAP (25 m x 0.2 mm x 0.3 u,m) column,

and a Model 7673A high-speed automatic liquid sampler

with a 10-u.l syringe was employed. The column temperature

was programmed from 70 to 200°C at a rate of 25°C/min.

The column inlet pressure was 150 kPa. The flow rate for

the makeup gas, He, was 30 ml/min, and the flow rates for

the detector gases were 40 ml/min H2 and 400 ml/min air.

The column flow rate was 1.0 ml/min and the septum purge

flow rate 1-2 ml/min. The split ratio was set at 1:20. Peak

areas were measured by using a Hewlett-Packard Model

3365A integrator.

Calculations

The relative amounts of fatty acids in total lipids and in

different lipid classes were determined as a percentage of

the total peak area. Absolute amounts of the individual fatty

acids in total lipids were calculated per one g of dry weight

of the sample by comparison of the peak area to that of the

internal methyl ester standard without any conversion fac

tors. The total amount of fatty acids was determined as the

sum of all individual fatty acids. Amounts of major lipid

classes per one g of dry weight of the sample were determined

by comparing the area of the fatty acids from a lipid class

to that of the corresponding standard. As an approximation,

it was assumed that the polar lipids consist of phosphadityl-

choline only. The distribution of different lipid classes was

determined as a percentage of total weights of lipid classes.

Assay of lipoxygenase activity

The lipoxygenase (LOX) activity was determined as the

rate of O2 consumption upon addition of linoleic acid (LA)

substrate using a Ysi Model 53 oxygen monitor. A 100-mg

sample of ground barley, malt or mash was suspended in

10 ml of 50 mM Na-phosphate buffer, pH 7.0, in a reaction

chamber attemperated to 25°C. After equilibration the reac

tion was initiated by the addition of 400 u.1 of LA substrate

prepared according to the method of Axelrod3. The LOX

activity unit was defined as the quantity of enzyme that

consumes one u,mol of O2 per minute under the assay con

ditions. The results are a mean value of two measurements.

Enzyme extraction

Five g of barley, malt or freeze-dried mash flour was

suspended in 50 ml of 50 mM Na-phosphate buffer, pH 7.0,

and extracted for one h in a shaking water bath (23°C,

160 rpm). A 10-ml portion of the suspension was centrifu

gated (3000 rpm) for 15 min and one ml of the supernatant

was mixed with 9 ml of 50 mM Na-phosphate buffer, pH

7.0, in the reaction chamber of the oxygraph and the LOX-

activity measured as above. The extracted flour was resus-

pended in 10 ml of 50 mM Na-phosphate buffer, pH 7.0,

and the LOX-activity was measured like before.

Results and Discussion

Lipid changes during malting

A Finnish malting barley, Kymppi, was malted and moni

tored for changes in lipid content and composition. For this,

the malting process was sampled throughout the three main

stages, steeping (44 h), germination (144 h), and kilning

(21 h) as shown in Fig. 1. The total lipid content of the

unmalted barley was 3.6% consisting of 69% TG, 27% PL,

2% FFA, and 2% DG. The content of steryl esters was too

low to be accurately detected. During malting the combined

amounts of the TG-, PL-, DG-, and FFA-classes were

reduced by 23% (Figure 1). FFA stayed at a relatively low

level reaching a maximum of 4% of total lipids at the end

of the germination period. The PL-fraction increased during

germination and was 42% of the total lipids at the end of

the germination period. The proportion of TG decreased

from 69% to 65% during malting. These observations are

in accordance with the results of Anness & Baxter1 and

indicate low overall changes in lipid composition during the

malting process. According to Anness2 the lipid content of

malt can be less than 70% of that of barley. In the present

study such a marked decrease applies mainly to the TG pool.

Changes in fatty acid compositions were also small. Com

parison of the composition in total fatty acids of barley and

malt showed slight increments in linoleic acid (LA) and

linolenic acid (LNA) in the middle of the germination period

but during kilning their proportions decreased again and

were 20% and 5% lower respectively in finished malt than

in barley (Table 1). In the PL fraction the proportion of LA

varied between 55 and 66% throughout malting. However,

the proportion of LNA increased by more than two-fold by

the end of the germination and was still distinctly higher

after kilning than in barley. The proportion of other fatty

acids remained constant (Table 2). In the TG fraction the

fatty acid composition also remained essentially unchanged

(Table 3). In the FFA-fraction the portion of LA increased

30%, but this does most probably not reflect any dramatic

lipid rearrangement as the contribution of FFA to total lipids


Vol. 99, 1993]

45.0

BARLEY LIPIDS 397

2S.0

_

-

_

r

.

p

. . n= =

D free fatty acids

I diglycerides

D triglycerides

■ polar lipids

steeping germination kilning

Fig. 1. Lipid content and composition of barley during malting.

TABLE 1. Changes in total fatty acid content and composition at different stages of malting

stage of sampling c 16:0

fatty acid content, mg/g (composition, %)

c 18:2 c 18:3

c 18:0 c 18:1 LA LNA others

total

mg/g

barley, Kymppi -90

after 1st wet steep

after 1st air rest

after 2nd wet steep

after 2nd air rest

after 3rd wet steep

germination, after 1st day

germination, after 2nd day

germination, after 3rd day

germination, after 4th day



kilning, after 6 h

kilning, after 12 h

kilning, after 18 h

malt with rootlets

lager malt after removal of rootlets

5.6

4.8

5.1

5.5

5.5

5.4

5.5

5.6

5.3

5.3

4.8

4.6

4.2

4.0

4.0

3.9

3.9

(21.6)

(21.9)

(21.9)

(22.3)

(22.5)

(21.8)

(21.9)

(21.3)

(20.9)

(21.1)

(20.1)

(20.8)

(20.1)

(21.0)

(22.3)

(19.4)

(20.3)

0.5

0.4

0.4

0.5

0.4

0.4

0.5

0.4

0.4

0.4

0.6

0.5

0.5

0.3

0.3

0.5

0.3

(2.0)

(1.9)

(1.6)

(2.0)

(1.6)

(1.7)

(1.8)

(1.5)

(1.4)

(1.6)

(2.3)

(2.0)

(2.2)

(1.6)

(1.8)

(2.3)

(1.6)

3.2

2.6

2.8

2.9

2.9

3.1

3.1

2.9

2.6

2.5

2.2

2.1

2.1

1.7

1.8

1.9

1.8

(12.5)

(11.9)

(12.0)

(12.0)

(12.0)

(12.5)

(12.2)

(10.8)

(10.4)

(10.0)

(9.1)

(9.4)

(10.0)

(9.0)

(9.9)

(9.3)

(9.3)

14.2

12.2

13.0

13.8

13.5

13.9

14.0

15.0

14.5

14.8

13.8

12.6

12.0

10.7

11.2

11.5

11.3

(55.3)

(55.8)

(55.3)

(56.1)

(55.4)

(55.6)

(55.3)

(56.7)

(57.3)

(58.7)

(57.7) ;

(56.7)

(56.8)

(57.0)

(62.0) (

(57.0)

(59.1)

.6

.2

.4

.4

.5

.4

.5

.8

.8

.9

>.l

.8

.8

.5

).l

.8

.5

(6.0)

(5.6)

(5.9)

(5.6)

(6.0)

(5.6)

(5.8)

(6.7)

(7.2)

(7.4)

(8.7)

(8.0)

(8.5)

(7.8)

(0.8)

(9.0)

(7.7)

0.6

0.7

0.8

0.5

0.6

0.7

0.8

0.8

0.7

0.3

0.5

0.7

0.5

0.7

0.6

0.6

0.4

(2.5)

(3.0)

(3.2)

(2.0)

(2.4)

(2.8)

(3.0)

(3.0)

(2.8)

(1.3)

(2.1)

(3.1)

(2.3)

(3.6)

(3.1)

(2.9)

(2.0)

25.7

21.9

23.5

24.5

24.4

25.0

25.2

26.5

25.3

25.2

23.9

22.1

21.1

18.8

18.1

20.1

19.2

was always low (Table 4). According to the literature the

lipid contents and comparisons vary from one variety to

another and seasonal variations can also be significant6. The

present malting results suggest that the lipid changes during

malting are probably minor.

Lipid changes during mashing

Samples taken during mashing differed markedly in lipid

composition from barley, malt or the samples taken during

malting. The samples taken immediately after mixing the

malt flour in water already contained high amounts of FFA;

20% of the total lipid (Figure 2). At the beginning of mashing

the proportion of TG fell slightly, from 65% to 62%, and

the proportion of PL decreased from 30% to 12%. However,

the fatty acid composition did not change in any of the lipid

classes. These observations all suggest that malt contains a

considerable potential for lipid hydrolysis which is stimulated

when malt is ground and soaked in water. The fact that the

LA content in malt is high and remains high even in the

increased FFA-fraction of the mashing samples, suggests the

absence of oxidative reactions. On the other hand, earlier

reports indicate that LOX activity increases during malting

and is still noticeable after drying at 65°C (Baxter, 1982).

This discrepancy could be due to the total inactivation of

LOX during the malting or kilning processes or at the begin

ning of mashing. Alternatively, the barley variety used may

have represented an exception with a very low LOX activity.

In order to evaluate each of these possibilities, LOX was

first assayed in seven Finnish barley varieties which were

each from two different harvests and in ten other varieties.

The LOX activities of the barley varieties varied from

450 U/100 mg to 1150 U/100 mg barley flour (Figure 3). The

experiments showed that the variety Kymppi was a typical

representative of the malting barleys with an activity of

715 U/100 mg (crop 1990) and 775 U/100 mg (crop 1991).

Following these experiments, possible changes of LOX dur

ing malting were monitored. At the beginning of steeping,

i.e. within the first 6 h, LOX activity dropped to about one

half of the activity of barley (Figure 4). Upon subsequent

steeping the activity was further reduced and by the beginning

of germination only 15-20% of the initial activity remained.

During germination the activity level remained low until the


398 BARLEY LIPIDS

TABLE 2. Changes in fatty acid content and composition of PL-fraction at different stages of malting

[J. Inst. Brew.

stage of sampling

barley, Kymppo -90

after 1st wet steep

after 1st air rest

after 2nd wet steep

after 2nd air rest

after 3rd wet steep







kilning, after 6 h

kilning, after 12 h

kilning, after 18 h

malt with rootlets


c

4.4

4.9

4.3

4.6

4.7

4.7

4.4

5.4

5.2

5.4

5.9

5.9

4.5

5.3

4.7

4.5

4.0

16:0

(21.9)

(23.6)

(22.3)

(22.1)

(22.8)

(23.1)

(22.7)

(21.6)

(21.9)

(21.7)

(22.3)

(21.2)

(21.9)

(21.7)

(24.1)

(23.9)

(23.8)

c

0.5

0.4

0.4

0.6

0.3

0.4

0.6

0.5

0.3

0.4

1.0

0.7

0.2

0.3

0.3

0.3

0.3

fatly acid

18:0

(2.6)

(2.0) (

(1.8)

(2.7)

(1.7)

(2.1)

(3.0)

(2.0)

(1.5)

(1.6)

(3.6)

(2.4)

(1.1)

(1.4)

(1.6)

(1.7)

(1.9) <

wntent,

c 18:1

1.2 (6.2)

).2 (1.0)

1.3 (6.6)

1.3 (6.4)

1.2 (6.0)

1.3 (6.4)

1.1 (5.6)

1.4 (5.5)

1.1 (4.6)

1.1 (4.5)

1.2 (4.7)

1.5 (5.3)

1.1 (5.4)

1.3 (5.4)

1.0 (5.1)

1.0 (5.2)

3.8 (4.9)

mg/g (composition, %)

c 18:2

(LA)

12.5 (62.3)

13.7 (66.2)

12.0 (62.4)

13.0 (62.1)

12.7 (61.8)

12.4 (61.5) 1

11.6 (59.7)

14.7 (58.3) :

13.8 (58.5) ;

14.6 (58.4) :

14.5 (54.8) :

15.6 (55.9) :

11.8 (56.8) :

13.9 (56.7) :

11.4 (58.2) ;

10.8 (57.1) ;

9.9 (58.9)

c 18:3

(LNA)

1.2 (6.0)

.3 (6.1)

1.2 (6.0)

1.2 (5.6)

1.4 (6.6)

1.2 (5.9)

1.6 (8.2)

5.9 (11.6)

'.9 (12.1)

U (12.5)

>.5 (13.3)

J.8 (13.6)

'.8 (13.5)

>.3 (13.4)

>.O (10.1)

>.O (10.5)

1.6 (9.7)

others

0.2 (1.0)

0.2 (1.0)

0.2 (1.0)

0.2 (1.1)

0.2 (1.1)

0.2 (1.0)

0.2 (0.9)

0.3 (1.1)

0.3 (1.3)

0.3 (1.3)

0.3 (1.2)

0.4 (1.6)

0.3 (1.3)

0.3 (1.3)

0.2 (0.9)

0.3 (1.5)

0.1 (0.8)

PL %

27.4

26.2

28.9

26.8

26.0

28.8

25.4

30.9

29.1

35.1

35.0

41.7

35.7

38.1

32.9

34.1

29.8

TABLE 3. Changes in fatty acid content and composition of TG-fraction at different stages of malting

stage of sampling

barley, Kymppi -90

after 1st wet steep

after 1st air rest

after 2nd wet steep

after 2nd air rest

after 3rd wet steep







kilning, after 6 h

kilning, after 12 h

kilning, after 18 h

malt with rootlets


c

13.7

15.2

12.9

15.2

15.4

13.0

14.6

14.5

13.5

11.5

11.2

9.3

8.1

8.8

8.7

8.4

8.9

16:0

(18.3)

(18.7)

(19.6)

(18.9)

(18.8)

(18.8)

(18.2)

(18.4)

(16.9)

(18.3)

(16.6)

(18.1)

(16.0)

(16.4)

(15.6)

(16.8)

(16.4)

c

0.6

0.5

0.5

0.5

0.5

0.0

0.6

0.5

0.5

0.6

0.5

0.6

0.4

0.4

0.5

0.5

0.4

fatty acid

18:0

(0.7)

(0.7)

(0.8)

(0.7)

(0.6)

(0.0)

(0.7)

(0.7)

(0-7)

(1.0)

(0.8)

(1.2)

(0.8)

(0.8)

(0-8)

(1.1)

(0.8)

content, mg/g (composition, '

c 18:2 c

c 18:1

8.9 (11.9)

8.8 (10.8)

8.4 (12.9)

9.1 (11.3)

9.3 (11.4)

7.9 (11.5)

9.8 (12.2)

8.6 (10.9)

8.3 (10.4)

7.4 (11.7)

6.6 (9.9)

5.8 (11.3)

5.2 (10.1)

4.9 (9.1)

4.8 (8.7)

5.2 (10.5)

5.2 (9.6)

(LA)

43.6 (58.0)

47.9 (58.8)

37.5 (57.2)

46.8 (58.2)

47.9 (58.6)

41.5 (60.3)

47.1 (58.7)

46.9 (59.6)

49.0 (61.2)

37.7 (60.0)

41.7 (61.8)

30.9 (59.8)

31.4 (61.5)

33.4 (62.0)

34.3 (61.6)

30.4 (60.9)

33.0 (60.6)

Vo)

18:3

(LNA)

6.9

7.5

5.0

7.2

7.3

6.0

6.5

6.8

6.9

4.3

5.9

3.8

4.9

5.3

6.0

4.3

5.7

(9.2)

(9.2)

(7.6)

(9.0)

(8.9)

(8.7) (

(8.1)

(8.6)

(8.6)

(6.8)

(8.8)

(7.4)

(9.6)

(9.9)

(10.9)

(8.6)

(10.4)

others

1.4 (1.9)

1.5 (1.9)

1.2 (1.9)

1.6 (2.0)

1.5 (1.8)

).4 (0.6)

1.7 (2.2)

1.4 (1.8)

1.7 (2.1)

1.4 (2.2)

1.4 (2.1)

1.2 (2.2)

1.0 (2.0)

1.0 (1.9)

.3 (2.4)

1.1 (2.2)

1.2 (2.3)

TG %

68.8

69.0

66.0

68.4

69.1

65.4

69.6

64.4

65.8

58.6

59.6

51.6

58.4

55.9

62.3

60.1

64.8

first hours of kilning when a sharp rise in activity up to the

original level of barley occurred. However, at the end of

kilning the level was low again, equivalent to only 5% of

the activity of barley (Figure 4).

Lipid changes during water soaking

According to the above data the net result of malting

seemed to be the reduction of LOX activity of barley, a

phenomenon which is likely to explain the oxidative stability

of the LA-rich FFA pool appearing in the early stages of

mashing. This was furhter tested by soaking the LOX-rich

barley flour and LOX-poor malt flour separately in water

under conditions simulating the early stages of mashing

(Figure 5). In both cases PL were reduced, in the barley

flour by 33% and in malt flour by 80% when compared to

the non-soaked controls. The reduction of TG was 22% in

the water suspended malt flour and 32% in the water sus

pended barley flour. It appears that both samples contained

hydrolytic activity attacking both PL- and TG-fractions.

Evaluation of the fatty acid compositions of PL and TG after

the water treatment suggests that all the fatty acids were

hydrolysed at the same rate (Tables S and 6). Only a slight

tendency for reduction of linoleyl residues was observed. In

both samples the DG fraction increased, about 1.6-fold in

the malt sample and 2.4-fold in the barley sample during

water soaking indicating some hydrolytic activity (Figure 5).

The observed lipolytic action would also be expected to

increase the FFA fraction during water soaking and this

appeared to occur in the malt flour suspension. The pro

portion of FFA in total lipids increased significantly and all

the major malt fatty acids showed some increase with LA

showing the greatest increase (Table 7). The soaking of the

malt flour was conducted for up to IS h without significant

changes in FFA content and composition. Therefore, it seems

that hydrolytic activity was present and LOX was absent in

the malt.

The proportion of FFA in the water-soaked barley flour

increased only slightly in spite of the observed loss of the

acyl lipid classes (Figure 5) and changes in FFA composition

were evident (Table 7). The proportion of LA decreased in

barley flour from 37% to 22%, while in the malt flour the

portion of LA increased slightly during the water-soaking.

This rapid loss of LA together with the presence of high

hydrolytic and LOX activity indicate that in barley flour the


Vol. 99, 1993] barley lipids 399

TABLE 4. Changes in fatty acid content and composition of FFA-fraction at different stages of malting

stage of sampling c 16:0

fatty acid content, |ig/g (composition, %)

c 18:2 c 18:3

18:0 c 18:1 (LA) (LNA) others FFA %

barley, Kymppi -90

after 1st wet steep

after 1st air rest

after 2nd wet steep

after 2nd air rest

after 3rd wet steep





germination, after Sth day


kilning, after 6 h

kilning, after 12 h

kilning, after 18 h

malt with rootlets


246 (44.6)

268 (44.0)

237 (41.5)

258 (41.7)

293 (44.6)

270 (43.3)

269 (42.0)

293 (41.8)

340 (41.3)

381 (40.0)

410 (40.8)

499 (39.2)

392 (39.4)

480 (42.2)

358 (42.4)

406 (42.6)

325 (40.6)

40 (7.2)

57 (9.4)

39 (6.8)

59 (9.6)

40 (6.1)

46 (7.4)

39 (6.1)

40 (5.7)

49 (5.9)

52 (5.5)

48 (4.8)

50 (3.9)

57 (5.7)

51 (4.5)

43 (5.1)

55 (5.8)

39 (4.9)

58 (10.6)

47 (7.7)

46 (8.0)

49 (8.0)

59 (8.9)

47 (7.5)

67 (10.5)

58 (8.3)

53 (6.4)

59 (6.2)

87 (8.6)

79 (6.2)

65 (6.5)

67 (5.9)

53 (6.2)

65 (6.8)

53 (6.6)

188 (34.2)

217 (35.7)

226 (39.6)

235 (37.9)

244 (37.2)

237 (38.1)

247 (38.6)

292 (41.7)

323 (39.3)

394 (41.4)

390 (38.9)

565 (44.4)

407 (40.9)

466 (41.0)

379 (44.8)

409 (42.9)

347 (43.3)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

0 (0.0)

44 (5.3)

43 (4.5)

48 (4.8)

59 (4.7)

49 (5.0)

56 (4.9)

0 (0.0)

0 (0.0)

24 (3.0)

18 (3.3)

19 (3.1)

24 (4.2)

18 (2.8)

21 (3.2)

23 (3.7)

18 (2.8)

18 (2.5)

14 (1.7)

23 (2.4)

20 (2.0)

20 (1.5)

25 (2.5)

17 (1.5)

12 (1.4)

18 (1.9)

14 (1.7)

1.5

1.5

1.7

1.6

1.7

1.8

1.7

1.7

2.0

2.7

2.7

3.8

3.4

3.5

2.8

3.4

2.9

1 free tatty acids

diglycerldes

triglycerides

polar lipids

temperature

mash samples

Fig. 2. The content of different classes of lipid in malt before mashing and in mash samples. The samples were taken at the end of thedifferent mashing periods, where temperature was kept constant. The temperature was increased by 1 centigrade degrees/min.

activities act together liberating fatty acids and oxidizing the

polyenic free acids.

Wort and spent grains

Both barley and malt was mashed in the laboratory.

After the separation of the wort from the spent grains less

than 0.5% of the total malt lipids remained in wort and

the amount was not dependent on whether the mashing

was carried out by using barley or malt flour (Table 8).This indicates that in addition to non-polar lipids most of

the free fatty acids also remain in the spent grains. These

findings are in accord with the results of Anness and Reed2who detected 0.3-4.5% of malt lipids in the wort depending

on the separation method. The oxidized lipids have been

shown to remain in the wort and are probably the main

reason for beer staling*-12. However, the difference

between barley and malt flour found in the water soaking

test (Figure 5) can clearly be seen in the spent grains. In

the spent grains of barley the total fatty acid content was

ca. 30% lower than that in spent grains of malt (Table 8)and this was due to an approximating correspondance in

the reduction of LA and LNA of barley flour. Despite

the high hydrolytic activity detected in the soaking tests

(Figure 5), the FFA content of barley spent grain was only

one tenth of the content of FFA found in the spent grains

of malt (Table 8).

The risk of undesirable lipid changes during mashing

could be largely eliminated by destroying the enzyme

activities in barley, or by eliminating the presence of oxy

gen. However, the data with other cereals have shown that

mild steam treatments may not be sufficient to destroy the

lipolytic activity and that lipid hydrolysis occurs during the

steam treatment8. The present findings also demonstrate

that the temperature stability of barley lipoxygenase may

be of interest.


400 BARLEY UPIDS [J. Inst. Brew.

1200 T

1000 ■

800 -

•I 600 •

400 '

200

+- I "I "I "I

i i i

ol iC

oi m a\ o>

a 09 .So

1 ii *!

en 5» In enII II

en enI i

."2 .'2

If

barley sample

Fig. 3. LOX activity in some Finnish (open bars) and other (black bars) malting varieties.

1200 t

■ activity with exogenous linolelc acid

D activity without exogenous linolelc

acid


Fig. 4. LOX activity during malting measured in flour suspensions.

Lipoxygenase during mailing

Song ei a/.,13 have shown that during germination of

soybean seeds LOX activity is reduced. In the present

study the monitoring of lipid changes in parallel with LOX

assays support the view that LOX activity is also consider

ably reduced during malting. However, the LOX profiles

(Figure 4) differ essentially from those of earlier reports

which indicated that the LOX activity increased during

germination412. This discrepancy calls for the re-evaluation

of the LOX assays used in malting experiments especially

as the present data shows that high lipolytic potential exists

in both barley and malt generating free fatty acid substrates

for LOX. Therefore, LOX was assayed directly in aqueous

barley and malt flour suspension and in particle free

extracts of the corresponding samples. The activity was

determined polarographically upon addition of exogenous

LA substrate in order to avoid problems caused by tur

bidity. LOX activities were invariably higher in the flour

suspensions than in the corresponding extracts but both

assays gave similar activity profiles indicating a reduction

of LOX during malting (Figure 4, 6). On the other hand,

the flour suspensions after extraction slowly assimilated

O: even without the exogenous LOX substrate (LA)

(Figure 7). Such oxidation can hardly be attributed to LOX

only, but interestingly the activity profiles that were

obtained resemble those of Baxter4 and Schwarz & Pyler12

determined by spectrophotometric methods in the presence

of exogenous LA.


Vol. 99, 1993] BARLEY LIPIDS 401

30.0 -r

□

■

□

■

free (any adds

diglycerWes

trtglycertdoa

polar Hptds

barley Oh barley 15h malt Oh malt iSh

Fig. 5. Changes in lipid content of barley and malt flour during 15-h soaking in water at 23°C.

TABLE 5. Changes in fatty add content and composition of PL-fraction in barley and malt samplesduring 15-h soaking in water at 23°C

c 16:0

fatty add content, mg/g (composition, %)

c 18:2 c 18:3

18:0 c 18:1 (LA) (LNA) others PL%

barley Oh* 3.9(30.4) 0.2 (1.7) 1.0(7.6) 7.1(55.5) 0.5(3.8) 0.1(0.9) 23.4barley 15h 2.8 (32.1) 0.3 (3.1) 0.6 (6.9) 4.4 (51.5) 0.3 (3.1) 0.3 (3.3) 20.8

malt Oh 3.4 (27.0) 0.3 (2.3) 0.6 (4.9) 7.2 (56.3) 1.0 (7.9) 0.2 (1.6) 27.4malt 15h 0.9 (33.3) 0.5 (17.8) 0.1 (4.8) 1.0 (40.0) 0.0 (0.0) 0.1 (4.1) 7.6

'soaking time at 23°C

TABLE 6. Changes in fatly acid content and composition of TG-fraction in barley and malt samplesduring 15-h soaking in water at 23°C

c 16:0

fatty acid content, mg/g (composition, %)

c 18:2 c 18:3

c 18:0 c 18:1 (LA) (LNA) others TG %

barley Oh*

barley 15h

malt Oh

malt 15h

12.1

9.0

8.1

5.6

(20.7)

(22.4)

(18.1)

(16.1)

0.4

0.4

0.4

0.5

(0.8)

(1.0)

(1.0)

(1.3)

7.6

5.4

4.4

3.0

(13.0)

(13.4)

(9.8)

(8.7)

32.6

21.5

26.8

21.0

(55.5)

(53.6)

(59.8)

(60.2)

4.4

2.8

4.0

3.7

(7.5)(7.0)

(8.8)

(10.6)

1.5

1.0

1.1

1.1

(2.6)

(2.6)

(2.4)

(3.0)

71.4

64.7

64.2

67.7


TABLE 7. Changes in fatly add content and composition of FFA-fraction in barley and malt samples

during 15-h soaking in water at 23°C

c 16:0

fatty add content, u.g/g (composition, %)

c 18:2 c 18:3

18:0 c 18:1 (LA) (LNA) others FFA

barley Oh*

barley 15h

malt Oh

malt 15h

300 (44.2)

740 (60.8)

490 (43.6)

1190(40.2)

50

70

70

90

(7.3)

(5.3)

(6.5)

(3.2)

50 (7.8)

100 (8.0)

60 (5.3)

180 (6.2)

250 (36.8)

270 (21.9)

440 (39.0)

1290 (43.7)

0.0 (0.0)

0.0 (0.0)

40 (3.9)

130 (4.4)

30 (3.9)

50 (4.0)

20 (1.7)

70 (2.2)

2.5

5.9

4.9

17.3



402 BARLEY LIPIDS [J. Inst. Brew.

■ I I ■ ■ ■ I li-

I activity with exogenous linoleic acid

D activity without exogenous linoleic

acid


Fig. 6. LOX activity during malting measured using the enzyme extract (See Enzyme extraction in Materials and Methods).

80 ■■

H 1 1 P»l ■ 'i I l| I l|

■ activity with exogenous linoleic acid

D activity without exogenous linoleic

acid

I I I*.1*.1*.1*;


Fig. 7. LOX activity during malting measured using the extracted flour (See Enzyme extraction in Materials and Methods).

Conclusions

Malting of barley did not influence significantly the kernel

lipids in spite of the presence of lipolytic activity. LOX

activity in malt was only about 5% of the activity of barley.

The data is consistent with the view that when malt is mashed

free fatty acids accumulate but oxidation occurs only to a

minor extent. Barley contains high lipolytic and LOX activi

ties, and its mashing also causes extensive lipid hydrolysis

without the accumulation of the corresponding free fatty

acids. A majority of barley fatty acids are polyunsaturated

and their liberation in the presence of LOX is likely to cause

oxidation and to influence the lipid quality of wort. Kilning

was considered to be the critical step in regulating the LOX

activity of malt.

Acknowledgement. Mrs. U. Ahman is thanked for technical

assistance.

References

1. Anness, B. J. & Baxter, E. D., European Brewery Convention

Proceedings of the 19th Congress London, 1983, 193.

2. Anncss, B. J. & Reed, R. J. R., Journal of Institute of Brewing,

1985. 91, 313.

3. Axelrod, B., Cheesbrough, T. M. & Laakso, S., Methods in

Enzymology, vol 71, 1981, 441-451.

4. Baxter, E. D., Journal of Institute of Brewing, 1982, 88, 390.

5. Drost, B. W., van den Berg, R., Freijee, F. J. M., van derVelde, E. G. & Hollemans, M., Journal of American Society

of Brewing Clients, 1990, 48, 124.

6. Fedak, O. & de la Roche, I., Canadian Journal of Plant Science,

1977, 57, 257.

7. Folch, J., Lees, M. & Sloane Stanley, G. H., Journal of Biologi

cal Chemistry, 1957, 226, 497.

8. Liukkonen, K., Montfoort, A. & Laakso, S., Journal of Agricul

tural and Food Chemistry, 1992, 40, 126.

9. Lulai, E. C. & Baker, C. W., Cereal Chemistry, 1976, 53, 777.

10. Lulai, E. C. & Baker, W. & Zommcrman, D. C, Plant Physi

ology, 1981, 68, 950.


Vol. 99, 1993] barley lipids 403

11. Morrison, W. R., Advances in Cereal Science and Technology, 13. Song, V., Love, N. H. & Murphy, P., Journal of the American

1978, II, 221. Oil Chemists Society, 1990, 57, 961.

12. Schwarz, P. B. & Pyler, R. E., Journal ofAmerican Society of 14. Suutari, M., Liukkoncn, K. & Laakso, S., Journal of General

Brewing Clients, 1984, 42, 47. Microbiology, 1990, 136, 1469.


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