use of chemiluminescencehplc for measurement of positional isomers of hydroperoxy fatty acids in...

J Sci Food Agric 1996,70,341-346

Use of Chemiluminescence HPLC for Measurement of Positional Isomers of Hydroperoxy Fatty Acids in Malting and the Protein Rest Stage of Mashing Martin D Walker,* Paul S Hughes and William J Simpson? BRF International, Nutfield, Redhill, Surrey, RH1 4HY, UK

(Received 9 March 1995; revised version received 4 July 1995; accepted 22 September 1995)

Abstract: Fatty acid hydroperoxides (9- and 13-hydroperoxides of linoleic acid and linolenic acid) were extracted from barley, malt and wort, and quantified by chemiluminescence HPLC. Although not detected in dried barley ( ~ 0 . 5 pmol kg- ' (dry wt)), the concentrations of hydroperoxides increased during germination (up to 156 pmol kg-' (dry wt) in the case of 9- hydroperoxylinoleic acid). Lipoxygenase (LOX) activity increased more than two-fold during germination. LOX activity and hydroperoxide concentrations were reduced considerably on kilning of malt. During mashing on a laboratory scale, malts with higher total LOX activities produced higher concentrations of hydroperoxides. The concentrations of 9-hydroperoxides were double those of the 13-hydroperoxides during malting and up to 10-fold greater during mashing, indicating a greater activity of LOX-1 in both processes.

Key words: beer, fatty acids, hydroperoxides, oxidation.

INTRODUCTION LOX catalyse reactions between molecular oxygen (0,) and polyunsaturated fatty acids with a cis,cis-1,4-

Stale flavours form in beer during storage, possibly as a pentadiene system to give optically active conjugated result of oxidation of unsaturated fatty acids (Graveland cis,trans-diene hydroperoxy derivatives. Substrates for et a2 1972). While oxidation reactions occur in the bottle LOX are readily available in barley, since this cereal or can, some of the propensity of beer to stale may be contains up to 44 g lipid kg-' (dry wt). Approximately determined earlier in the brewing process. For example, 580 g kg-' of this material is linoleic acid and lipoxygenases (LOX, linoleate: oxygen oxidoreductase 100 g kg-' is linolenic acid (Anness 1984). Much of it is EC 1.13.11.12) in barley and malt may catalyse oxida- esterified with glycerol (eg present as triglycerides). Free tion of fatty acids in the malting process and in wort fatty acids, including substrates for LOX, may be rel- production (Graveland et a2 1972; Lulai and Baker eased from such materials by the action of lipases. 1975; Kobayashi et a2 1993a-c). LOX-catalysed reac- Two types of LOX activity are present at low levels tions are specific with respect to formation of both posi- in barley and develop further during germination tional isomers and stereoisomers of hydroperoxy fatty (Baxter 1982). LOX-1 produces mainly 9- acids, in contrast to metal-catalysed auto-oxidation hydroperoxides from Iinoleic acid and linolenic acid; which is non-specific (Fig 1) (see Kochhar (1993) for a LOX-2 produces mainly 13-hydroperoxides (Doderer et review). a1 1992; Yang et a1 1993).

Here, we describe a chemiluminescence HPLC (CL- HPLC) method which allows individual 9- and 13- * Present address: Guinness Brewing GB, Park Royal,

London, NWlO 7RR, UK. hydroperoxide isomers of linoleic acid and linolenic t To whom correspondence should be addressed. acid to be quantified. Using this method, we have

J Sci Food Agric 0022-5142/96/$09.00 0 1996 SCI. Printed in Great Britain 341

342

R

M D Walker et a1

WH

OH 9-hydropemxylinoleic wid

OOH

OH

13-hydropsronylinoleic acid

Fig 1. Oxidation of linoleic acid. Auto-oxidation gives an equimolar mixture of 9- and 13-hydroperoxylinolenic acid. Oxidation by lipoxygenases (LOX) gives predominantly the 9-isomer in the case of LOX-1 and the 13-isomer in the case of LOX-2.

attempted to define the extent to which LOX-1 and LOX-2 are active in malting and mashing.

EXPERIMENTAL

Chemicals

All chemicals were analytical reagent grade except trifluoroacetic acid, acetonitrile and tetrahydrofuran which were HPLC grade. Tetrahydrofuran was distilled from copper(1) chloride before use to eliminate peroxides. Microperoxidase (MP-11) was from Sigma (Poole, UK). Individual hydroperoxy fatty acid isomers were from Cascade Biochem Limited (Reading, UK). N , N - Dimethyl-l,4-phenylenediamine (97% purity) was from Aldrich (Gillingham, UK). Solid CO, was from BOC (Manchester, UK). Isoluminol was from Sigma (Poole, UK).

Malting conditions

Batches (2 kg) of barley (Hordeurn uulgare var Blenheim) were malted at 16°C under the following conditions: steep (8 h); air rest (16 h); steep (24 h); steep volume 13.5 litres. After 4 days germination, the green malt was dried at 45°C for 8 h, then at 65°C for 16 h. Curing was at 80°C or 105°C. Malts used in mashing experiments were produced in the BRFI pilot-scale (50 kg) maltings using the following regime: steep (8 h); air rest (16 h); steep (8 h); air rest (10 h); steep (2 h); air rest (2 h); steep volume, 180 litres. The barley was germinated for 4 days at 16"C, then dried at 45°C for 8 h, followed by 65°C for 16 h, before curing at either 80°C or 105°C.

Laboratory-scale mashing conditions

A sample of malt (100 g) was ground in a disc mill (Type DLFU, Buhler-Miag (England), New Barnet,

UK) at a gap setting of 0.7 mm, mixed with distilled water (300 ml) then mashed at 52°C for 60 min using a mashing bath (Crisp Malting Ltd, Fakenham, UK) (Buckee et a1 1978).

Extraction of fatty acid hydroperoxides from barley and malt

Solid carbon dioxide (5 g) was added to samples (50 g) at various stages of the malting process. The mixture was milled for 30 s in a coffee mill then quantitatively transferred to a 250 ml stoppered conical flask and stirred for 30 min with diethyl ether/pentane (1 : 1, v/v; 200 ml) under N, in the dark. After filtration of the whole mixture through Whatman No 1 filter paper (Millipore, Watford, UK), solvent was removed under reduced pressure from a portion of the filtrate (120 ml). The residue was dissolved in methanol/chloroform (8 : 2, v/v; 10 ml) before analysis by CL-HPLC.

Extraction of fatty acid hydroperoxides from laboratory-scale malt mashes

At the time of sampling, the entire mash was poured into a beaker with addition of ice-cold distilled water (200 ml) and solid CO, (5 g). The mixture was extracted with diethyl ether/light petroleum (bp 40-60°C) 1 : 1, v/v; 500 ml: Kobayashi et al 1993a). Solvent was removed under reduced pressure from a portion (200 ml) of the extract which was then dissolved in methanol/chloroform (8 : 2, v/v; 10 ml) before analysis by CL-HPLC.

CL-HPLC analysis of hydroperoxides

Fatty acid hydroperoxides were separated by HPLC using a 100 x 8 mm C-18 4 pm Novapak cartridge (Waters, Watford, USA) under radial compression and a 20 pl injection loop. The mobile phase consisted of a

Oxidation of fatty acids 343

Activity / U g-' (dry wt)

600 -

500 -

400 -

300 -

barley cast day 1 day2 day3 day4 malt I I

germination

[Hydroperoxides] / pmol kg-' (dry wt)

b) T

100 '""1 A barley cast day 1 day2 day3 day4 malt

germination

Stage of malting

Fig 2. Lipoxygenase (LOX) activity and hydroperoxide production during the malting process. Hordeum vulgare var Blenheim was malted in 2 kg batches at 16°C using an inter- rupted steeping programme as described in the text. (a) LOX activities increased during germination and decreased as a result of kilning. (b) Hydroperoxides were formed during germination and destroyed on kilning. A, 9-Hydroperoxylinoleic acid was produced in excess over 0 , 13-hydroperoxylinoleic acid. Similarly, ., 9-hydroperoxylinolenic acid (.) was formed in excess over 13-hydroperoxylinolenic acid which was not detected (<0.5 pmol kg-' (dry wt)). Results shown represent single analyses. Similar results were obtained in replicate experiments. Error bars show the standard error of the

analysis.

mixture of distilled water (400 ml), tetrahydrofuran (200 ml), acetonitrile (200 ml), methanol (200 ml) and trifluoroacetic acid (1.0 ml), which was prepared daily and applied at a flow-rate of 2 ml min-l. (Care was taken to remove hydroperoxides from the tetrahydrofuran, to minimise background light emission from the CL assay.) After separation, the eluent was mixed with CL reagent via a zero-volume T-piece. The CL-reagent, which consisted of a mixture of methanol (700 ml), sodium tetraborate buffer (0.1 M, adjusted to pH 10 with 1 M NaOH; 300 ml), microperoxidase (27 mg) and isoluminol (180 mg), was applied at a flow- rate of 2 ml min-' and protected from light at all times.

Light output was measured using a Lumac M2010A luminometer (3M, St Paul MO, USA) equipped with a spiral flow-cell and connected to a Trivector Trio Com- puting Integrator (Trivector Inc, West Chester, USA).

Calibration plots were made of hydroperoxide concentration (0-200 p ~ ) against peak height of detector response.

Measurement of LOX activity

Total LOX activity (LOX-1 and LOX-2) was estimated as described by Baxter (1982), the method being based on that of Surrey (1964). This method assumes that oxidation of linoleic acid by LOC gives rise to production of conjugated dienes which absorb strongly ( E = 25 000; Doderer et al 1992) at 234 nm. The enzymes were extracted from barley or malt by grinding the grains in a coffee mill using solid CO, to cool the samples. After allowing the CO, to evaporate, sodium acetate buffer (0.1 M; pH 5.0; 40 ml) was added. The mixture was shaken using a reciprocal shaker (Griffin & George, London, UK) for 1 h at 4°C then clarified by centrifu- gation (3000 x g; 10 min). A portion (50 pl) of the clarified extract was added to sodium phosphate buffer (0.1 M; pH 6-8; 2.85 ml) which contained linoleic acid (50 pl). The substrate solution was prepared anoxically, using Tween 20 as a dispersant, as described by Surrey (1964). The increase in A, , , was monitored using a Philips P U 8720 spectrophotometer (Philips, Cam- bridge, UK) during incubation of the sample at 20°C. One unit of LOX activity represents an increase in A234

of 1.0 min - ' on incubation at 20°C. Control assays established that the 0, concentration did not become limiting in the assay. Samples of mash were analysed in essentially the same way. Portions of mash (50 g) were cooled with solid CO, (5 g) and freeze-dried before measurement of LOX activity.

RESULTS

Measurement of 9- and 13-hydroperoxides by CL-HPLC

The CL-HPLC method described here efficiently separated the positional isomers of linoleic acid and linolenic acid. The 13-hydroperoxide isomers of each fatty acid eluted before the 9-hydroperoxide isomer in each case. Replicate HPLC analyses had a coefficient of variation of no more than 4%. Overall, the coefficient of variation of the analytical method (extraction and assay) was of the order of 20%.

Total LOX activity and fatty acid hydroperoxide concentrations during malting

Total LOX activity increased markedly during germination on the laboratory scale, reaching a peak on day

344 M D Walker et a1

3 before falling on day 4. LOX activities were substan- tially reduced after kilning at 80°C (Fig 2a). No hydroperoxides were detected in dried barley ( ~ 0 . 5 pmol kg-' (dry wt)), but concentrations increased markedly during germination (Fig 2b). More 9-hydroperoxylinoleic acid and 9-hydroperoxylinolenic acid were present in the barley during malting than the 13-isomers. No hydroperoxides were detected after kilning (<0.5 pmol kg- (dry wt)).

Formation of fatty acid hydroperoxides during mashing

Concentrations of fatty acid hydroperoxides were measured during mashing at 52°C of two malts produced from the same batch of germinated barley in a pilot- scale malting facility. Final kilning temperatures were 80°C and 105"C, giving final LOX activities of 85.4 units g-' and 3.4 units g-', respectively. No hydroperoxides (< 0.5 pmol kg - (dry wt)) were detected in the milled malts before addition of water, but they could be detected after only 5 rnin mashing (Tables 1 and 2). After this initial increase, concentrations of hydroperoxides remained at a similar level, or declined

by up to 60%. More hydroperoxides were detected during mashing with malts kilned to 80°C than with those kilned to 105°C. In both cases, 9-hydroperoxylinoleic acid was found in larger amounts than 13-hydroperoxylinoleic acid. 9-Hydroperoxy- linolenic acid was formed in slight excess over 13- hydroperoxylinolenic acid.

DISCUSSION

A number of HPLC methods which allow the positional isomers of linoleic acid oxidation products to be measured (Aarle 1991; Doderer et al 1992; Laquet et al 1993) has been described. However, these methods suffer from poor sensitivity, since they employ uv detection. CL detection improves sensitivity and selectivity considerably, but the reagents used are incompatible with nonpolar solvents which constitute a large propor- tion of the mobile phase in some methods. The method described here, which is a hybrid of the methods of Yamamoto et al (1987) and Aarle et al (1991), over- comes these problems, allowing low level quantification of these isomers.

TABLE 1 Hydroperoxides produced during mashing at 52°C of malt kilned to

105°C (all measurements given as pmol hydroperoxide kg-' (dry wt))"

Hydroperoxylinolenic acid Hydroperoxylinoleic acid

13-Isomer 9-Isomer 13-Isomer 9-Isomer

Malt <0*5 < 0.5 < 0.5 < 0.5 5 rnin 0.8 f 0.16 1.1 f 0.22 1.8 f 0.36 9.2 & 1.84

15 rnin 1.0 f 0.20 1.2 f 0.24 3.2 f 0.64 8.4 f 1.68

60 min 0.8 f 0.16 1.1 f 0.22 3.0 f 0.60 4.0 f 0.80 30 min < 0.5 1.2 f 0.24 2.6 f 0.52 4.6 f 0.92

Results shown represent single analyses. Similar results were obtained in replicate experiments. Ranges illustrate the standard error of the analysis.

TABLE 2 Hydroperoxides produced during mashing at 52°C of malt kilned to 80°C

(all measurements given as pmol hydroperoxide kg- ' (dry wt))"

Hydroperoxylinolenic acid Hydroperoxylinoleic acid

13-Isomer 9-Isomer 13-Isomer 9-Isomer

Malt <0.5 <0.5 ~ 0 . 5 <0*5 5 rnin 4.0 f 0.80 3.4 f 0.68 4.0 k 0.80 30.5 f 6.10

15 rnin 3.7 f 0-74 2-9 f 0.58 3.0 k 0.60 24.8 f 4-96 30 rnin 2.9 f 0.58 4.5 f 0.90 3.6 0.72 26.6 k 5.32 60 rnin 2.7 f 0.54 3.9 f 0.78 4.9 f 0.98 22.1 f 4.42

a Results shown represent single analyses. Similar results were obtained in replicate experiments. Ranges illustrate the standard error of the analysis.

Oxidation of fa t ty acids 345

Formation of hydroperoxides during malting

Substantial formation of fatty acid hydroperoxides occurred during malting and especially during germination. LOX activity increased until day 3 of germination then fell on day 4 (Figs 2a and b). Even though LOX activity fell during the later stages of germination, the total amounts of hydroperoxides in the grain con- tinued to increase. The hydroperoxide measurements relate to concentration, whereas LOX measurements give instantaneous enzyme activities. Furthermore, the availability of free fatty acid substrate may have increased toward the latter stages of germination.

The total amount of hydroperoxy fatty acids produced was of a similar magnitude to that found previously using an assay based on the reaction of the hydroperoxides with xylenol orange (Bamforth et al 1993). More 9-hydroperoxide isomers were formed than the 13-isomers, suggesting that enzymic, rather than non- enzymic production was of greater importance under these conditions. In particular, LOX-1 appears to be more active than LOX-2. Interestingly, significant production of 9-hydroperoxylinolenic acid was detected, while 13-hydroperoxylinolenic acid was not detected. In uitro, barley LOX have been shown to display greater preference for linoleic acid than for linolenic acid (Doderer et a1 1992). However, as Doderer et a1 (1992) point out, the physical state of the fatty acids may be of critical significance in uiuo.

The concentration of hydroperoxides in malt was reduced to below the detection limit (<0-5 pmol kg- ' (dry wt)) on kilning. The fate of these hydroperoxides is of key importance, but presently unknown. They may be converted to volatile products and lost during kilning, which would mean that the extent of unsaturated fatty acid oxidation occurring during malting is irrelevant. However, they may remain in the malt in a modified form, imparting the potential for formation of stale flavours in beer. Which of these possibilities occurs is not yet established.

Formation of hydroperoxides during mashing

During mashing of lager malts, an initial temperature of 52"C, or lower, may be used to assist protein hydrolysis (Briggs et al 1981). After a period at 52"C, the temperature of the mash is raised to encourage saccharifi- cation. In the present study, we used a mash temperature of 52°C to model the processes occurring in the early stages of mashing.

During mashing, the concentrations of fatty acid hydroperoxides detected were greater with malt kilned to a final temperature of 80°C than with malt kilned to 105°C: this paralleled total LOX activities. In this respect, these results confirm those reported by Drost et al(1990) and Kobayashi et al(1993a-c) who found that,

under various mashing conditions 30-100 pmol hydroperoxide litre- ' were produced (note that our results are expressed as pmol hydroperoxide kg-' (dry wt)). However, we have extended the observation by showing that 9-hydroperoxide isomers are produced in excess over 13-hydroperoxide isomers.

Excess production of 9-hydroperoxide isomers may be indicative of enzymic oxidation, more specifically oxidation by LOX-1. Isomeric specificity was most apparent in the case of linoleic acid. It is possible, though less likely, that 9- and 13-hydroperoxides are produced in equal amounts, as a result of auto- oxidation or equivalent activity of the two LOX types, but consumed at unequal rates by hydroperoxide- degrading enzymes.

Kobayashi et a1 (1993a-c) used CL-HPLC to measure hydroperoxylinoleic acid and hydroperoxylinolenic acid in mashing. When LOX activity was suppressed, through the use of inhibitors, CO, purging, acidification of the mash, increasing mashing- in temperature or using malts of low LOX activity, production of hydroperoxides was suppressed. These findings, considered in the context of our own results suggest that, under some conditions, enzymic activity may be of significance in production of 9-hydroperoxy fatty acids in malting and mashing. However, a key question remains: are hydroperoxides converted to volatile products and lost before the later stages of beer production, or do they survive each stage to give rise to stale flavours in the packaged beer? Further work to identify the fate of such hydroperoxides is needed to establish the significance of this work.

ACKNOWLEDGEMENTS

The authors thank Ann Meacham, Louise Bolshaw and Anthony Heasman for technical assistance and Charlie Bamforth for many helpful discussions. Shaun Burke and Denise Baxter critically reviewed the manuscript, which is published with the permission of the Director- General of BRF International.

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use of chemiluminescencehplc for measurement of positional isomers of hydroperoxy fatty acids in...

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