Previous PageTable Of ContentsNext Page

Study of lipid-protein interactions in gluten using a 3-step solvent extraction and acetic acid fractionation techniques

T. McCann1, 3, D.M. Small1 and L. Day2, 3

1 RMIT University, Melbourne, VIC 3000, Australia (T. McCann was previously T. Vu)
2
Food Science Australia, Werribee, VIC 3030, Australia
3
Value Added Wheat CRC, North Ryde, NSW 1670, Australia

Introduction

Lipid constitutes 1.4–2.0% of the dried weight of wheat flour. After removal of starch, the remaining component, termed gluten, consists of 6-10% lipid. When gluten is used as a food ingredient, this level of lipid may have an adverse impact on product flavour and colour. The removal of lipids affects gluten functionality possibly due to the association of lipids with gluten proteins.

In flour and gluten, lipid exists in both free form (lipid that can be extracted with petroleum ether (PE), hexane or diethylether) and bound form (lipid that can be only extracted with ethanol or a mixture of an alcohol and water) (Cornell and Hoveling, 1998). Water addition and dough mixing during the gluten preparation process could promote the association between lipids and protein, thereby changing the solvent extractability of lipid. In comparison with flour, only a small proportion of lipids can be extracted from gluten using ether (Chung, 1986). The changes in the solvent extractability of lipid can be used to investigate the association of lipid and protein in gluten. In the present study, lipid from flour, gluten, PE-treated gluten and acetic acid gluten fractions were sequentially extracted with solvents of different polarity, followed by HPLC analysis. The aim was to investigate the association of lipids with wheat protein by comparing the solvent extractability of lipid between flour and gluten, and also between PE-treated gluten and acetic acid-treated gluten.

Materials and methods

Gluten preparation

Gluten was prepared from wheat flour cv. Lang provided by Allied Mills from the 2004 harvest. Flour (300 g) was mixed with water (180 mL) for 1 min at setting 1 using a Hobart mixer, and then at setting 2 for 2.5 min to form a dough. This was rested in water for 30 min and hand washed with water (3 5 L) to remove starch. Wet gluten was collected, freeze-dried and ground using a coffee grinder. All dried gluten, as well as other dry samples, were stored at −18C.

Lipid extraction

Lipid was extracted from flour and gluten samples using a sequential solvent extraction. PE was used as the first solvent, followed by chloroform and then ethanol. Hot ethanol (70C) was used for gluten to facilitate the lipid extraction. Flour or gluten was mixed with solvent in the ratio of 1:2 (w/v) for 15 min (MacRitchie 1985). The slurry was filtered with a Buchner funnel using a Whatman No 45 paper and refined by a normal filtration using a Whatman No 1 paper.

The solvent in lipid extracts was removed using a rotator evaporator at 40C for PE, 60C for chloroform and 75C for ethanol. The lipid extract was further dried under nitrogen and dissolved in chloroform for petroleum-extracted lipids and in chloroform/methanol mixture (1:1, v/v) for chloroform- and ethanol-extracted lipids. The extracts were stored at −20C until further analysis.

Lipid analysis by HPLC

Lipid classes were separated by HPLC using a polyvinyl alcohol chemically bonded stationary phase PVA Sil column (5 μm, 250 mm x 4.6 mm; YMC, Japan), eluted with a gradient solvent system containing 2,2,4 trimethyl pentane, iso-propanol, dichloromethane, methanol, N-ethyl morpholine and glacial acetic acid (Fagan et al 2004). Lipid compounds were detected using a PL-ELS 1000 detector operated at 40C for nebulization and 80C for evaporation with a gas flow of 1.0 mL/min. A range of lipids were obtained from Sigma (USA) for use as standards.

Gluten fractionation with acetic acid

Gluten (50 g) was firstly treated with PE (4 200 mL) to remove free lipids. The treated gluten was placed in a fume cupboard for 3 days to allow evaporation of solvent residue. Treated gluten (10 g) was mixed with 0.01 M or 0.1 M acetic acid (200 mL) for 2 min using an Ultra Turrax at speed of 9500 rpm followed by centrifugation at 24000 g, at 4C for 15 min (Brot et al 1994). The supernatant and pellet were collected, freeze dried, ground and stored.

Lipid extraction from PE-treated gluten and its fractions

Lipid was extracted using PE and followed by 70C hot ethanol (with 2 extractions). The extraction was performed at the ratio of 1:10 (w/v) for the gluten and 1:20 (w/v) for the fractions. The solvent was removed and lipid extracts were dissolved in chloroform or chloroform/methanol and stored at −20C until further analysis.

Results and discussion

Comparison of the e2xtractability of lipid in flour and gluten

Lipids in gluten were less readily extractable with PE compared with flour. This indicates that the level of free-form lipids was lower in gluten than flour (Table 1), consistent with earlier reports (Hoseney et al 1970). Consequently, the proportion of bound lipids (ethanol and chloroform extractable) was higher in gluten (72.5% of total lipid) than in flour (36.4%). The results demonstrate a likely association of lipids with gluten protein.

Table 1. Extractability of lipid from flour and gluten

 

Flour

Gluten

Total lipid extracted (g per 100g)

1.40

7.02

     

In each successive solvent

   

PE (%)

63.6

27.5

Chloroform (%)

15.7

10.4

Ethanol (%)

20.7

62.1

Of the lipids initially present in flour, a large proportion of the non-polar lipids remained in gluten (Table 2). However, non-polar lipids in gluten were less extractable with PE than in flour, demonstrated by only 35% of non-polar lipid extracted from gluten with PE, compared to 92% from flour (Table 2). Results showed that the large amount of non-polar lipids in flour had probably become associated with proteins during gluten preparation. These lipids could either be bound with proteins based on hydrophobic interactions or physically embedded in the protein matrix (Marion et al 1987). Approximately half of the glycolipids and only a small amount of phospholipids from flour were found in gluten (Table 2). Phospholipids have been known to be associated with starch granules (Morrison, 1988); therefore they may be removed along with the starch during gluten washing.

Table 2. Distribution of lipid classes in flour (cv. Lang) and gluten (values are g and results in parentheses are the proportion expressed as a percentage of the total)

 

Flour
(100 g of flour)

Gluten
(from 100 g of flour)

         

Total lipid

1.39

(100)

1.05

(100)

Total non-polar lipids

0.83

(60)

0.83

(79)

Total glycolipids

0.29

(21)

0.18

(17)

Total phospholipids

0.28

(19)

0.05

(5)

         

Total non-polar lipids

0.83

(100)

0.83

(100)

Free-form

0.76

(92)

0.29

(35)

Bound-form

0.07

(8)

0.54

(65)

         

Total glycolipids

0.29

(100)

0.18

(100)

Free-form

0.11

(38)

0.00

(0)

Bound-form

0.18

(62)

0.18

(100)

         

Total phospholipids

0.28

(100)

0.05

(100)

Free-form

0.02

(7)

0.00

(0)

Bound-form

0.26

(93)

0.05

(100)

         

Changes in lipid extractability after acetic acid fractionation

The amount of lipid in gluten treated with acetic acid was calculated by combining the data from separate analyses of supernatant and pellet. The results showed that the proportion of lipids extracted as free-form (PE-extractable) was higher in the acetic acid-treated gluten than the gluten control (Figure 1). This indicates that acetic acid was able to alter the gluten matrix to release additional lipids as a consequence of the protein structure modification. The higher acetic acid concentration (0.1 M) appeared to have a stronger effect on protein structure, as it generated a slightly higher amount of free-form lipids compared with the lower concentration of 0.01 M (Figure 1).

Figure 1. Free- and bound-form lipid levels in gluten before and after treated with acetic acid

Conclusion

Gluten contains high amounts of bound-form lipid compared to flour. Of the lipids initially found in the flour, a large proportion of non-polar lipids remained in gluten. These non-polar lipids became less readily extractable with PE and were more extractable with hot ethanol (70C). They could be bound to proteins or entrapped in the gluten matrix. Dilute acetic acid was able to change the protein conformation of gluten, thereby facilitating a dissociation of lipids from gluten proteins.

Acknowledgements

The authors are grateful to Dr I. L. Batey and Dr C. W. Wrigley, Value Added Wheat CRC and Food Science Australia, NSW for valuable discussions. This research is supported by funds from the Value Added Wheat CRC.

References

Brot, S., Gautier, S., Nicolas, M., Godon, B., and Popineau, Y. (1994) Inter. J. Food Sci. Tech. 29:489-502.

Chung, O.K. (1986) Cereal Foods World 31:242-256.

Cornell, H.J., and Hoveling, A.W. (1998) Wheat: Chemistry and Utilization. Penn.:Lancaster. 19-32.

Fagan, P., Wijesundera, C., and Watkins, P. (2004) J. Chromatogr. A 1054:251-259.

Hoseney, R.C.; Finney, K.F., and Pomeranz, Y. (1970) Cereal Chem. 47:135-140.

MacRitchie, F. (1985) J. Cereal Chem. 3:221-230.

Marion, D., Roux, C.L., Akoka, S., Tellier, C., and Gallant, D. (1987) J. Cereal Sci. 5:101-115.

Morrison, W.R. (1988) J. Cereal Sci. 8:1-15.

Previous PageTop Of PageNext Page