J. Agric. Food Chem. 1998, 46, 4475−4483
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Immunological Study of Hydrophobic Polypeptides in Beer E. N. Clare Mills,*,† Juliet A. Kauffman,† Michael R. A. Morgan,† J. Michael Field,‡ Jørn Hejgaard,§ Michael O. Proudlove,| and Akiko Onishi| Department of Biochemistry, Institute of Food Research Norwich Laboratory, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom; Advanta Seeds U.K., Docking PE31 8LS, United Kingdom; Department of Biochemistry and Nutrition, Building 224, Technical University of Denmark, DK-2800 Lyngby, Denmark; and Brewing Research International, Lyttel Hall, Nutfield, Surrey RH1 4HY, United Kingdom
A panel of 15 monoclonal antibodies (mAbs) has been raised to a fraction of lager beer protein precipitated with ammonium sulfate at 40% saturation. The antibodies have been characterized in terms of their binding to a set of Octyl-Sepharose fractions of lager beer foam with increasing hydrophobicity (groups 1-5) by enzyme-linked immunosorbent assay and immunoblotting methods. The epitopes recognized were present in all of the lager beer foam fractions to some degree, and many of the mAbs bound to both high molecular mass (MM) (>3000 Da) and low MM (5000 Da) have the greatest foaming potential (Anderson and Harris, 1963; Asano and Hashimoto, 1980), as do those of greater hydrophobicity (Slack and Bamforth, 1983; Onishi and Proudlove, 1994). The protein content of beer has been estimated to be ∼0.5 g L-1 (Kaersgaard and Hejgaard, 1979), although the figures obtained depend on the method of analysis and may in some instances also include nonprotein nitrogen (Bamforth, 1985). Beer proteins and polypeptides range in molecular mass from 90000 Da (Asano and Hashimoto, * Author to whom correspondence should be addressed (telephone 44-1603-255295; fax 44-1603-507723; e-mail
[email protected]). † Institute of Food Research Norwich Laboratory. ‡ Advanta Seeds. § Technical University of Denmark. | Brewing Research International.
1980), and many appear to be present as aggregates in beer (Sørensen and Ottesen, 1978; Dale and Young, 1992). Most beer proteins and polypeptides have been shown to originate from the malt by virtue of (1) being rich in the amino acids glutamine and proline, a characteristic of the barley storage proteins known as hordeins (Dale et al., 1989), and (2) immunological studies indicating the presence of barley-related proteins (Grabar and Daussant, 1971; Hejgaard and Sørensen, 1975; Kauffman et al., 1994; Sheehan and Skerritt, 1997). It has also been demonstrated that yeast proteins are present in beer, albeit as minor constituents (Hejgaard and Sørensen, 1975; Mohan et al., 1992). As the heterogeneity of barley proteins is further increased by modification during malting and brewing, it is not suprising that few beer proteins have been purified to homogeneity. The best characterized is protein Z (Hejgaard, 1977), an Mr 40000 protein found in barley, which remains virtually unchanged throughout the malting and brewing process and which has been estimated to make up ∼20-170 mg/L of beer protein. Another purified protein, associated with foam formation, is a barley lipid transfer protein 1 (LTP 1; Sørensen et al., 1993; Bech et al., 1995), which has an Mr of ∼9700, estimated by gel electrophoresis. In addition to classical biochemical methods of analysis such as chromatography and electrophoresis, researchers have also employed immunochemical techniques to study beer proteins. Polyclonal antisera against barley, malt, beer, and yeast proteins have been employed to investigate the origin of beer proteins, to remove polypeptides from beers to establish their effect on foaming (Hollemans and Tonies, 1989), to analyze beer for foam and haze proteins (Ishibashi et al., 1996; Mohan et al., 1992), and to measure the content of a
10.1021/jf9801163 CCC: $15.00 © 1998 American Chemical Society Published on Web 10/31/1998
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40000 Da foam protein (probably protein Z) in beer and its relationship with foam stability (Horiuchi et al., 1996). However, in general, the information that can be obtained using polyclonal antisera is limited partly because of the problem of polyspecificity, which results from the presence of immunodominant repetitive sequences from hordeins in beer (Mills et al., 1995); in some instances, polyclonal antibodies have to be affinity purified to remove anti-hordein antibodies (Bech et al., 1995). Such problems are circumvented by using monoclonal antibodies (mAbs) targeting a single epitope. They have been employed to examine modification of hordeins (Skerritt, 1988; Skerritt and Henry, 1988) and limit dextrinase (Sissons et al., 1992) during malting and also to characterize hordein-derived beer and foam polypeptides (Kauffman et al., 1994; Sheehan and Skerrit, 1997). Food proteins that undergo thermal and proteolytic processing, such as the storage proteins of soybean (Plumb et al., 1994, 1995) and pea (Quillian et al., 1990), have also been investigated using mAbs, but to date there are no reports of mAbs being raised to beer proteins. The present paper describes the production and characterization of a panel of mAbs developed to lager beer proteins; the data obtained show that many of the immunologically reactive beer polypeptides originate in malt. MATERIALS AND METHODS Materials. Bicinchoninic acid (BCA) protein assay kit was from Pierce and Warriner, Chester, U.K. Freund’s adjuvant was from Difco (Detroit, MI). Nunc Immunoplate I microtitration plates were from Gibco BRL Life Technologies Ltd. (Paisley, U.K.). Ultrafiltration membrane (YM3 DIAFLO with molecular weight cutoff of 3000 Da) was from Amicon Ltd. (Stonehouse, U.K.). Peroxidase and alkaline phosphatase labeled anti-rabbit IgG, plus anti-mouse IgG/IgM, avidinhorseradish peroxidase conjugate, nitroblue tetrazolium, 5-bromo-4-chloro-3-indolyl phosphate (BCIP), Nonidet NP-40, and bovine serum albumin (BSA) were from Sigma Chemical Co., Poole, U.K. Substrate based on 3,3′,5,5′-tetramethylbenzidine (TMB) was from Vetoquinol, Bicester, U.K. Kathon was from Rohm and Haas (Croydon, U.K.). Nitrocellulose membrane was from Sartorius Ltd., Epsom, U.K., Octyl-Sepharose CL4B was from Pharmacia Biotech (Uppsala, Sweden), and prestained molecular weight markers were from Bio-Rad (Hemel Hempsted, U.K.). All other reagents were of AR grade. Barley and Malt Samples. Lager beer for immunogen preparation and foam fractions, together with the pilsner and crystal malts used in its production, was obtained from a commercial brewery. Malting barley cultivars (cv.) Blenheim and Puffin were supplied by Advanta Seeds U.K. (Docking, U.K.), whereas the malted barley cv. Puffin was produced using pilot-scale maltings at BRI (Nutfield, U.K.). Lager Beer and Malt Fractions. A protein fraction of a commercial lager beer was prepared by precipitation at 4 °C with (NH4)2SO4 at 40% saturation (termed the ammonium sulfate fraction), followed by dialysis of the resulting precipitate using a 10000 Da cutoff membrane, against water prior to freeze-drying. A stock solution of this fraction was prepared in 4% (v/v) ethanol for use as the immunogen and stored at -20 °C. Foam fractions of differing hydrophobicity (groups 1-5) were prepared from the same lager beer by hydrophobic interaction chromatography on Octyl-Sepharose CL-4B as described by Onishi and Proudlove (1994) and fractionated further into high and low MM fractions by ultrafiltration through a 3000 Da cutoff membrane. The majority (pilsner-type) and minority (crystal-type) malts were ground, and 1 g was extracted with shaking in 2 × 10 volumes (i.e., 10 mL) of 1 M NaCl for 1 h at 25 °C. After centrifuging at 10000g for 30 min at 25 °C, the supernatants (containing the salt-soluble malt components and termed the
Mills et al. salt fraction) were decanted and pooled. The resulting pellets were rinsed with water and re-extracted with 2 × 10 volumes (i.e., 10 mL) of 50% (v/v) propan-1-ol, 2% (v/v) acetic acid, and 2% (v/v) 2-mercaptoethanol by shaking for 1 h at 25 °C. Extracts (essentially containing residual prolamins and termed the propanol fraction) were centrifuged as described above and the supernatants retained and pooled. Salt- and propanolsoluble materials were then dialyzed against water at 4 °C, freeze-dried, and stored at -20 °C until required. A saltsoluble fraction of barley (cv. Blenheim) was produced in the same way. The protein contents of extracts were determined using a BCA assay kit. Antibody Preparations. Polyclonal antisera were raised in rabbits to purified protein Z and β-amylase as described by Hejgaard (1982). mAbs were produced essentially as described by Mills et al. (1990) with the following modifications. Mice were immunized with the ammonium sulfate fraction of lager beer, which was also used for mAb selection. All cell culture procedures were performed using OptiMEM (Gibco, U.K. Ltd.) supplemented with 4% foetal calf serum (FCS; from Advanced Protein Products Ltd., U.K.) as the culture medium. mAbs were isotyped using an isotyping kit (Sigma Chemical Co.), and culture supernatants were concentrated as required using Centriprep centrifugal concentrators (30000 Da cutoff membrane, Amicon). ELISA Procedures. For selection of mAbs, Nunc Immunoplate I microtitration plates were coated (inner 60 wells only) with 0.3 mL/well of the immunogen (i.e., the ammonium sulfate fraction of lager beer at 1 µg/mL) in 0.05 M sodium carbonate-bicarbonate buffer, pH 9.6. After overnight incubation at 4 °C, plates were washed three times with water (using a Wellwash 5000, Life Sciences International U.K. Ltd., Billingshurst, U.K.), allowed to dry in air, and stored at -20 °C until required. For cross-reactions with lager beer fractions, plates were coated using a Biomek 1000 (Beckman Instruments, High Wycombe, U.K.) with a different fraction in each quarter, the lower right quarter always being coated with the 40% ammonium sulfate fraction of lager beer. For screening of hybridomas, culture medium (0.15 mL/well) was transferred from culture to assay plates and incubated overnight at 4 °C. Plates were then washed five times in phosphate-buffered saline containing Tween 20 [PBST; 0.14 M NaCl, 0.0015 M KH2PO4, 0.008 M Na2HPO4, 0.0027 M KCl; pH 7.4, containing 0.05% (v/v) Tween 20] prior to the addition of 0.2 mL/well of anti-mouse IgG horseradish peroxidase conjugate diluted 1:1000 (v:v) in PBST. Following a 3 h incubation at 37 °C, plates were washed a further five times in PBST before the addition of 0.2 mL/well of substrate based on 3,3′,5,5′-tetramethylbenzidine (TMB). After the color had been allowed to develop for 10 min at 37 °C, the reaction was stopped by the addition of 50 µL/well of 2 M H2SO4, and the absorbance values of the wells were determined at 450 nm using a Titertek MCC plate reader (Flow Laboratories, Thame, U.K.). For cross-reactions, identical titration curves were set up on each coated quarter plate using the Biomek 1000, with either culture supernatant diluted appropriately in PBST, or rabbit anti-protein Z or β-amylase antisera; plates were then developed as described above using anti-rabbit IgG labeled with horseradish peroxidase for those plates set up with the rabbit antisera. In this way comparable titration curves could be obtained for all of the fractions of interest. From these the cross-reactivities (as a percentage) were calculated as the ratio of binding observed to each lager beer fraction to the binding observed to the 40% ammonium sulfate fraction of lager beer, at a particular antibody dilution. For the polyclonal antisera cross-reactions were calculated relative to a salt-extract of barley (cv. Blenheim). Electrophoretic and Immunoblotting Procedures. Grain (barley or malt) was crushed and sonicated in 3.33% (w/v) SDS, 0.067 M Tris-HCl, pH 6.8, 10% (v/v) glycerol, and 0.001% (w/v) Pyronin Y containing 100 mM DTT and heated to 100 °C for 3 min. Beer and foam fractions were brought to the same buffer composition by the addition of 50% (v/v) glycerol and concentrated sample buffer [0.67 M Tris-HCl, pH 6.8, containing 33.3% (w/v) SDS and 0.01% (w/v) Pyronin Y
Immunological Study of Hydrophobic Polypeptides in Beer
Figure 1. SDS-PAGE analysis of barley, malt, and lager beer fractions: (A) track 1, total barley seed proteins cv. Puffin; track 2, total malt proteins cv. Puffin; track 3, 40% ammonium sulfate fraction of lager beer; track 4, lager beer foam group 5; (B) track 1, pilsner malt salt fraction; track 2, pilsner malt propanol fraction; track 3, barley from which pilsner malt was derived. Parts A and B show gels run at different times. Molecular weight markers were as follows for both gels: 1, bovine serum albumin, 66000; 2, hen egg ovalbumin, 45000; 3, rabbit muscle glyceraldehyde-3-phosphate dehydrogenase; 4, bovine erythrocyte carbonic anhydrase, 29000; 5, bovine pancreatic trypsinogen, 24000; 6, soybean trypsin inhibitor 20000; 7, bovine R-lactalbumin, 14000. containing 1 M DTT] and boiled as above. Barley, malt, and beer fractions were analyzed using a Tris-borate buffer system as described by Koenig et al. (1970) with a 15% acrylamide-0.1% bis(acrylamide) separating gel and a 3% acrylamide-0.08% bis(acrylamide) stacking gel using a vertical gel electrophoresis system (Hoefer Scientific, U.K.). Foam fractions were separated using a 15% polyacrylamide gel Laemmli system (Onishi and Proudlove, 1994) with prestained molecular weight markers. Immunoblotting was performed essentially as described by Mills et al. (1990) using a 0.2 µm pore nitrocellulose membrane and an alkaline phosphatase/BCIP-nitroblue tetrazolium detection method. RESULTS
A panel of 15 mAbs was developed to an ammonium sulfate fraction of lager beer, the composition of which was determined by SDS-PAGE (Figure 1A, track 3). The major components of this fraction were an Mr ∼ 20000 polypeptide and two low molecular mass polypeptides of
