Chemical Methods for the Characterization of Proteolysis in Cheese During Ripening Plh Mcsweeney, Pf Fox

Chemical methods for the characterization of proteolysis in cheese during ripening Plh Mcsweeney, Pf Fox

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Plh Mcsweeney, Pf Fox. Chemical methods for the characterization of proteolysis in cheese during ripening. Le Lait, INRA Editions, 1997, 77 (1), pp.41-76. hal-00929515

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Review

Chemical methods for the characterization of proteolysis in cheese during ripening

PLH McSweeney, PF Fox

Department of Food Chemistry, University College, Cork, Ireland

Summary - Proteolysis is the principal and most complex biochemical event which occurs during the maturation of most cheese varieties. Proteolysis has been the subject of much study and a range of analytieal techniques has been developed to assess its extent and nature. Methods for assessing proteolysis can he c1assified under two broad headings: non-specifie and specifie techniques, both of which are reviewed. Non-specifie techniques include the quantitation of nitrogen soluble in various extractants or precipitants and the Iiberation of reactive groups. Specifie techniques include those which resolve individual peptides or free amino acids, principally chromatography or electrophoresis. Techniques are often combined into a fractionation scheme for cheese nitrogen to facilitate the isolation of individual peptides. Methods for the identification of individual peptides are also reviewed. Strategies for assessing proteolysis in various cheese varieties are discussed.

proteolysis / cheese ripening / methodology

Résumé - Méthodes chimiques pour la caractérisation de la protéolyse des fromages durant l'affinage. La protéolyse, qui est le phénomène principal et le plus complexe intervenant dans la maturation de la plupart des fromages, a été le sujet de nombreuses études. Toute une gamme de techniques analytiques a été développée afin de permettre une meilleure compréhension de ce phénomène. Les méthodes de suivi de la protéolyse peuvent être classées en deux grands chapitres, traités dans cette revue: les techniques non spécifiques, et les techniques spécifiques. Les techniques non spécifiques incluent la quantification de l'azote soluble dans les divers extraits, et s'intéressent aussi à la libération des groupements réactifs. Les techniques spécifiques, principalement la chromatographie ou l'élec- trophorèse, sont celles permettant de séparer les peptides ou les acides aminés libres. Ces diverses techniques sont souvent combinées dans la stratégie de purification de la fraction azotée du fro-

Oral communication at the lOF Symposium 'Ripening and Quality of Cheeses', Besançon, France, February 26-28, 1996. 42 PLH McSweeney, PF Fox mage afin de pouvoir isoler individuellememt les peptides. Les méthodes d'identification des peptides, ainsi que les différentes stratégies de suivi de protéolyse dans diverses variétés de fromages sont discutées dans cette revue. protéolyse 1 affinage du fromage 1 méthodologie

INTRODUCTION Bockelmann, 1995) which contribute to proteolysis in chee se, particularly at the level of the The biochemistry of cheese ripening is normally production of short peptides and free amino acids considered under three general headings: pro- (Visser, 1977). teolysis, lipolysis and glycolysis. Of these pro- cesses, proteolysis is the most important for most ripened chee se varieties (Fox et al, 1995a). Pro- The adijun ct starter teolytic agents in cheese originate from five sources: 1) the coagulant; 2) indigenous and Proteinases and peptidases from the adjunct endogenous proteinases from the milk; 3) the starter are particuarly important for proteolysis starter; 4) the adjunct starter; and 5) non-starter in certain chee se varieties. Penicillium spp and lactic acid bacteria. microorganisms from the smear of bacteria! surface-ripened cheeses produce pote nt extracel- lular proteinases and peptidases (Gripon, 1993; The coagulant Reps, 1993).

Chymosin and bovine pepsin in the case of calf rennet or recombinant chymosin or fungal Non-starter lactic acid bacteria aspartyl proteinases in cheeses manufactured using rennet substitutes are principally respon- The microtlora of essentially ail chee ses is char- sible for the production of large peptides (Visser, acterized by the growth of adventitious non- 1977; McSweeney etai, 1994a). starter lactic acid bacteria, NSLAB (typically Lactobacillus spp but also Pediococcus spp and perhaps other genera). The contribution of lndigenous and endogenous proteinases NSLAB to cheese quality has been the subject of from the milk mu ch recent research (see Peterson and Mar- shall, 1990; Martley and Crow, 1993; The principal indigenous proteinase in milk is McSweeney et al, 1995, for references). The plasmin, but somatic cell proteinases and contribution of NSLAB enzymes to proteolysis enzymes from the native microfiora of the milk in Cheddar cheese appears to be relatively minor (particularly heat-stable proteinases from psy- and at the level of the production of short pep- chrotrophs) are also present (Fox et al, 1995a). tides and free amino acids (McSweeney et al, 1994b; Lane and Fox, 1996). The extent and nature of proteolysis show The starter considerable intervarietal variation caused by differences in production protocols and ripen- Lactococcus spp, Lactobacillus spp and Strep- ing conditions (particularly whether a secondary tococcus salivarius ssp thermophilus used as microtlora is encouraged). Proteolysis is very starters for cheesemaking have extensive pro- !imited in Pasta Filata varieties (eg, Mozzarella) teinase/peptidase systems (see Exterkate, 1995; due to denaturation of enzymes during high- Methods used to assess proteolysis in chee se 43

temperature stretching, while in mould-ripened required, the reader is referred to the above varieties (particularly blue mould cheeses) pro- reviews. teolysis is extensive due to the presence of patent fungal proteinases. The products of proteolysis vary in size from NON-SPECIFIC METHODS large peptides comparable to intact caseins (eg, FOR ASSESSING PROTEOL YSIS

Water on many of the peptides released by chymosin or plasmin. Water-insoluble peptides in Cheddar Water is perhaps the most commonly used chee se have been studied by McSweeney et al extractant for cheese N. The level of water-sol- (I994a), who found that the principal peptides in uble N (WSN) is a widely-used index of cheese this fraction were produced from u.sl-casein by ripening (see Rank et al, 1985; Fox et al, 1995b) chymosin or pepsin or from ~-casein by plas- and several procedures have been developed min and therefore that the complementary pri- (see Christensen et al, 1991). Water is frequently mary water-soluble peptides must be produced used as the primary extractant in the isolation via the action of these enzymes. This hypothesis of amino acids and peptides. has been strengthened by the results of Singh et Kuchroo and Fox (1982a) compared various aI (1994, 1995, 1996) who isolated and identified extraction techniques for Cheddar cheese. Ail a large number of water-soluble peptides from but one of the several homogenization tech- Cheddar cheese; with a few exceptions, these niques studied yielded essentially similar results: peptides corresponded directly to or were in the a stomacher was chosen for routine work. immediate vicinity of cleavage sites for lacto- Homogenization temperature (in the range coccal cell envelope-associated proteinase but 5-40 "C) had \ittle effect on the level of generally did not include the principal cleavage extractable N, which increased with the ratio of sites of chymosin on U.sl-or plasmin on ~-casein, water to cheese; a ratio of 2: 1 was recommended, suggesting that chymosin and plasmin act first. and 90% of the potentially water-extractable N WSN as a percentage of total N varies with was recovered in two extractions. The proce- variety and increases throughout ripening (fig 1); dure recommended is: grated chee se is homog- a typical value for mature Cheddar cheese is enized in a stomacher at 20 "C for 10 min with -25%. Water is a suitable extractant for inter- twice its weight of water; the slurry is held at naI bacterially-ripened cheese varieties, the pH 40 "C for 1 h, centrifuged and the supernatant fil- of which changes Iittle during ripening. How- tered through glass wool and Whatman no 113 ever, mould and bacterial surface-ripened vari- filter paper. The residue can be re-extracted to eties are characterized by an increase in pH dur- increase the yield of extract. This and similar ing ripening; and since the extractability of N procedures have been used by a number of work- varies with pH, an alternative method should be ers (eg, Gonzàlez de Llano et al, 1991; Tiele- adopted. man and Warthesen, 1991; Wilkinson et al, 1992; Bütikofer et al, 1993; Singh et al, 1995, Extraction with solutions containing salts 1996). The water-soluble extract (WSE) of Ched- Extraction and quantification of cheese N solu- dar cheese contains numerous small- and ble at pH 4.6 (pH 4.6-SN) is widely used as an medium-sized peptides, free amino acids and index of proteolysis. Since the pH of many inter- their degradation products, organic acids and nai bacterially-ripened varieties is -5.2, there is their salts; extraction with water efficiently sep- little difference between the levels extracted by arates the small peptides in cheese from pro- water or pH 4.6 buffers. However, in the case teins and large peptides. In many cheeses, the of cheeses characterized by an increase in pH principal proteolytic agents responsible for the during ripening, WSN may be considerably production of WSN are the coagulant (chymosin) higher than pH 4.6-SN. Like WSN, pH 4.6-SN and, to a lesser extent, plasmin (Visser, 1977; is produced mainly by rennet (O'Keeffe et al, Fox et al, 1995a). Starter proteinases and pepti- 1976) and increases during ripening (eg, fig 1). dases play a relatively minor role in the hydro- Whey proteins and proteose peptones (from plas- Iysis of intact caseins and large polypeptides to min action) are also soluble at pH 4.6, but their water-soluble peptides, although they are active contribution to pH 4.6-SN is relatively srnall; Methods used to assess proteolysis in cheese 45

40..----.------, and Venema et al (1987) homogenized cheese in

0,037 mol/L CaCI2, adjusted the homogenate to pH 7.5, followed by centrifugation. Visser (1977) 30 homogenized cheese in 4% NaCI containing

0,55% Ca (as CaCI2). Fat was removed and the z t:: pH of the homogenate was adjusted to 7.5, fol- z en 20 lowed by centrifugation. Noomen (1977b) used ;F. a method based on sensitivity to coagulation by pH and heat to fractionate cheese N. An extract 10 was prepared by homogenizing chee se in a

CaCI2 (0.137 mollL)lNaCI (0.684 mollL) solu- 0-- -0------0 tion; the homogenate was adjusted to pH 5.1.

10 20 30 40 The extract was fractionated by acidification

Ripening lime, weeks with 0.25 mol/L HCI to a final pH of -1 ,6 and holding at 55 "C for 30 min, and then overnight Fig 1. Formation of pH 4.6-soluble N (.), 12% at room temperature before filtration. trichloroacetic acid-soluble N (e) and 5% phospho- Kuchroo and Fox (1982a) found that only tungstic acid (PTA)-soluble N (....) in an Irish artisanal blue chee se and formation of water-soluble N 40% of the WSN was soluble in 0.1 mol/L (0) and PTA-soluble N (0) in a commercial Ched- CaCI2. Increasing the concentration of CaCI2 dar cheese. Modified from Folkertsma et al (1996) above 0.05% at or above pH 7.0 has Iittle influ- and Zarmpoutis et al (1996). ence on extractability (Noomen, 1977b). The Formation de N-soluble à pH 4,6 (_), N-soluble dans increase in CaClrsoluble N correlates with the TCA 12% (e) et N-soluble dans PTA 5% (""), dans un fromage bleu irlandais d'origine artisanale etforma- age of cheese and the extracts contain whey pro- tian de Nssoluble dans l'eau (0) et N-soluble dans teins, peptides and amino acids, while the CaClr PTA (0), dans un fromage de cheddar commercial. insoluble fraction con tains caseins and large Modifié d'après Folkertsma et al (1996) et Zarmpoutis peptides, similar to those in the water-insoluble et al (1996). fraction (Christensen et al, 1991). NaCI has also been used to fractionate chee se N (eg, Chakravorty et al, 1966; Gupta et al, 1974), but Reville and Fox (1978) claimed y-caseins are insoluble at pH 4.6 (see Rank et that> 90% of Cheddar cheese N was soluble in al, 1985). According to Kuchroo and Fox 5% NaCl, which was thus not sufficiently dis- (1982a), this method gives slightly lower val- criminating, ex ce pt perhaps for very young ues for soluble N than extraction with water; cheeses. NaCI (5% )-soluble N and unfractionated and although it is more difficult to perform, it cheese are indistinguishable electrophoretically, may be easier to standardize (Fox, 1989). Recent indicating that the parent caseins, as weil as pep- studies in which pH 4.6-SN has been used as tides, are extracted (Rank et al, 1985; Bican and an index of ripening include those by Lau et al Spahni, 199 J), Inclusion of CaCI2 in the NaCI (1991), Addeo et al (1992), Bütikofer et al solution should reduce the percentage N (1993), Picon et al (1994), Yun et al (1995) and extracted and thus increase the selectivity of the Zarmpoutis et al (1996). method (see Fox, 1989). Precipitation of casein-derived peptides by

CaCI2 has been used by sorne investigators. Extractionlfractionation Solutions containing CaCI2 have been used as with organic solvents the primary extractant or as a method to sub- fractionate WSN (Lau et al, 1991; Addeo et al, Harwalkar and Elliott (1971) developed an 1992; Bütikofer et al, 1993). Noomen (I977a) extraction technique with chloroform/methanol 46 PLH McSweeney; PF Fox

(2: l, v/v). Freeze-dried cheese was extracted ment of pH in the range 5.5-6.5 was found to using this solvent, the extract filtered and water be quite effective. added. The chloroform layer contained lipids Precipitation with ethanol may be used to and was discarded. Removal of methanol from fractionate cheese or to sub-fractionate WSEs, the aqueous methanol layer by evaporation and should be preferred to the largely equiva- resulted in the formation of a precipitate; the lent 12% TCA owing to the ease of removal of supernatant was very bitter and astringent. This ethanol by evaporation (Christensen et al, 1991). method has also been used by a number of Fractionation of WSEs of Cheddar using 70% authors (eg, Lemieux et al, 1989, 1990; Puchades ethanol is used routinely in the authors ' et al, 1989a, b, 1990; Tieleman and Warthesen, laboratory. 1991) using different fractions of cheese as the starting material. Poznanski et al (1966) fractionated 2 or 12% TCA-soluble extracts with 75% acetone acidified Chloroform/methanol extracts more N than to pH 4.6, while Aston and Creamer (1986) used water, which is understandable considering the methanol to fractionate a WSE of Cheddar. relatively hydrophobie nature of many casein- Butan-i-oi has been used to extract bitter pep- derived peptides, but chromatograms of both tides from casein hydrolysates (McSweeney and extracts on Sephadex G-25 were similar (Rank Fox, 1993) but does not appear to have been et al, 1985). However, Tieleman and Warthe- applied to the fractionation of cheese. This sen (1991) found that extracts prepared by the extractant may have potential for the isolation of method of Harwalkar and Elliott (1971) were bitter and/or hydrophobie peptides. significantly less heterogeneous, as indicated by HPLC, than extracts prepared by the methods Fractionation using trichloroacetic of McGugan et al (1979) or Kuchroo and Fox or trifluoroacetic acids ( 1982a). McGugan et al (1979) developed a fraction- Trichloroacetic acid (CCI3COOH; TCA) is a ation sc he me using methylene chloride: c1assical protein 'precipitant which has been used methanol:water which has also been used by widely for this purpose. Concentrations rang- Smith and Nakai (1990). ing from 2 or 2.5% (eg, Reville and Fox, 1978; Fractionation of proteins and peptides by O'Sullivan and Fox, 1990) to 12% (eg, Bican ethanol is a c1assical technique, and has been and Spahni, 1991; Folkertsma and Fox, 1992; used extensively to fractionate peptides in cheese Addeo et al, 1992; Bütikofer et al, 1993; Kat- (Christensen et al, 1991; McSweeney and Fox, siari and Voutsinas, 1994; Picon et al, 1994; 1993; Fox et al, 1995b). Ethanol concentrations Yun et al, 1995) have been used, depending on used have varied from 30 to 80% (Fox et al, the degree of fractionation required (Iarger pep- 1995b); 70% has been used widely (eg, Abdel tides being soluble at the lower TCA concen- Baky et al, 1987). Trichloroacetic acid, TCA trations). There is no 'precipitation threshold' (12%, see below) and 70% ethanol yield approx- relating peptide size to solubility in TCA, but imately equal extraction levels (Reville and Fox, ail peptides studied by Yvon et al (1989) con- 1978) and soluble fractions contain similar pep- taining less than seven amino acid residues were tides (Kuchroo and Fox, 1982b), although dif- soluble in 12% TCA; peptide solubility is related ferences are apparent in the peptides in the insol- to hydrophobicity. The majority of 12% TCA- uble fractions. soluble peptides isolated and identified from An ultrafiltration (UF) retentate (10 kDa) of Parmigiano-Reggiano cheese by Addeo et al a WSE of Cheddar was fractionated by Breen (1994) were phosphopeptides. Typically, in a et al ( 1995) using increasing concentrations of mature Cheddar about 15% of the total N ethanol (30-80%); fractionation of the UF reten- (Reville and Fox, 1978) and -90% of the WSN tate containing 30% ethanol by stepwise adjust- (Kuchroo and Fox, 1982b) is soluble in 2.5% Methods used to assess proteolysis in chee se 47

TCA; while 50-60% of a UF retentate (10 kDa acids. Samples for amino acid analysis (see below) membranes) and 100% of the UF permeate must also be rendered free of peptides. (0' Sullivan and Fox, 1990) is soluble in Phosphotungstic acid (tungstophosphoric 2% TCA. acid; 12W03.H3P04.xH10; PT A)-H1S04 is a Rennet is responsible for the production of very discriminating protein precipitant; only free sorne of the 12% TCA-soluble N, but starter amino acids (apart from lysine and arginine), proteinases and peptidases make a substantial and peptides less than about 600 Da are soluble contribution (Reiter et al, 1969; 0' Keeffe et al, in 5% PTA (Jarrett et al, 1982). PTA (1,2.5,5, 1976) to the formation of 12% TCA-soluble N. 6 or 6.5% )-soluble N has been used widely as an Venema et al (1987) reported that the level of index offree amino acids in cheese (eg, Wilkin- 12% TCA-soluble N is a better index of maturity son et al, 1992; Bütikofer et al, 1993; Picon et al, than WSN. The development of 12% TCA-sol- 1994; Guinee et al, 1995). The most widely- uble N in an Irish artisanal blue cheese is shown used PTA concentration is 5%. The short PT A- in figure 1. soluble peptides in sorne cheese varieties have A major disadvantage of TCA for peptide been studied (Jarrett et al, 1982; Bican and Spahni, 1991; Gonzâlez de Llano et al, 1991). fractionation is the need to remove it prior to further analysis of the fractions. Since small pep- The development of 5% PTA-soluble N in Ched- dar and an Irish artisanal blue cheese is shown in tides and free amino acids will be lost on dialy- figure 1. sis, other methods for removal of TCA, eg, repeated ether extraction or sorne form of chro- 5-Sulphosalicylic acid (2-hydroxy-5-sul- matography, are required and are laborious pro- phobenzoic acid; SSA), a discriminating procedures (Fox, 1989). The use of 70% ethanol, tein precipitant, has been used at 3% to prepare which gives similar precipitation levels, is prefer- extracts of cheese for amino acid analysis (Reiter able because ethanol can be readily removed by et al, 1969; Ramos et al, 1987), or as an index of evaporation (Fox, 1989). amino acid N (Cliffe and Law, 1991). However, Kuchroo and Fox (1982a) found that 2.5% SSA Trifluoroacetic acid (CF3COOH; TFA) as a precipitated only 10% of the WSN. The water- precipitant or extractant for cheese peptides may soluble peptides in the fractions obtained with be a useful alternative to TCA, and it has the SSA do not appear to have been characterized. major advantage of being easily removed by evaporation. Perhaps surprisingly, there are few Picric acid (2, 4, 6-trinitrophenol) is also a references to its use in the fractionation of very discriminating protein precipitant (Fox, chee se. Bican and Spahni (1991, 1993) used a 1989). Reville and Fox (1978) found that of the buffer containing TFA as an extractant for methods examined 0.85% picric acid-soluble Appenzeller cheese. The procedure involved ex tracts of cheese contained the lowest levels homogenization at 4 "C in TFA (1%, v/v)/formic of N. It was considered to be the most suitable acid (5% v/v)/1 % (v/v) NaCI/I mol/L HCI at extractant for amino acids, but small peptides 4 "C, fol1owed by centrifugation and applica- are also soluble in picric acid (Salji and Kroger, 1981). Picric acid interferes with the determi- tion to a Sep-Pak Cl8 cartridge. The use ofTFA as an ion-pair reagent in HPLC buffers is nation of N by Kjeldahl or spectrophotometric widespread (see below). methods (Reville and Fox, 1978; Fox, 1989). Free amino acids were extracted from Ched- Phosphotungstic acid and similar techniques dar by Hickey et al (1983) using Ba(OH)z/ ZnS04' Cheese (lOg) was macerated in 33 mL It is often desirabJe to precipitate ail peptides from 0.15 mol/L Ba(OH)z and 33 mL 0.14 mol/L a cheese extract in order to quant ify free amino ZnS04' filtered and residual fat removed by acids in the cheese since nitrogen soluble in such extraction with cholorform/ethanol (1: 1). The precipitants is a crude measure of free amino aqueous phase was tïltered and the filtrate freeze- 48 PLH McSweeney, PF Fox dried and dissolved in a sodium citrate buffer filtration and size-exculsion chromatography prior to ami no acid analysis. No data were given have been used as an alternative to techniques on extraction levels or whether peptides were based on peptide solubility in various reagents. soluble. This reagent has not been used exten- Kuchroo and Fox (1982b) found that exhaus- sively to fractionate cheese N nor to prepare tive dialysis (96 h, four changes) was a simple samples for amino acid analysis. and effective method for partitioning water-soluble peptides, and that it was suitable for rela- Miscellaneous techniques tively large samples. About 50% of the WSN for fractionating peptides in a mature Cheddar was dialysable; the diffusate was completely soluble and the retentate Phosphopeptides (or other peptides with a high 50% soluble in 70% ethanoI. This technique was negative charge) can he precipitated from a water included by Kuchroo and Fox (1983b) in their extract of cheese by addition of BaCI to a final 2 fractionation scheme. Sciancalepore and Alviti concentration of 10 to 50 mmol/L and adjust- (1987) dialysed unfractionated, grated cheese ing the pH to 6.7-7.0. Ba2+ complex with serine (1 g), dispersed in H 0 (1 mL), against H 0 phosphate residues of phosphopeptides (and 2 2 (10 mL) at 30 "C for 30 min. The A of the potentially other negatively charged groups) 28D dialysate correlated weil with age and with 12% which can then be precipitated by adding ethanol TCA-soluble and pH 4.6-soluble N. This method (20% v/v) and recovered by centrifugation was also used by Gonzâlez de Llano et al (1993) (3000 g, 30 min, 4 "C), The pellet can be redis- to study proteolysis in blue cheese. solved in water and adjusted to pH < 2.0 using While dialysis is a simple and useful tech- H2S04 to precipitate BaS04' This method, based on that of Peterson et al (1957), has been used in nique for peptide fractionation, it has been super- the authors' laboratory to prepare phosphopep- seded by ultrafiltration (UF) which is faster, tides from Cheddar. Kuchroo and Fox (l982b) capable of handling larger samples, uses mem- fractionated a WSE of Cheddar with 0.1 mol/L branes of known molecular mass eut-off and ethylenediamine tetraacetic acid (EDTA); reduces the problem of recovering peptides from approximately 30% of the WSN was precipi- a large volume of diffusate. Many authors have tated. However, EDT A does not appear to have used diafiltration to improve resolution, Mem- been used by other authors for this purpose. branes with nominal molecular weight cut-offs Fernândez and Fox (1997) fractionated pep- ranging from 0.5 to 10 kDa (most commonly) tides in the water-soluble and -insoluble frac- have been used. Most authors have used UF to tions of Cheddar cheese using chitosan (a poly- fractionate the larger peptides in cheese, although mer of N-glucosamine). Chitosan is polycationic Visser et al (l983a, b) isolated very low molec- at acid pH and thus can complex with nega- ular weight peptides from a bitter extract of tively-charged molecules, leading to their floc- cheese by UF on 0.5 kDa membranes, and Aston culation. Chitosan (0.02%) was unsuitable for and Creamer (1986) used ultrafiltration with the fractionation of water-insoluble peptides but 1 kDa eut-off membranes to fractionate WSN. fractionated water-soluble peptides quite effec- Ali peptides in a 10 kDa UF permeate are solu- tively at pH 4.0. Chitosan-peptide complexes ble in 2% TCA, but sorne peptides in the reten- were dissociated at pH 7.0. tate are insoluble (O'Sullivan and Fox, 1990); the permeate contains no peptides detectable by Fractionation techniques based polyacrylarnide gel electrophoresis (PAGE) on molecular mass when stained using Coomassie blue G250, and 40-50% of the WSN is permeable. UF was used Techniques for the fractionation of peptides by Singh et al (1994, 1995) as the principal frac- based on their molecular mass, ie, dialysis, ultra- tionation technique for water-soluble peptides. Methods used to assess proteolysis in cheese 49

Rejection of hydrophobie peptides by UF Methods based on the Liberation membranes and aggregation of small peptides ofreactive compounds or groups are potential disadvantages of this method. How- ever, UF allows fractionation of large samples Most of the techniques described above for the and does not require solvents, both of which preparation of various fractions and the quan- facilitate taste panel work (Fox, 1989). Despite tification of N which is soluble therein are quite sorne studies, the potential of UF membranes time-consuming, and it is desirable to develop with low molecular weight cut-offs « 1 kDa) more rapid methods to estimate proteolysis. Such to fractionate the smaller peptides from cheese techniques are based on the liberation of specifie compounds or reactive groups whieh are has not been extensively investigated. easily measured. A wide range of such tech- The use of size-exc1usion chromatography niques is available, and inc1udes the estimation to fractionate cheese peptides is discussed below. of Tyr and Trp in cheese fractions by absorbanee at 280 nm or by the Folin-Ciocalteau reagent, protein-dye binding, reaction of liberated amino Comparison of techniques to quantify groups with various reagents or the quantification nitrogen in fractions of ammonia or glutamic acid. Because of their relative simplicity, there is a growing demand The majority of studies described above have for such rapid methods for assessing cheese quantified nitrogen in various fractions by the maturity (Ardô and Meisel, 1991). macro-Kjeldahl method (Wallace and Fox, 1994). Although this method is highly repeat- Formation of ammonia able, it is a slow and potentially dangerous technique. Alternative methods have also been used Ammonia, formed in cheese on the deamina- for this purpose, most notably by absorbanee of tion of free amino acids, is an important product ultraviolet light at 280 nm (Vakaleris and Priee, of proteolysis in certain cheese varieties (eg, 1959) or the Folin-Ciocalteau reagent. The lat- Camembert or smear-ripened varieties; Fox et ter method was used by Citti et al (1963) to mon- al, 1995a) where it contributes to characteristic itorTCA-soluble peptides. The Lowry method (a f1avour and texture (by neutralization of the cheese). Several authors (eg, Furtado and Chan- combination of the Folin-Ciocalteau and biuret dan, 1985; Alonso et al, 1987; Zarmpoutis et al, reagents), which is widely used in biochemistry 1996) have indirectly monitored ammonia pro- and is simple and rapid, has been used to quan- duction by measuring the increase in the pH of tif Y peptides in various cheese fractions (eg, cheeses. However, it is perhaps surprising that Kaminogawa et al, 1986). In the hands of a com- the development of ammonia per se has not been petent analyst, the Lowry method is suitable for used more widely as an index of proteolysis. the routine analysis of proteinlpeptides in cheese Ammonia levels in a non-protein fraction of fractions (Wallace and Fox, 1994). According Ulloa chee se were measured by Ordo nez and to Gonzâlez de Llano et al (1993), the Kjeldahl Burgos (1977) using the reaction of ammonium and Lowry methods gave similar results for pro- salts with the Nessler reagent (alkaline solution teinlnitrogen in water-soluble extracts from blue of K2HgI4) with the formation of a red-brown cheeses. Protein can also be estimated by the colloidal precipitate (NH2Hg2I3) which was mea- Bradford method (which is based on a colour sured spectrophotometrically; the concentration change in Coomassie blue on binding to pro- of ammonia was - 4 mg g-l cheese dry matter tein) or by using erythrosin, but preliminary and increased slightly during ripening. Fernan- results from this laboratory suggest that these dez-Salguero et al (1989) quantified ammonia methods are unsuitable for analysis of chee se in a number ofblue chee se varieties by isother- fractions. mal distillation from a sample dispersed in a 50 PLH McSweeney, PF Fox

borate buffer, pH 10, followed by trapping in leading to poor separation and turbid filtrates or borie acid and back titration. An enzymatic assay centrifugai supernatants. for ammonia is available (Anonymous, 1989) A decrease in the dye-binding capacity of a which quantifies NH; by the glutamate dehy- dispersion of Cheddar cheese with age was drogenase-catalyzed conversion of 2-oxoglu- dernonstrated by Ashworth (1966). Kroger and tara te to L-glutamic acid, with the concomitant Weaver (1979) reported that a dye-binding stoichiometric oxidation of NADH to NAD+ method could be used as an index of proteolysis which is determined spectrophotometrically. in cheese, but Kuchroo et al (1983) concluded However, this technique does not appear to have that the dye-binding method of McGann et al been used in cheese analysis. (1972), using amido black, is not suitable for this purpose. Formation of soluble tyrosine and tryptophan The Bradford method for protein determination (Bradford, 1976) is based on the colour Measurement of the 'soluble' tyrosine and tryp- change in Coomassie blue G250 when it reacts tophan in cheese is a well-established method with proteins. However, Wallace and Fox (1994) for assessing proteolysis. These amino acids can found that this technique was not suitable for be quantificd by the use of Folin-Ciocalteau quantifying water-soluble peptides. reagent (acidic solution of Na tungstate and Na molybdate) or by measurement of absorbance Acid/base titration at 280 nm. lt is necessary to fractionate the cheese (eg, preparation of a water- or TCA-sol- The formol titration technique is a simple method uble extract) before using either of these tech- for estimating amino groups in milk and may niques. Hull (1947) used the Folin-Ciocalteau be used to measure the extent and rate of prote- reagent to assess proteolysis in milk. This reagent olysis. The method involves titration of a sample was also used by Singh and Ganguli (1972) to with NaOH to the phenolphthalein end-point, quanti l'y peptides in the TCA-soluble fraction of cheese, but it appears to be less sensitive than followed by the addition of formaldehyde whieh 2, 4, 6-trinitrobenzenesulphonic acid, TNBS con verts free amino groups to less basic sec- (Samples et al, 1984). As described above, Vaka- ondary and tertiary amines whieh dissociate at a lower pH; therefore the solution becomes more leris and Priee (1959) measured the A280 of a sodium citrate-HeI extract (pH 4.6) of cheese as acidic, and it is necessary to re-titrate to the phe- an index of proteolysis. nolphthalein end-point (Davis, 1965). This procedure has been used to estimate proteolysis in Dye-binding cheese (eg, Vakaleris et al, 1960; El Soda et al, 1981) but il is now considered obsolete owing to At pH values below their isoionic point, pro- variations in the buffering capacity of cheese teins have a net positive charge and can inter- (Ardô and Meisel, 1991). Sciancalepore and act with anionic dyes (eg, amido-black, acid Longone (1988) compared a dialysis method orange 12 or orange G) leading to the forma- (Sciancalepore and Alviti, 1987) with formol tion of an insoluble protein-dye complex. This titration and the method of lnikhov (1951). The complex can be removed by centrifugation or latter method involved homogenization of finely filtration and the excess dye in the supematant or ground chee se in water, followed by filtration. filtrate measured spectrophotometrically. The Samples of the filtrate were diluted and titrated am ou nt of dye bound by the protein is propor- with NaOH to thymolphthalein and phenol ph- tional to the concentration of protein (and thus thalein end points, the index of proteolysis being the excess dye is inversely proportion al to the the difference in the titration values. protein concentration). However, low molecular The buffering capacity of cheese increases weight proteins and peptides react only slowly, during ripening owing to the formation of Methods used to assess proteolysis in cheese 51

ammonia, amino, imino and carboxyl groups. mophore which remains attached to the amino This principle was used by Ollikainen (1990) to acid, peptide or protein and ab sorbs maximally assess proteolysis in a Swiss-type cheese; it was at 420 nm (fig 2a). The reaction is performed at claimed that the method is rapid and convenient an alkaline pH and stopped by lowering the pH. and as accurate as colorimetrie methods. TNBS also reacts slowly with OH-, a reaction Changes in the buffering capacity of Emmental which is catalyzed by Iight. Since ammoniacal around pH 9 have been attributed to proteoly- nitrogen produces only 20% of the absorbance of sis (Lucey et al, 1993). amino groups when reacted in equimolar concentrations with TNBS (Clegg et al, 1982), this Free amino groups-colorimetrie and f1uorimetrie methods reagent may underestimate proteolysis in cheeses which have undergone significant deamination. The c1eavage of a pepitde bond results in the Clegg et al (1982) concluded that although liberation of a new amino group which can react TNBS is not as sensitive as ninhydrin for assay- with several chromogenic or fluorogenic ing proteolysis in cheese, it is preferable owing reagents. A number of techniques have been to the simple analytical procedure; they pro- developed to assay proteolysis in cheese based posed a correction factor for ammoniacal nitro- on this principle. gen. A disadvantage of the TNBS method is that 2,4,6- Trinitrobenzenesulphonic acid (TNBS) the dry powder is explosive, and prolonged stor- is one such reagent which reacts stoichiometri- age leads to high blank values (Ardô and Meisel, cally with primary amines, producing a chro- 1991).

(a) (c) HOOC-rNH'~

o ""'"w Auoresc:lmine (Ilon-tluoresc.onl/ "'< 5&- "'\ 5!-.~ Auorophore NO: NO: 2 ... k6-Trinilro\>t"117ene StliphLllü"A,,;d

HA Hydrolysis Prudu<:ls tnon-tluoresceru r (b)

0r\OH

~OH o (d)

»-Phrhaldialdehyde 1SDS. pH '.0

Ninhydrin

Chromophore

Fig 2. Reaction of (a) 2, 4, 6-trinitrobenzenesulphonic acid, (b) ninhydrin, (c) f1uorescamine and (d) o-phthaldialdehyde with the œ-arnino group of an amino acid. Réaction de (a) l'acide 2,4,6 -trinitrobenrenesulphonique, (b) de la ninhydrine, (e) de la fluorescamine et (d) de l'o-phthaldialdéhyde avec le groupe a-aminé d'un acide aminé. 52 PLH McSweeney, PF Fox

Habeeb (1966) and Adler-Nissen (1979) mea- who compared a flow-injection technique for sured the absorbance of the TNBS-amino group the TNBS and o-phthaldialdehyde (see below) complex at 340 nm, whereas in the procedure assays with c1assical methods. Bouton and Grap- of Fields (1971) the absorbance of a sul- pin (1994) found a good relationship between phite- TNBS-amino group complex was mea- PTA-soluble N values (by Kjeldahl) and the sured at 420 nm, thus avoiding the maximum TNBS method. of absorbance of TNBS itself at 335-340 nm. Ninhydrin is also widely used to monitor the Barlow et al (1986) reported that the latter is Iiberation of free amino groups in cheese dur- preferable to the method of Habeeb (1966) in ing ripening. The principle of the ninhydrin reac- which an unstable coloured complex is formed tion is the spectrophotometric estimation of a and the procedure is liable to operator error. chromophore formed when ninhydrin reacts with Alder-Nissen (1979) developed a procedure to free ami no groups (fig 2b). The purple chro- assess the degree of hydrolysis of food proteins mophore, named Ruhemann' s purple after its using TNBS; a linear relationship was found discoverer, does not remain attached to the pro- between the concentration of œ-amino groups tein or peptide and therefore is not precipitated and A420 but the relationship varied between during procedures necessary to c1arify the sam- proteins owing to variations in the concentra- pIe prior to spectrophotometric analysis at tion of lysine. 570 nm (Ardë and Meisel, 1991), which is a The method of Hull (1947) was compared major advantage of the technique (Pearce et al, with the TNBS method by Samples et al (1984), 1988). The "max of the chromophore is shifted to who found that the latter was a better index of a shorter wavelength (-507 nm) when the water proteolysis in cheese. Jarrett et al (1982) used content of the reaction mixture is reduced (Doi the TNBS assay to quantify free amino acids in et al, 1981). Ninhydrin reacts with ammonia 5% PTA-soluble fractions of cheese. The TNBS almost as readily as with amino groups and method was found to be reproducible for mon- therefore levels of proteolysis found by ninhydrin itoring proteolysis in cheese (Kuchroo et al, assays are consistently higher than those found 1983) and while unfractionated cheese could be by the TNBS procedure, the discrepancy being assayed, it is more sensitive if applied to pH 4.6- due to ammonia (Clegg et al, 1982). Ninhydrin or 12% TCA-soluble extracts owing to a lower is more sensitive than TNBS, but the latter was background colour caused by reaction of TNBS with e-amino groups. A strong correlation was preferred by Clegg et al (1982) because the ana- found by Madkor et al (1987) between WSN Iytical procedure is simpler. Ninhydrin is widely used to quantify amino groups in chromato- and the A420 of the TNBS complex for water- soluble extracts of Stilton cheese. graphie eluates, particularly in amino acid analysis by ion-ex change chromatography followed Barlow et al (1986) claimed that analysis of by post-column derivatization (see below). Cliffe a water-soluble extract prepared according to et al (1989) used ninhydrin to monitor fractions Kuchroo and Fox (1982a) by the TNBS method obtained by reversed-phase chromatography. of Fields (1971) would provide a simple routine method for quantifying soluble N in cheese. Pearce et al (1988) developed a Li-ninhy- Who le cheese, dispersed in 0.1 rnol/L Na2B407' drin assay for proteolysis in cheese based on 0.1 mollL NaOH, pH 9.5, was analyzed by Poly- that of Friedman et al (1984): an aliquot of chroniadou (1988), who found a good correlation cheese dispersed in a citrate buffer was mixed between the results of the TNBS method and with aqueous solutions of dimethyl sulphoxide, WSN determined by the Kjeldahl procedure; ninhydrin and lithium acetate at pH 5.2, heated, fractionation of the cheese prior to analysis was diluted and A570 determined. Total free amino considered to be unnecessary. The TNBS acids determined by this method correlated weil method was also used by Lemieux et al (1990), with conventional amino acid analyses. Methods used to assess proteolysis in cheese 53

Doi et al (1981) described a number of ninhydrin technique, is nonnally expressed as mg hydrin-based assays for peptidase activity; two Leu g-l cheese (by reference to a leucine stan- were modifications of the methods of Moore dard curve) and increases during ripening (fig 3). and Stein (1948, 1954b) and two were based on The Cd-ninhydrin procedure of Folkertsma and the Cd-ninhydrin assay of Tsarichenko (1966). Fox (1992) is a simple, useful method for esti- The Cd-ninhydrin reagent was found to he more mating total free amino acids in WSEs of cheese selective for the amino group of free amino acids and is used routinely in this laboratory; for best than for the amino groups of peptides or pro- results, a set of samples should be analyzed teins and was the most sensitive of several nin- together. hydrin reagents, inc1uding Sn-ninhydrin. Folk- Fluorimetric reagents are also available for ertsma and Fox (1992) applied the Cd-ninhydrin quantifying free amino groups. Such techniques assay of Doi et al (1981) to the assessment of are more sensitive than the colorimetrie methods proteolysis in chee se; the reagent was found to described above but have not been used as be about five times as sensitive as TNBS for the widely. The HPLC separation of fluorescent measurement of amino acid nitrogen and could derivatives of ami no acids and amines (pre-col- be performed on citrate-, water- or PT A-solu- umn derivatization) is common (see below). ble fractions but not on TCA-soluble fractions, Fluorescaminc (4-phenylspiro [furan-2 (3H), as the latter appeared to interfere with colour 1'-phthalan]-3,3'-dione) is used to quantify amino development. The release of total free amino acids and peptides in the picomole range. Fluo- acids in cheese, as detennined by the Cd-nin- rescamine, introduced by Weigele et al (1972), reacts with primary amino groups to produce a 2.0,------, fluorophore which is assayed at 390 nm excita- tion and 475 nm emission (fig 2c). It reacts at room temperature with water or primary amines, 1.5 the latter reaction being far faster (Udenfried et

E al, 1972). Fluorescamine has been used to mon- c: .... itor the hydrolysis of x-casein by chymosin 0 1.0 "'

1ight -sensiti ve. 9-Fluorenylmethoxy-carbony 1 acids, since peptides, which react to a greater (FM OC) may also have potential for use in or lesser extent in most other direct-assay pro- cheese analysis as a fluorescent labelling reagent cedures for free amino acids, are not degraded by for amino groups. L-amino acid oxidase. However, this enzyme is not equally active on ail amino acids; it exhibits Enzymatic techniques high activity on Leu, Phe and Trp but His, Ile and Cys are poor substrates (Puchades et al, L-Glutamic acid is an important free amino acid 1990). in many cheese varieties. An enzyme assay for Glu using flow injection analysis and glutamate dehydrogenase immobilized on activated glass TECHNIQUES WHICH RESOLVE was developed by Puchades et al (1989b); PEPTIDES enzyme activity was determined based on the reduction of NAD+. These authors measured Although the above non-specifie techniques can free Glu in Cheddar cheese extracts, prepared provide valuable information about the extent by the method of Harwalkar and Elliott (1971), of proteolysis and the activity of proteolytic which were freeze-dried, resuspended in a phos- agents, they provide little information on which phate buffer, sonicated, filtered and passed peptides accumulate or are degraded during through a Sep-Pak C'8 cartridge prior to analysis. ripening. For this reason, techniques which This technique was reported to be sensitive, resolve individual peptides have received much rapid and accu rate. attention in recent years. McSweeney et al (1993a) measured free Glu in water extracts of Cheddar using a commer- cially available assay kit containing glutamate Electrophoresis dehydrogenase (Boehringer-Mannheim, Mannheim, Germany). Unlike the procedure of Since only proteins and large peptides can be Puchades et al (1989b), in which NADH was visualized by staining, electrophoresis in gel measured directly, this procedure uses pig-heart media is Iimited to monitoring hydrolysis of the diaphorase to catalyze the reaction of iodonitrote- parent caseins and the formation and subsequent trazolium chloride with NADH, forming for- hydrolysis of the primary proteolytic products mazan which is assayed spectrophotometrically of caseins and thus it has been used widely to at 492 nm. The free Glu content of the chee ses monitor the primary proteolysis of caseins in increased with age and varied between cheeses cheese. More recently, it has been shown that with different non-starter populations. capillary electrophoresis (CE) has considerable Puchades et al (1990) developed an enzy- potential for resol ving casein-deri ved peptides matie assay with flow injection analysis for total with a wide range of molecular masses. free amino acids in water-soluble extracts of Although c1assical free-boundary techniques Cheddar cheese, prepared by the method of Har- have been used (eg, Lindqvist and Storgards, walkar and Elliott (1971) and filtered through 1959), the overwhelming majority of elec- Sep-Pak C'8 cartridges. L-Amino acid oxidase trophoretic analyses of cheese and other dairy (immobilized on glass beads) acted on free products involve zonal electrophoretic tech- amino acids, producing stoichiornetric amounts niques in which the electrophoresis buffer is sta-

of H202 which then reacted with luminol, a bilized in various media and the proteins and chemiluminescent agent, in the presence of peptides migrate as discrete bands under the

K3Fe(CN)6' emitting light, which was quanti- influence of an electric field. Zonal elec- fied. This technique was reported to be rapid trophoresis on paper (eg, Lindqvist et al, 1953; and reproducible and specifie for free amino Kuchroo and Fox, 1982b), porous cellulose Methods used to assess proteolysis in cheese 55 acetate or in agar or agarose gels are now rarely mended the direct stammg procedure of used. Blakesley and Boezi (1977) with Coomassie Electrophoresis in starch gels, which was blue G250 in the presence of TCA, although first applied to cheese by Melachouris and amido black was preferred for larger peptides. Tuckey (1966), has been used by sorne work- However, since only relatively large peptides ers to separate caseins or their degradation prod- stain under these conditions, the procedure is ucts in cheese or other products (eg, Vreeman limited to the detection of the primary degrada- and van Reil, 1990; van den Berg and de Koning, tion products in cheese. Q'Sullivan and Fox 1990; de Koning et al, 1992) and is quite effec- (1990) found that the peptides in a 10 kOa UF tive. Although starch gel electrophoresis gives far permeate did not stain with Coomassie blue on better results than earlier zonal techniques and urea-PAGE, but the retentate of the WSE con- MOIT(1971) c1aimed that it gave superior reso- tained several detectable peptides, as did 2% lution of lower-mobility and cationic peptides TCA-soluble and insoluble fractions of the reten- than polyacrylamide gels, starch gels are brittle tate. Low molecular mass peptides can be visu- and opaque after staining (Creamer, 1991) and alized using a silver staining technique involving have been largely replaced by polyacrylamide extensive fixing with glutaraldehyde, although gels. such stains have not been used widely to study Electrophoresis in polyacrylamide gels, first cheese peptides. applied to chee se by Ledford et al (1966), has Although urea-containing buffers at acid pH become the standard technique. The Iiterature have been used (eg, Creamer, 1991), the major- was reviewed by Shalabi and Fox (1987), Fox ity of authors have used urea-containing buffers (1989), Creamer (1991), Strange et al (1992), at alkaline pH. Shalabi and Fox (1987) com- McSweeney and Fox (1993) and Fox et al pared seve rai electrophoretic procedures for the (1995b ). Continuous buffer systems have been analysis of cheese and strongly recommended used to separate caseins and are reported to have the stacking gel system of Andrews (1983) in certain advantages over discontinuous buffers Tris buffers (pH 8.9) containing 4.5 mol/L urea. (see Creamer, 1991). However, nearly ail one- Electrophoresis in alkaline urea-containing gels dimensional PAGE techniques used in recent with direct staining by Coomassie blue G250 is studies on chee se have employed discontinuous widely used in this and other laboratories to buffer systems containing urea or sodium dode- monitor proteolysis in various cheese varieties cylsulphate (SOS) as a dissociating agent. Non- (fig 4). denaturing buffers are not suitable for caseins, Electrophoresis in SOS-containing buffers although they are used for the analysis of whey is a standard technique for protein analysis in proteins. general biochemistry; electrophoretic mobility in Sample preparation usually involves dis- the presence of SOS is inversely proportional solving cheese in a buffer (which usually con- to the logarithm of the molecular weight of the tains a reducing agent, usually 2-mercap- peptide. However, it is less widely used for toethanol) prior to electrophoresis; the solution cheese since the caseins have similar molecu- may be centrifugally defatted, and sucrose or Jar weights (20 000 to 25 000) and are therefore glycerol added to increase the density of the not as weil resolved by SOS-PAGE as by alka- sample to facilitate loading into gel slots line urea-PAGE. Shalabi and Fox (1987) con- (Creamer, 1991). cluded thatSOS-PAGE was inferior to Direct or indirect staining (followed by urea-PAGE for cheese analysis, and did not rec- destaining using acetic acid; eg, Laemmli, 1970) ommend its use. However, Creamer (1991) and using Coomassie blue is usually used. Shalabi Strange et al (1992) considered that SOS-PAGE and Fox (1987), who compared a number of provides valuable information on cheese ripen- staining techniques for PAGE gels, recom- ing, and has been widely used (eg, Marshall et al, 56 PLH McSweeney, PF Fox

Origin

Fig 4. Alkaline urea-polyacrylamide gel electrophoretograms (Andrews, 1983) of casein (lane 1), Cheddar cheeses made from high heat-treated milk (90 "C x 10 min) at one day and l, 2, 4 and 6 months of age (lanes 2---{) and water-soluble extracts therefrom (lanes 7-11). Direct staining using Coomassie brilliant blue 0250 was by the method of Blakesley and Boezi (1977); (Folkertsma and Fox, unpubl results). Électraphorégrammes sur gel alcalin urée-polyacrylamide (Andrews, 1993) de caséine (bande 1). deframages cheddar fabriqués à partir de lait traité à température élevée (90 oC x 10 min) à 1jour et 1. 2, 4, 6 mois (bandes 2-6) et leurs extraits solubles dans l'eau (bandes 7-11). Fixation directe par du bleu brillant de Coomassie G250 avec la méthode de Blakesley et Boezi (1977); (Folkertsma et Fox. non publié).

1988; Basch et al, 1989; Bhowmik et al, 1990; Isoelectric focusing (IEF) is a powerful elec- Tunick et al, 1993). Shalabi and Fox (1987) con- trophoretic technique for resolving proteins and sidered that the 20% acrylamide gel system of peptides based on separation according to dif- Tegtmeyer et al (1975) was more satisfactory ferences in their isoelectric points. It has been than the more widely used method of Laemmli particularly valuable in studies on genetic poly- (1970), in which 12% acrylamide gels are used. morphism in milk proteins (see Creamer, 1991; However, Creamer (1991) reported that 20% and Strange et al, 1992, for references). Sorne acrylamide gels give poor reso1ution of the of the applications of IEF in dairy chemistry caseins (ail caseins migrate as two bands), but are include the detection of adulteration of ovine very satisfactory for peptides with molecular milk cheeses with bovine or caprine milks or weights in the region of 10 000. the study non-bovine caseins (Addeo et al, 1983, Two-dimensional electrophoresis has been 1990a,b; Conti et al, 1988; Moio et al, 1989, used by sorne authors; eg, Trieu-Cuot and 1992; Amigo et al, 1992). Trieu-Cuot and Gripon Gripon (1982) used SOS-PAGE in one dimen- (1982) used IEF to study the pH 4.6-insoluble sion and isoelectric focusing in the other to study fraction of Camembert. Kim and Jimenez-Flores proteolysis in Camembert. Although 2-D elec- (1994) obtained excellent resolution of milk pro- trophoresis may give good resolution, it is time- teins by preparative IEF followed by urea- or consuming and there are difficulties with repro- SOS-PAGE. This technique could easily be ducibility and in obtaining quantitative data adapted for the study of proteolysis in cheese. (Creamer, 1991). Bican and Spahni (1991) sep- Electrophoresis of cheese peptides has been facil- arated peptides in extracts from Appenzeller itated by the recent introduction of pre-poured cheese by thin-layer chromatography in one gels and by rapid semi-automated systems such dimension, followed by electrophoresis at 90°. as the Phast system" (Pharmacia, Uppsala, Methods used to assess proteolysis in chee se 57

Sweden; Strange et al, 1992; Van Hekken and Thompson, 1992). After staining, electrophoretograms are usually recorded photographically, although den- sitrometry or excision and elution of the stained bands, followed by spectrophotometric quantitation have also been used. Difficulty in obtaining quantitative data is a serious limitation of electrophoresis. Creamer (1991) recommended that several control samples should be included in each gel and that comparisons should he made only between samples on the same gel. He emphasized that band dimensions are critical for densitometry, and that dye uptake is a function of the protein as weil as staining and destaining pro- tocol. However, de Jong (1975) reported that electrophoresis in alkaline urea gels (pH 8.9), stained with ami do black lOB and scanned by densitometry, gave good quantitative results. Identification of peptides produced during cheese ripening has posed difficulties. Peptides can be isolated from PAGE gels by excision of the bands or by electroblotting. The latter technique is preferable because of higher recover- Fig 5. Urea-polyacrylamide gel electrophoretograms ies of protein and because the size of PAGE gels of casein (eN) and the water-insoluble fraction (WISF) of 3-month-old Cheddar cheese made with Lactococ- can change on staining which makes accurate eus lactis subsp lactis SKII, indicating the positions excision of unstained regions difficult. Elec- of the caseins and the N-terminii of the polypeptides troblotted peptides can be identified by N-ter- identified by McSweeney et al (1994); * undetermined minai amino acid sequencing (see below), but C-terminus. they are more difficult to analyze by mass spec- Électrophorégramme sur gel urée-polyacrylamide de trometry (MS) because they cannot be stained caséine (CN) et de la fraction insoluble dans l'eau (WISF) d'unfromage de cheddar de 3 mois fabriqué prior to MS and must he eluted from the blotting avec Lactococcus lactis subsp lactis SKI l, avec indi- matrix. However, the location of ail the caseins cation des positions des caséines et des extrémités and sorne major degradation products (eg, N-terminales des polypeptides identifiés par y-caseins and asl-CN f24-199) on most PAGE McSweeney et al (1994) .. * extrémité Csterminale systems is known (Creamer, 1991; Strange et indéterminée ou non identifiée. al, 1992). The majority of the peptides in a urea-PAGE electrophoretogram of Cheddar cheese were partially identified (fig 5) by McSweeney et al (1994a). Addeo et al (1995) A technique described as high performance used immunoblotting (rabbit polyclonal anti- electrophoresis chromatography was used by bodies raised against the caseins and peroxi- Girardet et al (1994) to resolve component 3 dase-Iabelled immunoglobulins as secondary glycoproteins from bovine milk. Proteins were antibodies) to detect bands in electrophore- separated in a gel-fi lied column under the influ- tograms of a number of cheese varieties. This ence of an electric field and separated proteins technique permitted the identification of the exiting the bottom of the gel tube were eluted casein from which a number of peptides origi- by a continuous flow of buffer and passed nated. through a UV spectrophotometric detector. To 58 PLH McSweeney, PF Fox

date, this technique does not appear to have been visualized by staining and also means that CE is used to study proteolysis but may have applica- quantitative, unlike other electrophoretic tech- tion in the fractionation of large peptides in niques. It can be used in a number of modes, cheese. including free solution capillary electrophoresis, FSCE (although problems have been encoun- Capillary electrophoresis (CE) resolves pep- tered with interations between peptides and the tides in a buffer-filled capillary under the influ- capillary wall), micellar electrokinetic chro- ence of an electric field (fig 6) and as such is a matography (where electrophoresis is performed free-boundary technique although, as described in the presence of micelles of SDS and peptides below, zonal CE is also possible. CE separates partition between the micelles and buffer), cap- on the basis of the net charge on the peptides, illary isotachophoresis (where the analyte is their mass and Stokes' radius (Young et al, 1992) 'sandwiched' between a leading and terminating and sometimes on sorne other property of the electrolyte), capillary gel electrophoresis (which peptide (see below). The use of CE in food uses a molecular sieving effect), capillary elec- analysis has been reviewed (Zeece, 1992; trochromatography (which uses a capillary Lindeberg, 1996a,b). packed or coated with a stationary phase) or iso- Capillary electrophoresis is reported to have electric focussing (in which analytes may be great potential for the resolution of complex eluted from the capillary by a pump after they mixtures of peptides. It has a number of advan- have reached equilibrium at their pl) (Zeece, tages over traditional electrophoretic techniques, 1992; Lindeberg, 1996a). There are also CE including the choice of running buffer and the techniques for resolving racemic mixtures by use of automated, high-performance instru- diasteriomeric interaction of analytes with a chi- mentation. The composition of running buffer ral mobile or stationary phase (Gordon et al, can be changed easily and separation times are 1988). relatively short, although only one sample at a The capital cost of CE instrumentation at pre- time can be analyzed. Unlike conventional elec- sent is higher than that of equivalent high per- trophoresis, in which proteins and peptides in a formance liquid chromatographs (HPLC), gel are visualized by staining, CE uses continu- although costs are likely to fall. Sample size in ous monitoring by UV absorbance, and poten- CE is extremely small and therefore the tech- tially by the range of other techniques used for nique is not suitable for preparative-scale work. detection in HPLC. UV absorbance allows the CE may be limited to producing peptide pro- detection of small peptides which cannot be files, although CE coupled with in-line mass

Capillary electrophoretograrn

Capillary

Fig 6. Schematic diagram of a capillary electrophoresis unit. Représentation schématique d'une Electrolyte butTer unité d'électrophorèse capillaire. Methods used to assess proteolysis in cheese 59

1.S0E-02 T

1.30E-02

1.10E-02 E e 'Ol' 9.00E-03 ~...

3.00E-03

1.00E-03 0 2 4 6 8 10 12 14 16 18 20 Time min

Fig 7. Free solution capillary electrophoretogram of the water-soluble extract (WSE) from a 6-month-old Ched- dar cheese made using Lactococcus lactis subsp cremoris SKII as starter. WSE was dissolved in 1 mL 8% (v/v) CH3CN in water containing 0.1 % (v/v) tritluoroacetic acid. Electrophoresis was performed using a Beckman Model P/ACE 2050 capillary electrophoresis unit (Beckman, San Ramon, CA, USA) equipped with a 57 cm fused silica capillary (50 cm to detector; internai diameter, 75 um). Electrophoretic buffer was 0.1 mollL Na phosphate buffer, pH 2.5. Electrophoresis was performed al 20 kV and spectrophotometric detection was al 214 nm (Singh, unpubl results). Capillaire en solution libre d'un extrait soluble dans l'eau (WSE) de fromage de cheddar de 6 mois fabriqué avec Lactococcus lactis subsp cremoris SKI 1 comme levain. WSE était dissous dans 1 ml: de CH3 CN à 8 % (vlv) dans l'eau, contenant 0,1 % (vlv) d'acide trifluoracétique. L'électrophorèse était réalisée avec une unité d'élec- trophorèse capillaire Modèle Beckman PIACE 2050 (Beckman, San Ramon, CA, États-Unis) équipé d'un capillaire en silice fondue de 57 cm (50 cm jusqu 'au détecteur; diamètre intérieur, 75 pm). Le tampon pour l'élec- trophorèse était un tampon phosphate de Na 0,1 mol/L, à pH 2,5. L'électrophorèse était réalisée à 20 kV et la détection spectrophotométrique à 214 nm (Singh, non publié).

spectrometry gives much information on the slices and dips (Pant and Trenerry, 1995), detec- identity of peptides (Zeece, 1992). It is unlikely tion of chloramphenicol in milk (Blais et al, that CE will replace chromatographie and other 1994) and quantification of seleno amino acids electrophoretic methods, and it is now viewed as in human milk (Michalke, 1995). complementing other separation techniques The potential of this technique for isolating (Lindeberg, 1996a). peptides from cheese was demonstrated by Zeece CE has received considerable attention in (1992), who separated peptides in a tryptic digest general food analysis (Zeece, 1992; Lindeberg, of native and dephosphorylated asl- and 1996a,b). To date, FSCE has been the form of ~-caseins. The authors are aware of a number CE most widely used in dairy chemistry. Appli- of laboratories investigating the potential of CE cations have included ion analysis in milk, milk to study proteolysis in cheese, although no powders and cheese (Morawski et al, 1993; reports of capillary electrophoretograms of Schmitt et al, 1993; Prestwell et al, 1995), mea- cheese peptides appear to have been published to surement of hippuric and orotic acids in whey date. A free-solution capillary electrophore- (Tienstra et al, 1992), fractionation of whey pro- togram of a water extract from a mature Cheddar teins (Cifuentes et al, 1993; OUe et al, 1994), cheese is shown in figure 7 (Singh, unpubl measurement of sorbate and benzoate in chee se results). 60 PLH McSweeney, PF Fox

Chromatography of cheese peptides sieving properties of cation-exchanger Dowex 50 resins to fractionate cheese peptides (eg, Tokita Paper chromatography (PC) was used by many and Nakanishi, 1962; Huber and K1ostermeyer, early workers to quantify free amino acids in 1974; Biede and Hammond, 1979). cheese (eg, Kosikowsky, 1951a; Storgards and Mooney and Fox (unpubl results) fraction- Lindqvist, 1953; Clemens, 1954) or to study ated peptides in the water-insoluble fraction of cheese peptides (eg, Q'Keeffe et al, 1978; Cheddar cheese by high-performance hydropho- Kuchroo and Fox, 1982b, 1983a). Paper chro- bic interaction chromatography on a matography is cheap and straightforward, but Phenyl-Sepharose column in a 0.48 mol/L Na suitable only for assessing the complexity of phosphate buffer, pH 6.3, containing 2.5 mol/L systems containing only small peptides (Ardë urea; elution was by means of a gradient from and Gripon, 1991) and has been large1y super- 0.48 to 0.037 rnol/L Na phosphate (fig 8). seded by other techniques. Thin layer chromatography (TLC) on silica gel, using different 0.4 solvents, eg, n-propanol/water (70:30, v/v) or n-propanoUacetic acid/water (5: 1:3), has been used to characterize peptides in cheese fractions (Kuchroo and Fox, 1982b, 1983a, b; Edwards 0.3 and Kosikowski, 1983; Visser et al, 1983b). Nin- hydrin is usually used to develop the plates, = 15 although UV fluorescence of the spots was used ~ 0.2 il by Edwards and Kosikowski (1983). Prepara- '" -c tive TLC has been used to isolate bitter peptides 50 from Cheddar (Edwards and Kosikowski, 1983). Bican and Spahni (1991) combined TLC on sil- 01 ica gel plates (chloroforrnlammonialethanol as solvent) with electrophoresis in the perpendicular dimension to resolve peptides in Appenzell / cheese: plates were developed by spraying with fluorescamine and viewed under UV Iight. TLC is a simple, straightforward method for separat- Fraction no. ing peptides and is normally superior to paper Fig 8. Hydrophobie interaction chromatogram of the chromatography, although Kuchroo and Fox water-insoluble fraction of a 20-week-old Cheddar (1983a) obtained better resolution of peptides cheese on Phenyl-Sepharose High Performance" (Pharmacia, Uppsala, Sweden). Elution was by a gra- by PC than by TLC. Both paper and TLC chro- dient of Na phosphate buffer from 0.48 (buffer A) to matographic techniques are now rarely used. 0.037 mol/L (buffer B), pH 6.3 to 6.6, containing Column chromatography on silica gels (eg, 2.5 mol/L urea and 0.1 % dithiothreitol. Flow rate was Visser et al, 1975), metal chelating media (eg, 1 mL/min and UV spectrophotometric detection was at 280 nm (Mooney and Fox, unpublished). Cu-Sephadex; Ney, 1985; Mojarro-Guerra et al, Chromatogramme d'interactions hydrophobes de la 1991) or on hydrophobie interaction media (eg, fraction insoluble dans l'eau d'un fromage de Kuchroo and Fox, 1983a; Visser et al, 1983b) cheddar de 20 semaines sur Phenyl-Sepharose High have been used to fractionate peptides from Performance' (Pharmacia Uppsala, Suède). L'élu- cheese or casein hydrolysates. Mulvihill and Fox tion était réalisée avec un gradient de tampon phos- (1979) used phosphocellulose to fractionate pep- phate de Na de 0,48 (tampon A) à 0,037 mol/L (tampon B), pH 6,3 à 6,6, contenant 2,5 mol/L d'urée et tides produced from Ust-casein by chymosin, but 0,1 % de déthiothreitol. La vitesse d'écoulement était this medium does not appear to have been used de 1 mUmin et la détection en spectrophotométrie UV for cheese. A few authors have used the molecular était à 280 nm (Mooney et Fox, non publié). Methods used to assess proteolysis in cheese 61

The most popular forms of chromatography chee se solubilized in urea by HPSEC on TSK for analysis of cheese peptides have been ion- 3000SW and 2000SW columns in series. This exchange and size-exclusion techniques and technique gave sorne information about primary reverse-phase high performance liquid chro- and secondary proteolysis during ripening, but matography (RP-HPLC). these authors considered that the size-exc1usion Molecular weights may be estimated by size- columns were not suitable for the separation of exclusion chromatography on a calibrated col- the components present in water- or TCA-solu- umn. The majority of workers have used ble fractions. Vijayalakshmi et al (1986) reported Sephadex" gels (Pharmacia, Uppsala, Sweden) an interesting method for resolving peptides of of various pore sizes. Cheese preparations chro- molecular mass between 250 and 6000 Da on a matographed have included cheese or high TSK-SW 2000 column using 50 mmollL phos- molecular weight peptides (Lindqvist, 1962; phate buffer containing 0.1 % trif1uoroacetic acid Tokita and Hosono, 1968; Creamer and Richard- and 35% methanol as mobile phase. HPSEC has son, 1974; Foster and Green, 1974; Gripon et also been used to characterize caseins, whey al, 1975), water-soluble or similar extracts (Nath proteins and peptides derived therefrom (van and Ledford, 1973; Desmazeaud et al, 1976; Hooydonk and Olieman, 1982; Bican and Blanc, O'Keeffe et al, 1976; Santoro et al, 1987), dia- 1982; Vreeman et al, 1986). Iyzable or UF permeable fractions from water Ion-exchange chromatography is used widely extracts (Kuchroo and Fox, 1983b; Singh et al, to fractionate milk proteins but has only had 1994, 1995) or PTA-soluble peptides (Gonzâlez limited use in cheese analysis (Fox, 1989). de Llano et al, 1987, 1991). Anion-exchange chromatography on diethy- Numerous eluents have been used, although laminoethyl (DEAE)-cellulose was used to iso- water and dilute buffers are most corn mon and late asl-CN f24-199 (asl-I casein) from Ched- chromatograms are usually quantified by spec- dar (Creamer and Richardson, 1974) and to trophotometry at UV wavelengths. Generally, fractionate water-insoluble peptides (Breen, 280 nm is used but for fractions containing 1992; McSweeney et al, 1994a; Mooney and smaller peptides, absorbance of the carbonyl Fox, unpubl results) and the 2% TCA-soluble group in the peptide bond at a lower wavelength, and insoluble fractions of a 10 kDa UF reten- eg, 220 nm (Foster and Green, 1974; Green and tate of a water-soluble extract of Cheddar Foster, 1974) is preferable, since small peptides (0'Sullivan and Fox, 1990). High-performance may not contain aromatic residues. Measure- ion-exchange columns (eg, Mono-S" or Mono- ment of the amino group by reaction with nin- QTM,Pharmacia) give better resolution of bovine hydrin (eg, Gripon et al, 1977) or sorne other milk proteins than c1assical chromatography on reagent is an alternative. DEAE-eellulose (Barrefors et al, 1985; St Mar- Low-pressure, preparative HPLC techniques tin and Paquin, 1990). Anion exchange chro- (eg, FPLCTM, Pharmacia, Uppsala, Sweden) on matography of a urea solution of Gouda cheese size-exc1usion media have simplified this form on a Mono-QTM column was used by Haasnoot of chromatography. Wilkinson et al (1992) et al (1989) to study proteolysis. It was con- resolved press juice from Cheddar by high-per- c1uded that the ratio y- to ~-casein peak areas is formance size-exclusion chromatography a good indicator of the maturity of Gouda cheese (HPSEC) on a Superose-12 column; changes in ripened for up to about 10 months, whereas the the peptide profile during ripening were evident. ratio of the peak areas representing asl-casein HPSEC on a Superose-12 column is also valu- and ast-CN f 24-199 was a suitable index for able for characterizing peptides in various frac- very young cheeses « four weeks). Urea-con- tions from Cheddar (Breen et al, 1995) and is taining buffers have usually been used to dis- suitable for preparing peptide fractions. Haas- solve cheese prior to chromatography and chro- noot et al (1989) fractionated peptides in Gouda matograms are monitored by A280, although 62 PLH McSweeney, PF Fox

ninhydrin has been used by sorne workers (Fox has greatly reduced the workload involved in et al, 1995b). these forms of column chromatography, while ln our experience, anion-exchange chro- increasing speed and reproducibility. Thus, it matography on DEAE-cellulose or equivalent seems likely that these techniques will achieve high-performance medium (eg, Mono-QTM) in more widespread application in the future for urea-containing buffers is very suitable for the monitoring proteolysis in cheese. fractionation of large casein-derived peptides. Reverse phase-HPLC has been used exten- This form of chromatography is used routinely sively to characterize peptides in casein in this laboratory to fractionate the water-insol- hydrolyzates (eg, Le Bars and Gripon, 1989; uble peptides from cheese (fig 9; Mooney and McSweeney et al, 1993b) and the technique is Fox, unpubl results). Fractions from the anion- also very valuable for resolving shorter peptides exchange chromatogram can be collected and in cheese fractions. Water-soluble extracts have analyzed by urea-PAGE. usually been used for RP-HPLC analysis (eg, The introduction of high performance ion- Cliffe and Law, 1991; Gonzâlez de Llano et al, exchange chromatography (HPIEC) and HPSEC 1991; McSweeney et al, 1993a) but other fractions have also been studied, including pH 4.6-

% AU soluble and -insoluble extracts (eg, Kamino- 100 i 040 gawa et al, 1986; Christensen et al, 1989), 10 kDa UF permeate (Singh et al, 1994),70% 1 0.30 ethanol soluble and insoluble fractions (Zarm- poutis et al, 1996) and fractions from size-exclusion chromatography (Cliffe et al, 1993). 0.20 Gradient elution with water/acetonitrile (eg, Amantea et al, 1986) or water/methanol (eg, 1 0.10 Cliffe et al, 1993) is usually used; but isocratic conditions using a phosphate buffer as eluent ==- --' 0.00 have also been used by sorne workers (eg, Pham ml and Nakai, 1984). TFA is the most widely used Fig 9. High-performance anion-exchange chro- ion-pair reagent. Detection is generally by UV matogram of the water-insoluble fraction (WISN) of Cheddar cheese on a Mono-Q 5/5 column (Pharmacia, spectrophotometry, usually at a wavelength in Uppsala, Sweden). WISN (500 ilL of 15 mg WISN the range of 200-230 nm (which measures the mL-1 dissolved in 50 mmollL Tris-HCl buffer, pH 8.0, carbonyl in the peptide bond), although 280 nm containing 4.5 mollL urea and 1.2 x 10-3 mol/L dithio- has been used in cases where larger peptides, threitol, OTT) was applied to the column and eluted at which are more likely to contain aromatic a flow rate of 1 mL min-I using the above buffer con- residues, are expected (eg, Christensen et al, taining 8 x 1()-4 mollL OTT and an NaCI gradient (0 to 0.5 mollL). UV spectrophotometric detection was at 1989). Fluorescence detection has also found 280 nm (Mooney and Fox, unpubl results). . limited use (eg, Gonzâlez de Llano et al, 1991). Chromatogramme d'échanges d'anion haute perfor- Reverse-phase chromatography on a semi- mance de la fraction insoluble dans l'eau (W/SN) de preparative CZ/C1s-coated silica was used by fromage cheddar sur colonne Mono-Q 5/5 (Pharma- Cliffe et al (1989) to fractionate cheese peptides. cia Uppsala, Suède). W/SN (500 pl: de 15 mg de WISN mL -t dissous dans un tampon Tris-HCI à 50 mmol/L, Although RP-HPLC essentially remains a pH s,a, contenant 4,5 mol/L d'urée et 1,2 x 10-3 mol/L research tool, Smith and Nakai (1990) discussed de dithiotreitol, DIT) était injecté dans la colonne et its potential for the routine assessment of chee se élué avec une vitesse d'écoulement de 1 mlrmin:', en quality. Difficulties remain with the interpreta- utilisant le tampon ci-dessus contenant S X /()-4 tnol/L tion of RP-HPLC data and the development of DIT et un gradient NaCI (0 à 0,5 mol/L). Détection en spectrophotométrie UV à 2S0 nm ( Mooney et Fox, a solvent system which will permit tasting of non publié). the fractions (Ardô and Gripon, 1991). As var- Methods used ta assess proteolysis in chee se 63

ious columns, elution conditions and detection long analysis times (- 10 h) are likely to militate wavelengths have been used, comparison of against the widespread use of 2D- HPLC. chromatograms from different studies is difficult. Although RP-HPLC is widely used to purify individual peptides from cheese (eg, Singh QUANTIFICA TION OF et al, 1994, 1995, 1996), the authors caution FREE AMINO ACmS AND against the identification of most peptides in THEIR DEGRADATION PRODUCTS chromatograms based solely on their retention time. However, two major peptides, as/-CN Ultimately, the hydrolysis of caseins by the corn- fI-9 and fI-13 (produced by the action of Lac- bined action of proteinases and peptidases in tococcus cell envelope-associated proteinase on cheese leads to the liberation of free amino acids ast-CN fI-23, which is produced rapidly by which are considered to be important flavour chymosin) are characteristic of the chro- compounds in man y cheeses; their concentra- matograms of Cheddar cheese (fig 10a). tion varies widely with variety. In addition, RP-HPLC is a very valuable technique for volatile compounds formed from amino acids the resolution of water-soluble peptides in by decarboxylation, deamination, transamina- cheese, It hasbeen our experience that water- tion and other transformations can make sub- soluble extracts resolve weil on Cg wide-pore stantial contributions to cheese tlavour (see Fox (300 À) columns with a shallow acetonitrilel et al, 1995a). water gradient with TFA as the ion-pair reagent It is generally necessary to deproteinize sam- (eg, McSweeney et al, 1994b ). However, due to pIes prior to amino acid analysis to reduce inter- the complexity of the WSE from many cheeses, ference from peptides; reagents used have il is often desirable to fractionate the WSE. Chro- incIuded sulphosalicylic acid (Reiter et al, 1969; matography of 10 kDa UF permeates is ade- Omar, 1984), ethanol (Visser, 1977; Polychro- quate but time-consuming. For this reason, we niadou and Vlachos, 1979), picric acid (Weaver now fractionate with 70% ethanol prior to et al, 1978; Shindo et al, 1980), TCA (Zarm- RP-HPLC. Typical chromatograms of the poutis et al, 1996) or Ba(OH)z/ZnS04 (Hickey ethanol-soluble and insoluble fractions from et al, 1983). An internaI standard (eg, norleucine) Cheddar are shown in figure lOa, b (Tobin et is normally added to facilitate quantitation of al, unpubl results). amino acids. Elution of peptides from a C 19-reverse phase Early attempts to quantify free amino acids in cartridge using a stepwise acetonitrile gradient cheese used paper chromatography developed was used by Singh et al (1994, 1995) to resolve », using ninhydrin (eg, Kosikowsky, 1951a, b) but peptides in a fraction obtained by size-exclu- such techniques were only semi-quantitative. sion chromatography of a 10 kDa UF permeate Likewise, classical ion-exchange column chro- of a WSE of Cheddar cheese, and by Bican and matography (eg, Mabbitt, 1955, who used Spahni (1993) to sub-fractionate extracts of Dowex-50 resin) is now obsolete and has been Swiss-type cheees. This technique gave consid- superseded by automated amino acid analyzers, erably simpler sub-fractions from which indi- which are less laborious. vidual peptides were isolated. Dedicated ami no acid analyzers based on Two-dimensional HPLC (2D-HPLC) was ion-exchange chromatography are used widely used by Lagerwerf et al (1995) to fractionate and have greatly facilitated the analysis of free the peptides in the WSE of Cheddar cheese; the amino acids in chee se, which is now relatively eluate from the first dimension (ion-exchange) simple, accurate, quantitative and requires Iit- was directed to a reverse-phase column (C1g). tle sample preparation. Amino acids are usually The resolution obtained was impressive, detected by post-column derivatization, often although increased equipment requirements and using ninhydrin. Recent studies in which this 64 PLH McSweeney, PF Fox 0.30 a 0.25 x

0.15 y

0.05

0.16· b

0.12

0.02-

0.00-

0.'00 5.'00

Fig 10. Reverse-phase (Cs) high-perforrnance liquid chromatograms of the 70% ethanol-soluble (a) and insoluble (b) fractions of the water-soluble fraction of a 9-month-old Cheddar cheese. The locations of the peptides Œs1-CN fl-9 (X) and fl-13 (Y) are indicated (Tobin et al, unpubl results). Profil obtenu par chromatographie liquide haute performance en phase inverse (Cs) des fractions (a) solubles dans l'éthanol à 70% et (h) insolubles de lafraction soluble dans l'eau d'un/ramage de cheddar de 9 mois. Les localisations des peptides asrCN fl-9 (X) etfl-13 (Y) sont signalées (Tobin et al, non publié). Methods used to assess proteolysis in cheese 65 technique was used incIude those by Ardô and umn derivatization (eg, Bütikofer et al, 1990). Gripon (1995) and Zarmpoutis et al (1996). Gas chromatography with mass spectrometrie Reverse-phase HPLC of pre-column-deriva- detection (GC-MS) allows the quantification tized tluorescent-Iabelled amino acids is increas- and identification of volatiles from cheese (eg, ingly being used to measure free amino acids in Urbach, 1993), incIuding those resulting from cheese, and has the advantage of using standard amino acid catabolism. A discussion of GC-MS equipment and being amenable to automated is outside the scope of this review. derivatization. Amino acids have been separated as their dansyl (eg, Polo et al, 1985), OPA (eg, Ramos et al, 1987; Bütikofer et al, 1991) or A FRACTIONATION SCHEME FMOC (Bütikofer et al, 1991) derivatives. FOR CHEESE PEPTIDES Bütikofer et al (1991) compared the results of amino acid analysis of an acid hydrolysate of The peptide system in most cheese varieties is cheese pro teins by pre-column derivatization extremely complicated. For example, > 200 pep- with OPA or FMOC, followed by RP-HPLC, tides have been isolated and identified from the with the results obtained using an amino acid WSE of Cheddar (Singh et al, 1994, 1995, 1996). analyzer fitted with an ion-exchange column The detailed study of proteolysis in cheese often and using post-column derivatization with nin- requires that peptides in the initial extract be hydrin. HPLC gave rapid, simple and sensitive fractionated by a combination of the techniques determination of amino acids and yielded nar- discussed above. A number of fractionation rower, better-resolved peaks with shorter reten- schemes have been proposed (Kuchroo and Fox, tion times and a more stable baseline. Although 1983b; Aston and Creamer, 1986; Fox, 1989; the HPLC method used had a slightly lower O'Sullivan and Fox, 1990; Cliffe et al, 1993; repeatability for sorne amino acids, it had good Singh et al, 1994, 1995, 1996; Breen et al, 1995). reproducibility and correlated weil with ion- The scheme used at present in this laboratory is exchange chromatography. shown in figure 11. This fractionation scheme Amino acids can be also quantified by gas was developed for Cheddar cheese and may need chromatography (GC) after derivatization with to be modified for other varieties. heptatluorobutyric anhydride (HFBA) to yield n-heptatluorobutyryl isobutyl derivatives. Capil- liary GC is most suitable (eg, Wood et al, 1985; ISOLATION AND IDENTIFICATION Laleye et al, 1987; Bertacco et al, 1992). This OF INDIVIDUAL PEPTIDES technique is reported to give good recovery of added amino acids; ail protein amino acids can Individual small peptides are usually isolated be resolved on a single chromatogram, and speed by RP-HPLC. Fractions of eluate may be col- and accuracy are comparable to those of auto- lected and freeze-dried, Il is desirable to check mated amino acid analyzers and at far lower the homogeneity of fractions by re-chromatog- equipment costs. The technique has good repro- raphy under the same or preferably different ducibility and high accuracy, although the coef- elution conditions. Peptides eluted in salt- ficients of variation for Met and Gly are some- containing buffers may be desalted by re- what high (Bertacco et al, 1992). However, GC chromatography using an acetonitrile or has not been used as widely as alternative methanol gradient, the components of which are techniques. easily volatilized. Ammonia is produced by deamination of In our experience, RP-HPLC does not ade- amino acids, and techniques for its quantification quately resolve larger casein-derived peptides. have been discussed above. Amines in cheese Such peptides may be resolved by PAGE and may be quantified by RP-HPLC using pre-col- isolated by electroblotting onto a suitable mem- 66 PLH McSweeney, PF Fox

brane (eg, polyvinylidine difluoride; McSweeney stained gels, the dimensions of which change et al, 1994a; Singh et al, 1995). Alternatively, on staining. The N-terminal sequence (see unstained regions of the gel may be excised, and below) of electroblotted peptides can be deter- peptides extracted from the macerated gel pieces mined readily, but it is necessary to remove pep- by electrophoresis. However, the precise location tides from the blotting membrane prior to mass of peptides on unstained gels may be difficult spectrometrie analysis. since the y cannot be compared directly with

GRATED CHEESE

Watcr Extraction

Pcllel Fat Re-Exlracl ~------t.~WSN (Discard)

WISN

Ion Exchangc Chromutography Urea-PAOE Diafiltrution, 10 kOa membranes or 70'k Ethanol

~ RETENTATE PERMEATE or or INSOLUBL FRACTION SOLUBLE !FRACTION Scphadcv 025 DEAE PAGE, Electroblotting HPLC+ ~Plides l ...... Il III IV V VI VII VIII IX X " V J Amine Acids

HPLC

Fig 11. Fractionation scheme for chee se N (modified from Fox et al, 1995). WSN: water-soluble N; WISN: waler-insoluble N; DEAE: ion-exchange chromalography on DEAE--œllulose; PAGE: alkaline urea-polyacrylamide gel electrophoresis; HPLC: reverse-phase (Cg) high performance liquid chromatography. Schéma de fractionnement de l'azote (N) du fromage (modifié à partir de Fox et al 1995). WSN: azote soluble dans l'eau .. W1SN: azote non soluble dans l'eau .. DEAE : chromatographie échangeuse d'ions sur cellulose DEAE .. PAGE: électrophorèse sur gel alcalin urée-polyacrylamide .. HPLC: chromatographie liquide haute performance en phase inverse (C8). Methods used to assess proteolysis in cheese 67

The amino acid composition of a peptide, tides in Cheddar cheese (Gouldsworthy et al, determined after hydrolysis, can pro vide useful 1996). Most MS techniques used to identify pep- information as to its identity (eg, Kaminogawa tides involve the production of ions of the intact et al, 1986). Computer software can be written peptide. However, if high energies are used, the (eg, Petrilli, 1982) or is available from com- peptide is fragmented and the masses obtained mercial sources to aid in locating a peptide of a are those of the resulting fragments. Cyclic given ami no acid composition in the protein dipeptides and y-glutamyl peptides have been chain. However, more than one casein-derived identified using this technique (Roudot-Algaron peptide may have the same amino acid compo- et al, 1993, 1994), which yields more informa- sition and thus identification of peptides in a tion on the structure of the compound under complex mixture from their amino acid compo- study than just its mass; il is likely to be useful sition, alone is not recommended. Identification for studying very short peptides or those with of the N-terminal residue or sequence (eg, Visser an unusual structure. et al, 1977; Gonzâlez de Llano et al, 1991) improves the probability of correct identification of peptides of a gi ven composition. STRATEGIES FOR Amino acid sequencing (either manual, eg, ASSESSING PROTEOL YSIS Mojarro-Guerra et al, 1991 or automated, eg, McSweeney et al, 1994a; Singh et al, 1994, The choice of a technique for assessing prote- 1995, 1996) by Edman degradation can also be olysis in cheese depends on a number of fac- used to identify peptides. However, unless the tors, including availability of equipment and peptide is relatively short, it may not be possible resources, chee se variety and objectives of the to determine its full sequence. The N-terminal study. Since proteolysis is one of the principal sequence (5-7 residues) is normally adequate biochemical events during cheese ripening, it is to locate the origin of a peptide in the caseins, but desirable to include an assay for proteolysis in other techniques must be used to determine its most chee se ripening studies. The formation of length. C-terminal sequencing (eg, Iiberation of soluble N or liberation of reactive groups may be ami no acids over time by carboxypeptidase Y, adequate if a detailed investigation of proteoly- eg, McSweeney et al, 1993b) could be used for sis is not warranted. However, if a thorough this purpose but is not popular. Mass spectrom- study of proteolysis is deemed worthwhile, it is etry (MS) is more convenient for identifying desirable to consider the entire process from the peptides of known N-terminal sequence (eg, initial hydrolysis of the caseins to the develop- Singh et al, 1994, 1995, 1996), although mass ment of free amino acids and to use techniques analysis alone is often insufficient to identify which resolve individual peptides. peptides since different casein-derived peptides Certain cheese varieties have characteristics can have the same ami no acid composition (and which influence the choice of method. The hence mass). An interesting application of MS increase in pH of mould-ripened varieties means involves determining the residual mass of a pep- that pH 4.6 buffers are more suitable as primary tide after each cycle of a manual Edman degra- extractants than water. Likewise, since ammonia dation. The mass of the residue (Mf) re1eased is a major product of proteolysis in mould and can be calculated from Mf = Mn-Mn_1, where bacterial surface-ripened varieties, it may be Mn and Mn-1 are the masses of the peptide before desirable to quantify ammonia. The rate and/or and after removal of the N-terminal residue, pattern of proteolysis may be influenced by loca- which can thus be identified. Sequencing of the tion within a cheese, eg, surface-ripened or first few amino acids, coupled with its mass, young, brine-salted cheeses. A suitable sam- permils precise identification of the peptide. pling scheme should take account of such Such an approach has been used to identify pep- differences. 68 PLH McSweeney, PF Fox

If one or more of the proteolyic agents is REFERENCES modified in a cheese, then the methods used to study proteolysis should be chosen so as to Abdel Baky AA, Abo Ella WM, Aly ME, Fox PF emphasize the level of proteolysis caused by (1987) Improving the quality of Ras-type cheese that agent. For example, if the influence of coag- made from recombined milk containing high levels of total solids. Food Chem 26, 175-188 ulant is studied, the formation of WSN and peptide profiles by urea-PAGE are suitable meth- Addeo F, Chianese L, Di Luccia A, Petrilli P, Mauriello R, Anelli G (1983) Identification of cds. Since the coagulant does not produce free bovine casein variants by gel isoelectric focusing. amino acids, their determination does not directly Milchwissenschaft 38, 586-588 measure the contribution of the coagulant. In Addeo F, Moio L, Chianese L, Stingo C, Di Luccia A contrast, since the contribution of cheese (1990a) Improved procedure for detecting bovine microflora (natural or modified) to cheese ripen- and ovine milk mixtures in cheese by isoelectric ing is mainly in the production of short peptides focusing of para-K-casein. Milehwissensehaft 45, and free amino acids, RP-HPLC and amino acid 221-224 analysis are likely to be most effective. How- Addeo F, Moio L, Chianese L, Stingo C, Resmini E, ever, it is also important to include sorne index Berner l,Krause D, Di Lucci A, Bocca A (1990b) Use of plasmin to increase the sensitivity of of primary proteolysis (eg, PAGE) to ensure that the detection of bovine milk in ovine cheese it is typical. by gel isoelectric focusing of y2-caseins. ln this laboratory, routine assessment of pro- Milehwissensehaft 45,708-711 teolysis in Cheddar cheese during ripening Addeo F, Chianese L, Salzano A, Sacchi R, Cappuc- involves the determination of WSN (Kuchroo cio U, Ferranti P, Malorni A (1992) Characteriza- and Fox, 1982a) and peptide profiles of the tion of the 12% trichloroacetic acid-soluble oligopeptides of Parmigiano-Reggiano cheese. cheese and WSEs therefrom by alkaline J Dairy Res 59, 401-411 urea-PAGE (Blakesley and Boezi, 1977; Addeo F, Chianese L, Sacchi R, Spagna Musso S, Fer- Andrews, 1983). These assays pro vide impor- ranti P, Malorni A (1994) Characterization of the tant information on primary proteolysis. Water- oligopeptides of Parrnigiano-Reggiano cheese sol- soluble extracts are fractionated using 70% uble in 120 g trichloroacetic acidIL. J Dairy Res 61, ethanol and the soluble and insoluble fractions 365-374 analyzed by RP-HPLC (Cg; Singh et al, 1995). Addeo F, Garro F, Intorcia N, Pellegrino L, Resmini P, The 70% ethanol-insoluble fraction can also be Chianese L (1995) Gel electrophoresis for the analyzed by urea-PAGE. Total free amino acids detection of casein proteolysis in cheese. J Dairy Res 62, 297-309 are determined using the Cd-ninhydrin assay (Folkertsma and Fox, 1992) and individual Adler-Nissen 1, (1979) Determination of the degree of hydrolysis of food protein hydrolyzates by trini- amino acids by an automated amino acid ana- trobenzene-sulphonic acid. J Agrie Food Chem Iyzer with post-column derivatization using nin- 27,1256-1262 hydrin. This strategy provides information on Alonso L, Juârez M, Ramos M, Martin-Alvarez Pl the level and type of proteolysis from the initial (1987) Effects of changes during ripening and hydrolysis of the caseins to the Iiberation of free frozen storage on the physicochemical and sen- amino acids; and is suitable for most internai, sory characteristics of Cabral es cheeses. lnt J Food bacterially-ripened cheese varieties. Sei Teehnol22, 525-534 Amantea GF, Skura Bl, Nakai S (1986) Culture effect on ripening characteristics and rheological behaviour of Cheddar cheese. J Food Sei 51, 912-918 ACKNOWLEDGMENT Amigo L, Ramos M, Calhau L, Barbosa M (1992) Comparison of electrophoresis, isoelectric focus- The authors wish to express their thanks to ing, and immunodiffusion in determination of A Cahalane for assistance in preparing cow' s and goal' s milk in Serra da Estrela cheeses. the manuscript. Lait 72, 95-10 1 Methods used to assess proteolysis in chee se 69

Andrews AT (1983) Proteinases in normal bovine Bican P, Blanc B (1982) Milk protein analysis, milk and their action on caseins. J Dairy Res 50, a high performance chromatography study. 45-55 Milchwissenschaft 37, 592-593 Anonymous (1989) Ammonia, UV Methodfor the Bican P, Spahni A (1991) Low molecular mass nitro- Determination of Ammonia in Foodstuffs and gen components in ripening cheese. Lebensm Wiss Other Materials and for the Determination of TechnoI24,315-322 Nitrogen After Kjeldahl Digestion. Boehringer- Biede SL, Hammond EG (1979) Swiss cheese. 1. Mannheim, Mannheim, Germany Chemical analysis. J Dairy Sei 62, 227-237 Ardô Y, Gripon JC (1995) Comparative study of pep- Blais BW, Cunningham A, Yamazaki H (1994) A tidolysis in sorne semi-hard round-eyed cheese novel immunofiuorescence capillary elec- varieties with different fat contents. J Dairy Res trophoresis assay for the determination of chi or- 62,543-547 amphenicol in milk. Food Agric lmmunol 6, Ardô Y, Gripon JC (1991) Chromatographie methods 409-417 used to measure proteolysis in cheese. Bull lnt Blakesley RW, Boezi JA (1977) A new staining tech- Dairy Fed 261,29-34 nique for proteins in polyacrylamide gels using Ardô Y, Meisel H (1991) Methods for direct mea- Coomassie brilliant blue G250. Anal Bioehem 82, surement of peptide bond cleavage in cheese. Bull 580-582 IntDairyFed261,1O-13 Bockelmann W (1995) The proteolytic system of Ashworth VS (1966) Determination of protein in dairy starter and non-starter bacteria: components and products by dye-binding. J Dairy Sei 49,133-137 their importance for cheese ripening. lnt Dairy J 5, Aston JW, Crea mer LK (1986) Contribution of the 977-994 components of the water-soluble fraction to the Bouton Y, Grappin R (1994) Measurement of proteo- flavour of Cheddar cheese. NZ J Dairy Sei Technol Iysis in cheese: relationship between phospho- 21,229-248 tungstic acid-soluble N fraction by Kjeldahl and Barlow lE, Lloyd GT, Ramshaw EH (1986) The rnea- 2,4, 6-trinitrobenzenesulphonic acid-reactive surement of proteolysis in Cheddar cheese: a com- groups in water-soluble N. J Dairy Res 61, 437-440 parison of trinitrobenzene sulphonic acid proce- Bradford M (1976) A rapid and sensitive method for dures. Aust J Dairy Technol41, 79-81 the quantitation of microgram quantities of pro- Barrefors P, Ekstrand B, Fagerstam L, Larson- tein utilizing the principle of protein-dye binding. Raznikiewicz M, Scharr J, Steffner P (1985) Fast Anal Bioehem 72, 248-254 protein liquid chromatography (FPLC) of bovine Breen ED (1992) Fractionation and identification of caseins. Milchwissenschaft 40,257-260 water-soluble and water-insoluble cheese nitro- Basch 11, Farrell HM Jr, Walsh RA, Konstance RP, gen. MSc thesis, Nat Univ Ireland, Cork Kumosinski TF (1989) Development of a quanti- Breen ED, Fox PF, McSweeney PLH (1995) Frac- tative model for enzyme-catalyzed, time-depen- tionation of water-soluble peptides from Cheddar dent changes in protein composition of Cheddar cheese. Ital J Food Sei 7, 211-220 cheese during storage. J Dairy Sei 72, 591-603 Bütikofer V, Fuchs D, Murni D, Bosset JO (1990) Beeby R (1980) Use of fluorescamine at pH 6.0 to Beitrag zur Bestimmung biogener Amine in Kâse. follow the action of chymosin on x-casein and to Vergleich einer verbesserten HPLC-mit einer IC- estimate this prote in in milk. NZ J Dairy Sei Methode und Anwendung bei verschiedenen Kas- TechnoI15,99-108 esorten. Mitt Geb Lebensmittelunters Hyg 81, Bertacco G, Boschella 0, Lercker G (1992) Gas chro- 120-133 matographic determination offree amino acids in Bütikofer V, Fuchs D, Bosset JO, Gmur W (1991) cheese. M ilchwissenschaft 47, 348-350 Automated HPLC-amino acid determination by Bhowmik T, Riesterer R, van Boekel MAlS, Marth precolumn derivatization with OPA and FMOC EH (1990) Characteristics of low-fat Cheddar and comparison with classical ion-exchange chro- cheese made with added Micrococcus or Pedio- matography. Chromatographia 31, 441-447 coccus species. Milchwissenschaft 45,230-235 Bütikofer V, Ruegg M, Ardo Y (1993) Determination Bican P, Spahni A (1993) Proteolysis in Swiss-type of nitrogen fractions in cheese, evaluation of a col- chee ses: a high performance liquid chrornato- laborative study. Lebensm Wiss Teehnol 26, graphie study. lnt Dairy J 3,73-84 271-275 70 PLH McSweeney. PF Fox

Chakravorty SC. Srinivasan RA. Babbar Il. Dudani Creamer LK (1991) Electrophoresis of cheese. Bull AT. Burde SD. Iya KK (1966) Comparative study Int Dairy Fed 261. 14-28 of proteolysis during ripening of Cheddar cheese by Creamer LK. Richardson BC (1974) Identification of bacterial milk clotting enzymes and rennet extract. the primary degradation product of

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