Proteinaceous Surfactants Produced from Gelatin by Enzymatic Modification: Application to Preparation of Food Items

Proteinaceous surfactants produced from gelatin by papain-catalyzed incorporation of L-leucine n-alkyl esters were used as ingredients replacing conventional surfactants for food use. The incorporation of leucine Cz-C6 alkyl esters gave surfactants effective in making snow jelly. In ice cream making, a leucine C₁₂ alkyl ester-incorporated product used as the surfactant gave a high degree of overrun even in a few minutes from the start of whipping. This surfactant was applicable also to making mayonnaise, with formation of a tine emulsion having favorable hardness and adhesiveness. In bread making as well, the same surfactant was usable and found preferable to monostearin. The use of this surfactant resulted in satisfactory loaf quality as well as slow staling of bread over a long period of storage.     

Structures and pesticidal activities of derivatives of dinitrophenols. X. Effects of substitution od dinitrophenols with C3 to C13 alpha-branched alkyl groups and of esterification on the eradication of barley mildew

The studies of the eradication of barley mildew due to Erysiphe graminis Mérat with alkyldinitrophenols reported earlier¹ were continued. The degree of eradication was highest with 4-(C₉ α-branched alkyl)-2,6-dinitrophenols. 4-Alkyl-2,6-dinitrophenols containing a heptyl or higher alkyl branch were significantly less active, and compounds containing the C₁₂ or C₁₃ alkyls were not active since the most compact of the C₁₂ α-branched alkyls is 1-pentylheptyl. 2-(1-Methylheptyl)- and 2-(1-propylpentyl)-4,6-dinitrophenols had high activity, but their esters showed reduced activity. Esterification of 4-(α-branched alkyl)-2,6-dinitrophenols to methyl carbonates did not affect the activity shown by the compounds, but when certain 4-C₁₀ and C₁₁ alkyl-2,6-dinitrophenols were esterified to ethyl carbonates the activity of the products was reduced. Also activity was diminished by conversion of some 4-C₉ and C₁₀ alkyl-2,6-dinitrophenols to crotonates. Whereas the methyl- and ethyl- carbonates of 4-(1-ethylhexyl)-2,6-dinitrophenol gave consistently high degrees of eradication of barley mildew, the performance of the crotonate of this phenol was not consistent. Lower aliphatic esters of the active C₈-alkyl phenols were themselves active. Esterification of 4-(1-ethylhexyl)-2,6-dinitrophenol to the benzoate, isopropyl carbonate or S-methyl thiolocarbonate gave compounds with substantially reduced activity. 2-t-Butyl-4,6-dinitrophenyl or 4-t-butyl- or t-octyl-2,6-dinitrophenyl esters gave little or no significant eradication.     

Synthesis of aroma compounds by apples supplied with alcohols and methyl esters of fatty acids

Ester synthesis by apples supplied with alcohols (C2–C8) and methyl esters of short chain fatty acids (C4–C8) was studied using gas chromatographic analysis of the products. The substrates were supplied as vapours to whole fruits stored in 2% O₂ at 3°C. The alcohols were converted to the corresponding acetate ester; butanol, pentanol and hexanol were converted most rapidly. The methyl esters of short chain fatty acids (C_n) were converted to esters with an alkyl group (C_n-2, C_n-4) confirming the presence in whole fruits of an active β-oxidation pathway for fatty acids. Ester synthesis was stimulated when apples were supplied with methyl octanoate at different periods during long term storage in 2% O₂. Treatment of the fruit immediately postharvest did not enhance ethylene synthesis.     

Structures and pesticidal activities of derivatives of dinitrophenols. IX. Effects of substitution of dinitrophenols with C4 to C13 alpha-branched alkyl groups and of esterification on the eradication of apple mildew

The eradication of apple mildew caused by Podosphaera leucotricha (Ell. and Everh.) Salm. with 2-(C₄ to C₁₃ α-branched alkyl)-4,6-dinitrophenols and 4-(C₄ to C₁₃ α-branched alkyl)-2,6-dinitrophenols was examined. 4-(1-Ethylbutyl)- and most 4-(C₇ to C₁₂ α-branched alkyl)-2,6-dinitrophenols were significantly more active than their 2-alkyl-4,6-dinitrophenol analogues. Compounds containing 4-C₁₃ alkyls did not show significant activity. Activity generally increased as the α-alkyl branch lengthened. Methyl carbonates did not show lower activity than the parent phenols, but esterification of 4-(C₁₂ or C₁₃ α-branched alkyl)-2,6-dinitrophenols to ethyl carbonates or crotonates gave compounds with reduced activity. Methyl-, ethyl- or isopropyl-carbonates and crotonates of 4-(1-ethylhexyl)-2,6-dinitrophenol had much higher activity than the corresponding esters of the 4-(1-methylheptyl) isomer or of 2-(1-ethyl-hexyl)-4,6-dinitrophenol. Compounds containing long ester chains (C₇ or C₈) had less activity than 4-(1-ethylhexyl)-2,6-dinitrophenol. O-Methylation produced compounds with less activity than the parent 4-(1-ethylhexyl)- and 4-(1-propyl-pentyl)-2,6-dinitrophenols.     

GC evaluation of flavour compound sorption from water solutions by corn starch cryotextures obtained by freezing

Sorption of essential oil aroma components, n-alcohols and linalool by starch corn cryotexture was studied. Results show that terpene hydrocarbons of Rosmarinus officinalis L. essential oil are sorbed quantitatively from 0.05% water solution by cryotexture due to hydrophobic interactions with starch polysaccharides. Aroma compounds with oxygen atoms within the molecule are sorbed two times less. A templating effect with glucose, sucrose, maltose and some essential oil components was observed. Sorption of n-alcohols C₄, C₆, C₈, linalool and their mixture with alkyl acetates in concentration 0.5 — 15 mmol/l was carried out. Individual octanol is sorbed by cryotexture on 88%, hexanol on 20%, linalool on 25%. Butanol is not sorbed at the chosen concentration. Synergism in sorption of hexanol from mixture with octanol and alkyl acetates was observed, while a suppression effect of octanol sorption was found for the same mixture.     

Characteristics and functional properties of gelatin from seabass skin as influenced by defatting

Gelatins from nondefatted and defatted seabass skins were characterised and evaluated for their functional properties in comparison with commercial fish skin gelatin. All gelatins contained α₁‐ and α₂‐chains as the predominant components and showed a high imino acid content (199–201 residues/1000 residues). All gelatins had a relative solubility greater than 90% in the wide pH ranges (1–10). Foaming properties of all gelatins increased with increasing concentrations (1–3%, w/v). Gelatin from defatted skin had higher foam expansion and stability than that extracted from nondefatted skin. Emulsion containing gelatin from defatted skin had smaller oil droplet size (d₃₂, d₄₃), compared with that having gelatin from nondefatted skin (P < 0.05). After 10 days of storage at room temperature (28–30 °C), emulsion stabilised by gelatin from defatted skin showed the higher stability as indicated by the lower increases in d₃₂ and d₄₃, and lower flocculation factor and coalescence index. Coincidentally, emulsion stabilised by gelatin from defatted skin had higher zeta potential than that containing gelatin from nondefatted skin. Thus, defatting of seabass skin directly affected characteristics and functional properties of resulting gelatin.     

Binding of aroma compounds with legumin. III. Thermodynamics of competitive binding of aroma compounds with 11S globulin depending on the structure of aroma compounds

This paper considers the prominent features in competitive binding of aroma esters from their mixtures to 11S globulin of broad beans (legumin) in aqueous medium at pH 7.2 and ionic strength of 0.05 mol dm⁻³. Series of alkyl acetates (C₄–C₈) and methyl esters of carbonic acids (C₅–C₉), differing in the length of hydrocarbon chain, have been under our studying. To accomplish the ends of the study, a combination of ultrafiltration and gas–liquid chromatography (GC) has been used. An increase in the length of hydrocarbon chain of the aroma esters brought about greater binding affinity for the protein, the occurrence of some structural restrictions in the interior of the protein molecule, preventing binding, and the change in the binding mechanism of the aroma compounds at the specific critical length of hydrocarbon chain. Differential scanning microcalorimetry data suggested that the revealed changes in the binding mechanism of the studied aroma compounds were attributable to the conformational modification of the protein globule as a result of binding with the aroma compounds. A distinguishing feature in binding of methyl esters of carbonic acids with legumin was their greater binding affinity for the protein as compared with alkyl acetates. The mutual effect of aroma compounds on binding from their equimolar mixtures to the protein made itself evident, firstly, as a drastic increase in the binding extent of aroma esters, having rather long hydrocarbon chain and, secondly, as a dramatic change in the binding mechanism of the aroma esters with relatively short hydrocarbon chain.     

Emulsifying Property and Antioxidative Activity of Cuttlefish Skin Gelatin Modified with Oxidized Linoleic Acid and Oxidized Tannic Acid

Cuttlefish skin gelatins modified with oxidized linoleic acid (OLA) and oxidized tannic acid (OTA) were characterized and determined for emulsifying properties and antioxidative activity. Modification of gelatin with 5% OTA increased the total phenolic content and 1,1-diphenyl-2-picrylhydrazyl, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activity and ferric reducing antioxidant power of gelatin–OTA. Incorporation of OLA into gelatin (OLA-to-free amino group molar ratio of 10:1) increased surface hydrophobicity of gelatin from 17.39 to 32.38 and reduce surface tension at air/water interface of gelatin solution from 53 to 32 mN/m. Gelatin–OLA had the increase in emulsion activity index, compared with gelatin without modification and was capable of producing a fine emulsion (d ₃₂?=?0.79 μm, d ₄₃?=?0.82 μm). Modification of gelatin–OLA complex with OTA at different concentrations (2.5%, 5%, and 10%) increased antioxidative activity but decrease emulsifying properties. However, gelatin–OLA modified with 5% OTA had higher emulsifying properties than the commercial gelatin (bovine gelatin). The presence of an alkyl group and a hydroxyl group in gelatin after modification with OLA and OTA, respectively, was revealed by Fourier transform infrared study. Coincidental decrease in free amino group was also noticeable in modified gelatin. Menhaden oil-in-water emulsion stabilized by gelatin modified with OLA and 5% OTA was more resistant to lipid oxidation and phase separation as evidenced by the lower thiobarbituric acid reactive substances value and smaller oil droplet size, compared with that stabilized by commercial bovine gelatin. Thus, the modification of gelatin by both OLA and OTA was able to improve antioxidative and emulsifying properties of cuttlefish skin gelatin.     

Structural Characteristics of Tilapia (Oreochromis mossambicus) Bone Gelatin: Effects of Different Liming Methods

Fish bone is a good source of gelatin. In this study, gelatins were prepared from tilapia bone after the bone was pretreated with alkali protease, desalted immediately by 0.6 mol L^?1 HCl, and hydrolyzed by papain or limed by Ca(OH)₂. Gelatins extracted from papain-treated tilapia bone exhibited space structures similar to those of alkali-treated tilapia bone. Despite this similarity, many differences were observed between these gelatin samples. Compared with alkali-treated gelatin, papain-treated gelatins showed higher values for imino residue content, molecular weight proportion, bloom strength, and viscosity. The bloom strengths of the second and third papain-treated gelatins were 163 and 94 bloom, respectively, which were lower than the bloom strength of the first papain-treated gelatin (189 bloom). The viscosities of the three papain-treated gelatin samples were 4.18, 2.81, and 0.51 mPa.s^?1. The first papain-treated gelatin achieved the highest gelling (16 °C) and melting points (23.9 °C). The yields of the first (5.40%) and second (6.71%) papain-treated gelatins were higher than those of the alkali-treated gelatins (3.33 and 5.76%, respectively). However, the yield of the third papain-treated gelatin (2.27%) was lower than that of the third alkali-treated gelatin (5.42%). More importantly, papain hydrolysis can prevent destruction by Ca(OH)₂ in the bone structure and effectively reduce the denaturation temperature of tilapia bone collagen. Moreover, papain hydrolysis can dramatically reduce the time required for liming (0.8% of traditional liming process spent). Papain hydrolysis is a clean production method that can replace traditional liming.     

Development and verification of a quantitative real-time PCR method to identify and quantify gelatin derived from animal hide

The species origin of hide gelatin is a crucial issue with respect to health concerns and religious restrictions. Analysis of the animal-derived ingredients of gelatin by reliable methods is necessary to ensure its authenticity. However, due to the highly processed nature of gelatin, it remains a challenge to identify gelatin end products accurately and robustly. Our study established and verified a quantitative real-time PCR (qPCR) method based on careful selection of target genes and a DNA extraction method. The middle products of the gelatin production streamline were investigated to explore the influence of each critical processing step on the method. Gelatin reference samples were used to quantify the levels of target species. Commercial gelatin commodities were surveyed to highlight the mislabeling situation. In summary, the qPCR method was demonstrated to be highly specific and sensitive, with limits of detection (LOD) of 0.1 to 1 pg/µL and gelatin LODs of 0.1% to 5% (w/w). The transition from decoction to concentrated gel was found to have the most severe effect on the qPCR. Intensification of pressure or temperature or employment of enzyme hydrolysis aggravated the DNA damage, resulting in elevated Cq values. Quantitation of gelatin products was feasible; gelatin products produced from 5% target hide and 95% matrix hide mixtures showed 2.9% to 5.2% target species. The 26% relative error for low gelatin content is acceptable for semiquantitation purposes. A market survey showed that 52.6% of the gelatin products were mislabeled as being of animal origin.     

Structures and pesticidal activities of derivatives of dinitrophenols. XII.—In vitro activity of C3 to C13 α-branched alkyldinitro-phenols and their esters and ethers against the spores of Venturia inaequalis and other fungi

The activity in vitro of 2-(C₃ to C₁₃ α-branched alkyl)-4,6-dinitrophenols and 4-(C₄ to C₁₃ α-branched alkyl)-2,6-dinitrophenols and of their various esters and ethers was determined against the spores of Venturia inaequalis (Cooke) Wint., Botrytis cinerea Pers., Fusarium bulbigenum, Cooke & Massee, var. lycopersici (Brushi) Wollenw. and Cercospora melonis Cooke. Several alkyldinitrophenols were highly active against Venturia and the 2-alkylphenols were generally more active than the 4-alkylphenols. The alkyldinitrophenols were not effective against the spores of Botrytis, Fusarium and Cercospora.. Esterification of several lower alkyldinitrophenols to methyl carbonates gave compounds with enhanced activity, but methyl carbonates of C₇ to C₁₁ alkyl dinitrophenols showed considerably reduced activity. Esterification to ethyl- or other alkyl-carbonates gave compounds with considerably reduced activity against Venturia. Esterification of 2-isopropyl- and 2-t-butyl-4,6-dinitrophenols to crotonates gave compounds with enhanced activity against Venturia, but crotonates of other phenols showed reduced activity. The acrylates of several C₆ to C₈ alkyl dinitrophenols proved highly active. Ethers of active phenols showed considerably reduced activity.     

Influence of bulk and interfacial properties and operating conditions on continuous foaming operation applied to model media

Continuous foaming operation was investigated on model food as a function of rotation speed N and of the gas-to-liquid flow ratio G/L. Newtonian glucose syrup solutions exhibiting various viscosities were studied using additives in order to modify their elasticity and interfacial properties. Without surfactant, gas dispersion experiments showed that a total incorporation of the gas phase could not be achieved at constant liquid flow rate (L = 30 mL/min) for G/L higher than 5/30; the Sauter diameter d₃₂ of the bubbles could not be predicted using a simple approach based on a constant critical value of the laminar Weber number We = We_C. The addition of whey proteins gave abundant foams with total gas incorporation. d₃₂ could be predicted using We = 0.3 when We was defined using the process viscosity measured during foaming. Gelatin increased We_C, while gelatin–whey proteins mixtures decreased We_C by reinforcing the rigidity of the interfacial film; PAA–whey protein mixtures exhibited similar trends, but We_C decreased when G increased. Conversely, We_C varied significantly with operating conditions when emulsifiers or proteins/emulsifiers mixtures were used as foaming agents.     

Improvement of gel strength and melting point of fish gelatin by addition of coenhancers using response surface methodology

Abstract: Fish gelatin is a potential alternative to mammalian gelatin. However, poor gel strength and low melting point limit its applications. The study was aimed at improving these properties by adding coenhancers in the range obtained from response surface methodology (RSM) by using Box–Behnken design. Three different coenhancers, MgSO₄, sucrose, and transglutaminase were used as the independent variables for improving the gel strength and melting point of gelatin extracted from Tiger‐toothed croaker (Otolithes ruber). Addition of coenhancers at different combinations resulted gel strength and melting point in the range of 150.5 to 240.5 g and 19.5 to 22.5 °C, respectively. The optimal concentrations of coenhancers for predicted maximum gel strength (242.8 g) obtained by RSM were 0.23 M MgSO₄, 12.60% sucrose (w/v), and 5.92 mg/g transglutaminase and for predicted maximum melting point (22.57 °C), the values were 0.24M MgSO₄, 10.44% sucrose (w/v), and 5.72 mg/g transglutaminase. By addition of coenhancers at these optimal concentrations in verification experiments, the gel strength and melting point were improved from 170 to 240.89 g and 20.3 to 22.7 °C, respectively. These experimental values agreed well with the predicted values demonstrating the fitness of the models. Results from the present study clearly revealed that the addition of coenhancers at a particular combination can improve the gel strength and melting point of fish gelatin to enhance its range of applications. Practical Application: There is a growing interest in the use of fish gelatin as an alternative to mammalian gelatin. However, poor gel strength and low melting point of fish gelatin have limited its commercial applications. The gel strength and melting point of fish gelatin can be increased by incorporation of coenhancers such as magnesium sulphate, sucrose, and transglutaminase. Results of this work help to produce the fish gelatin suitable for wide range of applications in the food industry.     

ESTERASES OF BAKER'S YEAST. II. SUBSTRATE SPECIFICITIES TOWARDS ESTERS FORMED DURING SUGAR FERMENTATIONS

Ethyl esters of fatty acids (C₂–C₁₂), isoamyl acetate and 2-phenethyl acetate were studied as substrates for yeast esterases and compared with the synthetic substrates, p-nitrophenyl esters and β-naphthyl esters. Intact yeast, the 55% and 55–75% ammonium sulphate precipitate of centrifuged yeast homogenate, and partly purified esterases were used for the determination of the hydrolysation activity towards the esters. The results showed that the yeast esterases prefer to hydrolyse the ethyl esters with acyl chain length of C₅ to C₁₂. The acetate esters, ethyl acetate, isoamyl acetate and 2-phenethyl acetate are only very slowly hydrolysed or remain unaffected. The substrate specificity of different esterases varies and can be used for their characterisation. Investigating pH optimum curves using intact yeast and a crude esterase preparation and different substrates confirmed the earlier result that there are esterases on both sides of the plasma membrane. The specificity of intracellular and periplasmically located esterases is, however, different.     

Structures and pesticidal activities of derivatives of dinitrophenols. XI. Effects of substitution of dinitrophenols with C3 to C13 alpha-branched alkyl groups and of esterification on the eradication of cucumber mildew

The eradication of cucumber mildew caused by Sphaerotheca fuliginea (Schlecht. ex Fr.) Poll. with 2-(C₄ to C₁₃ α-branched alkyl)-4,6-dinitrophenols and 4-(C₄ to C₁₃ α-branched alkyl)-2,6-dinitrophenols was examined. 2-(C₈ to C₁₃ α-Branched alkyl)-4,6-dinitrophenols were highly active, but O-methylation produced compounds with no activity. 4-(C₇ to C₁₀ and several C₁₁ to C₁₃ α-branched alkyl)-2,6-dinitro-phenols gave a significant degree of eradication. 2-(C₆ to C₁₃ α-Branched alkyl)-4,6-dinitrophenols were significantly more active than their 4-alkyl-2,6-dinitrophenol analogues. Esterification of C₄ to C₇ α-branched alkyl dinitrophenols to methyl- or ethyl- carbonates or crotonates gave compounds with enhanced activity. Generally, the methyl- or ethyl- carbonates or 2-C₈-alkyl-4,6-dinitrophenols were as active as the parent phenols, but the higher alkyl carbonates or crotonates had lower activity. Esterification of 2-(C₉ to C₁₃ α-branched alkyl)-4,6-dinitrophenols to ethyl carbonates and crotonates gave compounds with reduced activity.     

Development of antipeptide enzyme‐linked immunosorbent assay for determination of gelatin in confectionery products

The gelatin sources have become a controversial issue with regard to religious and health concern. Thus, the aims of this study were to develop and evaluate the efficiency of polyclonal antibodies against peptide immunogen of collagen α2 (I) chain for determination of gelatin sources in confectionery products by competitive indirect enzyme‐linked immunosorbent assay (ELISA). Collagen α2 (I) chain protein showed resistance against heat treatment and detectable in certain commercial products when analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS‐PAGE). The established ELISA exhibited low cross‐reactivity to fish and chicken gelatin. The IC₅₀ value was 0.39 μg mL^?1, and the limit of detection (IC₁₀) was 0.05 μg mL^?1. There were no false‐positive results from forty‐eight commercially processed products. The present method is useful for determination of gelatin in confectionery products.     

Gelatin hydrolysates from sliver carp (Hypophthalmichthys molitrix) improve the antioxidant and cryoprotective properties of unwashed frozen fish mince

In this study, gelatin extracted from silver carp skin was hydrolysed by different enzymes (alcalase, papain and flavourzyme), aiming to improve the storage stability of unwashed fish mince during freeze-thaw cycles. Flavourzyme hydrolysates exhibited the highest DPPH• scavenging activity (1.2-fold of alcalase, 1.6-fold of papain) and reducing ability (1.2-fold of alcalase, 2.3-fold of papain), while the alcalase hydrolysates demonstrated the highest Fe²⁺ chelating ability. Moreover, fish mince treated with the mixture of flavourzyme hydrolysates and sucrose exhibited higher sulfhydryl and salt-soluble protein contents, greater Ca²⁺-ATPase activity and better water holding capacity. Hence, the gelatin hydrolysates of flavourzyme from silver carp skin are promising candidates as natural and high-performance antioxidant and cryoprotectants in fish mince processing.     

Gastrointestinal stability and bioavailability of (poly)phenolic compounds following ingestion of Concord grape juice by humans

The in vitro gastrointestinal stability of (poly)phenolic compounds in Concord grape juice was compared with recoveries in ileal fluid after the ingestion of the juice by ileostomists. Recoveries in ileal fluid indicated that 67% of hydroxycinnamate tartarate esters, and smaller percentages of the intake of other (poly)phenolic compounds, pass from the small intestine to the colon. The juice was also ingested by healthy subjects with an intact functioning colon. Peak plasma concentrations (C_max) ranged from 1.0 nmol/L for petunidin‐3‐O‐glucoside to 355 nmol/L for dihydrocoumaric acid. Urinary excretion, as an indicator of bioavailability, varied from 0.26% for total anthocyanins to 24% for metabolites of hydroxycinnamate tartarate esters. The C_max times of the anthocyanins indicated that their low level absorption occurred in the small intestine in contrast to hydroxycinnamate metabolites which were absorbed in both the small and the large intestine where the colonic microflora appeared responsible for hydrogenation of the hydroxycinnamate side chain. The bioavailability of the complex mixture of (poly)phenolic compounds in Concord grape juice, was very similar to that observed in previous studies when compounds were either fed individually or as major components in products containing a restricted spectrum of (poly)phenolic compounds.     

Improvement of foaming properties of cuttlefish skin gelatin by modification with N-hydroxysuccinimide esters of fatty acid

Conformation and foaming properties of cuttlefish skin gelatin modified by N-hydroxysuccinimide esters of different saturated fatty acids including capric acid (C10:0), lauric acid (C12:0) and myristic acid (C14:0) at different molar ratios (0.25, 0.50, 1.00 and 2.00) were investigated. Covalent attachment of fatty acids into gelatin was observed as evidenced by the decrease in amino groups. Fourier transform infrared spectroscopic study indicated the presence of alkyl group of modified gelatin. The higher increase in surface activity with coincidental increase in surface hydrophobicity was observed in gelatin modified with fatty acid ester having a longer chain, especially at the higher molar ratio. The increase in foam expansion was related with the improved surface activity mediated by the modification by N-hydroxysuccinimide esters of fatty acid.     

Purification of Brussels sprout isoperoxidases

Extracts of Brussels sprouts contain a complex mixture of isoperoxidases. Purification of the anionic and cationic groups of isoenzymes by ion-exchange chromatography has resulted in identification of four distinct isoperoxidases (A₁; A₂, C₁ and C₂) of different molecular size. Each preparation showed single staining bands for isoperoxidase activity and by the more sensitive silver staining technique for protein. However, only the C₂ isoperoxidase preparation showed a single band by Western blotting against polyclonal horseradish antibody, whereas the other isoenzymes still showed cross contamination with other isoperoxidases although minor in the instances for the a₁ and A₂ isoperoxidases. Also these results show that, even after extensive chromatographic purification of peroxidases, it is still necessary for assessment of homogeneity, prior to structural studies, to use the more sensitive Western antibody technique. Amino acid sequencing yielded a short identical sequence for A₂ and C₁ which was also 95% identical to some previously detected drought/salt stress proteins.