Formation of protein bound lysine‐derived galactosyl and glucosyl pyrroles in heated model systems

Residues of lysine‐substituted AGEs (advanced glycosylation end‐products) arising from the Maillard reaction and containing the carbon backbone of lactose or maltose, thus deriving from 1‐desoxy‐ketose degradation, were produced when casein was heated in the presence of the corresponding U¹⁴C labelled disaccharides. The enzymatic hydrolysates of the washed casein were purified by SPE and submitted to HPLC on a C18 column flushed with diluted acetic acid. A specific chromatographic peak (λ_max, 288.1 nm; MW, 416.2 Da) with a different retention time was obtained for each disaccharide reacted. On the basis of the value of the specific radioactivity, the two compounds appeared to contain the whole carbon backbone of the parent sugar. Analyses by MS/MS and NMR performed on the same two compounds extracted at preparative scale from lysine‐lactose and lysine‐maltose model systems allowed the structure assignment of 6‐[2‐acetyl‐3‐(β‐D‐galactopyranosyloxy)‐1‐pyrrolyl] 2‐amino hexanoic acid and6‐[2‐acetyl‐3‐(α‐D‐glucopyranosyloxy)‐1‐pyrrolyl] 2‐amino hexanoic acid, respectively. Both compounds submitted to enzymatic deglycosylation by specific α‐ or β‐ glucosidases produced the lysine‐derived acetyl pyrrole 6‐(2‐acetyl‐3‐hydroxy‐1‐pyrrolyl) 2‐amino hexanoic acid(λ_max, 288.1 nm; MW, 254.1 Da). Galactosyl‐ and glucosyl‐ isomaltoles, extracted from the lysine‐containing systems, identified with the reference molecules and heated in the presence of lysine under slightly alkaline conditions, gave the expected lysine‐derived glycosyl pyrroles as identified above. The HPLC conditions were optimized by adjusting the composition of the eluting solvent and temperature of the column to achieve the best separation and identification of the AGEs in mixtures such as foods with possible interfering molecules like Trp and lysyl pyrrole aldehyde. Because of the reported presence of the two precursors isomaltol glycosydes in some foods, the corresponding lysine‐derived glycosyl pyrroles can occur as both protein bound and in free form.     

Effect of pH on the growth,nodulation and nitrogen fixation of Centrosema pubescens and Stylosanthes gracilis

Centrosema pubescens and Stylosanthes gracilis were grown on two soils of different texture adjusted to pH values ranging from 5–7 to 8–0 by the addition of either dilute sulphuric acid or calcium carbonate. Centrosema grew best on a soil having final pH values of 6–1, while Stylosanthes grew best on a soil having a final pH value of 5–7. Nodulation followed a similar trend with dry matter yields but was increasingly suppressed by increases in pH, with complete suppression in some cases at pH8. Maximum nitrogen fixation in both cases occurred at a pH value of about 6.     

Investigations on the Guanidination of Lysine in Proteins

The present study was conducted to compare the efficacy of five different reaction conditions on the guanidination of lysine in casein and to establish optimum lysine: O -methylisourea (OMIU) for maximum guanidination of lysine in casein and soya bean meal. The results indicate that the presence of glycine–NaOH buffer is not required for guanidination of proteins at pH 10·5. A OMIU concentration of 0·4 M was found to be as effective as 0·6 M for guanidination. Both OMIU–hydrogen sulphate and free OMIU were equally effective reagents in terms of conversion of lysine to homoarginine. The use of OMIU–hydrogen sulphate for guanidination and the use of ethanol to recover guanidinated protein, however, resulted in the formation of crystalline sodium sulphate, a known purgative agent, in the guanidinated material, and therefore are not recommended if the guanidinated protein is to be used in animal trials. The molar ratio of lysine: OMIU required for efficient lysine conversion to homoarginine varied for different protein sources. Ratios required for maximum conversion for casein and soya bean meal were determined to be 1:10 and 1:16, respectively. A simple procedure developed for the large-scale guanidination (5–10 kg batches) of proteins is also described. The results showed that guanidination of proteins can be easily scaled up from 20 g to 5–10 kg and that large-scale guanidination is feasible and efficient.     

Colour improvement of common carp (Cyprinus carpio) fillets by hydrogen peroxide for surimi production

The preferred colour for surimi is white, but surimi prepared from light fillets of common carp (Cyprinus carpio) is slightly pink. Hydrogen peroxide (H₂O₂; 1–3% v/v) with and without sodium tri‐polyphosphate (STP; 1–2% w/v) was added to a sodium carbonate bath (pH 7.0–11.5) resulting in a final pH range of 4.4–10.1 which was injected into carp fillets. After soaking and tumbling for 30 min at 4–10 °C, the fillets were evaluated for colour and water holding capacity (WHC). Fillets tumbled with treatment solution with different pH levels (7.0–11.5), but with no H₂O₂ or STP added, had improved colour with significantly (P < 0.05) higher L* compared with untreated fillets as the control. However, the colour improvement [(L* and colour deviation (ΔE)] was not significantly different (P > 0.05) within the pH levels (7.0–11.5) trialled. With increasing H₂O₂ levels (1–3%), fillets became lighter and ΔE increased significantly (P < 0.05), especially with a 3% H₂O₂ treatment at pH of 10.5 (adjusted pH before H₂O₂ addition, actual pH after H₂O₂ addition was 8.2). The whiteness (L*?3b*) of kamaboko produced from treated (3% H₂O₂, pH 10.5) common carp light fillets was not significantly different to that of kamaboko from Alaska pollock and threadfin bream. Treatments combining H₂O₂ (3%) with STP (1–2%) significantly reduced the L* value obtained in comparison with fillets treated with only H₂O₂ (3%). Similarly, fillets treated with STP (1%) alone, resulting in lower L* values, irrespective of treatment pH (7.0–11.5). WHC, an indicator of the quality of the fillet texture, increased from 816 g/kg at pH 7.0 without STP to 841 g/kg at pH 11.5 with 1% STP. Treatment with H₂O₂ (without STP) decreased the WHC of the fillets.     

Characterisation of a trypsin/chymotrypsin inhibitor from jack fruit (Artocarpus integrifolia) seeds

Jack fruit seed (Artocarpus integrifolia Hook f) trypsin inhibitor (JSTI) was found to be rich in acidic amino acids and devoid of free thiol groups. The N-terminal and C-terminal amino acids of JSTI were aspartic acid and scrine, respectively. The inhibitor was stable under conditions of extremes of pH (3·0–12·0), at high temperatures and in the presence of denaturing agents. JSTI showed a non-competitive type of inhibition with K_i values of 0·48 ± 0·17 nM and 0·16 ± 0·04 nM for trypsin and chymotrypsin, respectively. The JSTI–trypsin complex exhibited chymotrypsin inhibitory activity suggesting the ‘double-headed’ nature of the inhibitor. Chemical modification of lysine residues resulted in loss of trypsin and chymotrypsin inhibitory activities of JSTI indicating that amino groups are essential for activity.     

Effect of Water on the Production of Cooked Beef Aroma Compounds

Aroma compounds isolated from cooked fresh ground beef, freeze-dried, defatted and rehydrated to 58% and 17% water, respectively, were less meaty and contained higher relative concentrations of hydrocarbons, ketones, lactones, esters, benzenoids, pyrroles, pyridines and thiazol(in)es (58% H₂O), and of aliphatic ketones, lactones, esters, aliphatic sulfur compounds, pyrroles and pyrazines (17% H₂O) than those obtained from untreated beef. Maximum production of volatiles at water activity (a_w)?0.7, described previously for a model simulated meat flavor system, did not occur. Suggested contributory components to the meatier aroma analyzed from untreated beef were 2-methyl-3-(methylthio)furan, 3-(and 2-)methylcyclopcntanones, cyclopent-2-enones and cyclohex-2-enones.     

Identification and quantification of α-dicarbonyl compounds produced in different sugar-amino acid Maillard reaction model systems

A mixture of α-dicarbonyl compounds generated from Maillard reaction (MR) model systems, comprising of fructose with glycine (Fru-Gly) and lysine (Fru-Lys); glucose with glycine (Glu-Gly) and lysine (Glu-Lys); ribose with glycine (Rib-Gly) and lysine (Rib-Lys), were identified and quantified. α-Dicarbonyl compounds generated in the hexose models were predominantly glucosone and 3-deoxyglucosone (3-DG), with 3-deoxypentosone (3-DP) and pentosone being the major α-dicarbonyls produced in the pentose models. Ethyl acetate extraction of model MRPs resulted in poor recovery of 2,3-diaminonaphthalene benzoquinoxalines of glucosone, 3-DG, pentosone, 3-DP and methylglyoxal (MGO), respectively. The temporal profiles of pentosone, 3-DP, glyoxal (GO) and MGO were similar in the pentose models, with maximum concentration occurring within 5 min. These four α-dicarbonyl compounds were higher (P < 0.05) in Rib-Gly than in Rib-Lys MR systems. The quantity of α-dicarbonyl compounds was affected by the interaction among the type of sugar and amino acid and the reaction time. The type of sugar is the most important factor that affected both the quantity and quality of α-dicarbonyl compounds produced in MR mixtures.     

Influence of Fermentation Time and an ‘Indigenous Tenderiser’ (Kanwa) on the Microbial Profile,Chemical Attributes and Shelf-Life of Rice Masa (a Nigerian Fermented Product)

Investigations on the influence of fermentation time, ‘Kanwa’ (an indigenous tenderiser and a flavouring agent) and potassium sorbate (0·5, 1·0 or 1·5 g kg⁻¹) were conducted by examining the microbial profile, chemical attributes and shelf-life of rice ‘Masa’ (a Nigerian fermented product). Various types of microorganisms occurred at the initial stages of rice slurry fermentation and these included fungi (Aspergillus spp, Penicillium spp, Saccharomyces spp, Rhizopus spp) and bacteria (Lactic acid bacteria, Bacillus spp, Enterobacter spp and Acetobacter spp). But beyond 8 h fermentation time, fewer types of micro-organisms (Lactobacillus spp, Saccharomyces spp and Rhizopus spp) were isolated.Total aerobic counts increased significantly (P=0·05) as fermentation progressed (0, 6 or 12 h), with lactic acid bacteria (LAB) and yeasts dominating and probably contributed to the sour–sweet spongy characteristic of the rice Masa. But after fermentation and baking process, the rice Masa became dominatedand spoilt by Bacillus spp, Lactobacillus spp and Saccharomyces spp withstorage time. Longer fermentation time resulted in a less acceptable product,being more acidic. Rice Masa produced from a 6 h fermentation showedbetter sensory qualities (aroma and visual appearance). Use of Kanwa(Na₂CO₃.NaHCO₃.2H₂O) improved aroma but was detrimental to visualappearance and spoilage was accelerated, probably due to higher pH. Potassium sorbate at higher concentrations (1·0 and 1·5 g kg⁻¹) induced beneficial antimicrobial effects and reduced the microbial load thereby prolonging the shelf life of the rice Masa (treated with 1·5 g kg⁻¹) by about 3 days.     

Interaction of lysine with pectin

The interaction of l-lysine with citrus pectin was studied in a model system to determine under what conditions it would occur and whether it would be of physiological significance. Thirty-nine per cent of the lysine was non-diffusible at pH 7, when the pectin concentration was 0·5% and the lysine concentration was $\text{2 × 10}{}^{\mathrm{?3}}\text{M}$. Of this, 29% was restricted due to the Donnan effect and the remainder was specifically bound. Increasing the pH (in the range 6–8) and the pectin concentration both increased the non-diffusible lysine. Increasing the ionic strength and the degree of esterification both decreased the non-diffusible lysine. It was concluded that lysine would not bind to pectin in the intestine because of the high salt concentration there.     

Functionality of freeze-dried L. casei cells immobilized on wheat grains

Lactobacillus casei cells were immobilized on wheat grains and the effect of nine cryoprotectants during freeze-drying was investigated. Survival and fermentative activity of the freeze-dried immobilized biocatalysts was studied by monitoring pH, lactic acid and lactose content in successive fermentations batches of both synthetic lactose medium and milk. Freeze-dried L. casei cells immobilized on wheat grains without using cryoprotectants resulted in high cell survival and metabolic activity. The same biocatalysts were stored at room temperature for 9 months and at 4 °C and −18 °C for 12 months. Reactivation of the stored biocatalysts was carried out in synthetic lactose medium. Storage at room and low temperatures (4 °C and −18 °C) resulted in about 5.11, 4.9 and 4.3 final pH respectively during fermentations, indicating the suitability of the immobilized biocatalysts for the production of mild and low pH dairy products. The immobilization of a probiotic microorganism, such as L. casei, on boiled wheat which contains prebiotic compounds might provide a potential synbiotic preparation.     

Factors affecting in vitro formation of tannin-protein complexes

In interaction of condensed tannins from Desmodium intortum and Lotus pedunculatus and tannic acid (hydrolysable tannin) with salivary mucoproteins (from sheep and goats), plant leaf proteins and bovine serum albumin were evaluated. These studies were carried out over a pH range of 2-0-9-0 and different inorganic ion conditions to simulate conditions in which dietary proteins would interact with tannins in a ruminant digestive tract. Insoluble tannin-protein interactions were found at pH 4–5–5–5 for bovine serum albumin and 3–5–5–5 for plant leaf protein. The present study showed that pH alone was not the sole determinant for tannin-protein complex formation, since tannin-protein complexation was found in the pH range 6-0–6-5 when different inorganic ions were added to the solutions. Insoluble complexes were not formed with salivary proteins, although precipitation by tannic acid was achieved at 5°C. This suggests that tannins may form soluble rather than insoluble complexes with salivary proteins. It was concluded that purified F1 leaf protein (the major protei occurring in leaf tissue) ought to be used as the test protein for evaluating tannin-protein interactions for in vitro assay procedures. Using this method it was calculated that 27–43% and 19–40% of available plant protein may interact with condensed tannins from Desmodium intortum and Lotus pedunculatus, respectively.     

Identification and quantitative evaluation of the lysine‐arginine crosslinks GODIC,MODIC, DODIC,and glucosepan in foods

The presence of the various protein crosslinks GOLD 2 , MOLD 3 , GODIC 4 , MODIC 5 , DODIC 6 , and glucosepan 7 in foods has been established for the first time by liquid chromatography – mass spectrometry (LC‐MS) with electrospray ionization (ESI). In compounds 2 and 3 two lysine moieties, in 4 – 7 a lysine and an arginine side chain are joined by the crosslink. Unequivocal identification of 2–7 was achieved with independently synthesized reference material. The quantitative results for the investigated foodstuffs show MODIC 5 to be the most important Maillard crosslink. The concentrations of 5 and GODIC 4 are 5–10 fold higher than those of the corresponding imidazolium compounds 3 and 2 , establishing 5 and 4 as the major food protein crosslinks derived from methylglyoxal and glyoxal, respectively. The maximum value of 151 mg MODIC 5 /kg protein (equivalent to 0.42 mmol/kg protein) was found in a butter biscuit sample which also shows the highest overall Maillard crosslink content with 0.71 mmol 47 /kg protein. These first quantitative results suggest that compounds 4 – 7 can be jointly responsible for protein polymerization in the course of food processing.     

Injury,Inhibition and Inactivation ofEscherichia coli O157:H7 by Potassium Sorbate and Sodium Nitrite as Affected by pH and Temperature

The effect of potassium sorbate (0–2 g litre⁻¹) and sodium nitrite (0–1 g litre⁻¹) on the growth of four strains of Escherichia coli O157: H7 in tryptic soya broth at various pH levels (pH 4·0–7·0 for sorbate, pH 5·0–8·0 for nitrite) were determined at 37°C and 4°C. Among the pH levels tested, sorbate and nitrite exhibited the highest antimicrobial activity at pH 4·0 and 5·0, respectively. At pH 5·0 and 37°C, the presence of 500 mg litre⁻¹ sorbate or 200 mg litre⁻¹ nitrite completely inhibited the growth of E coli O157: H7. While at higher pH levels, 2 g litre⁻¹ sorbate or 1 g litre⁻¹, nitrite, the highest concentration tested, did not show significant antimicrobial action against the test organisms. At 4°C and pH 5·0, the inoculated test organisms did not showed any significant growth in preservative-free control media. Different degree of inactivation and injury was observed when E coli O157: H7 strain 933 was stored in TSB (pH 5·0) containing 1 g litre⁻¹ sorbate or nitrite at 37°C. At 4°C, inactivation and injury of E coli O157: H7 cells was not observed in the medium containing sorbate or nitrite throughout the 24 h experimental period.     

The importance of the furanones HDMF and HEMF in the flavour profile of Japanese barley miso and their production during fermentation

All 13 samples of Japanese barley miso accepted for the Kumamoto Prefecture Miso Competition of 1995 were assessed by tasting and by GC analysis using FID, SIM and olfactometric detection. Forty three compounds were separated by GC, of which 15 had a detectable aroma. Consideration of the chemical and sensory analyses of this latter group showed that only the two furanones, 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) and 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF), correlated significantly with the quality of miso flavour (P < 0·05). Experimental fermentations of salted barley koji and full barley miso mash with Zygosaccharomyces rouxii resulted in alcoholic fermentation in all cases investigated. In contrast, production of the three furanones, HMMF (4-hydroxy-5-monomethyl-3(2H)-furanone), HDMF and HEMF, was very sensitive to the conditions used. Negligible amounts were found in the absence of soy beans and the highest levels occurred when the full mash was incubated at 37°C for 14 days before inoculation of the yeast. When pre-incubation was carried out, the concentration of HMMF was maximal at the end of mashing and declined during fermentation whilst the amount of both HEMF and HDMF increased. The time courses were consistent with synthesis of HEMF, and then HDMF, by the yeast from HMMF. © 1998 Society of Chemical Industry.     

In vitro studies on chelating agents as potential iron absorption promoters

Ligands which can bind iron to keep it in solution with good stability at the neutral or alkaline pH of the small intestine can serve as useful absorption promoters of dietary iron. A number of biologically compatible compounds were screened in vitro for this ability at pH 7·5, employing three different systems.Studies carried out with iron-amino acid complexes have shown that amino acids and their derivatives were stable only in the acid pH range with stability constants of 10·9 or lower. Compounds which could solubilize insoluble ferric orthophosphate at pH 7·5 exhibited efficiencies in the order: hydroxamate of lysine > nicotinic acid > EDTA > ascorbic acid > picolinic acid > cysteine. A large number of compounds were tested for their ability to keep iron in solution at pH 7·5 at different iron: ligand ratios. Of these, ascorbic acid, inorganic polyphosphates, hydroxamates of nicotinic acid and lysine, EDTA, caffeic acid, citric acid, xanthurenic acid and phytic acid showed high chelating ability. Some of these compounds can be further exploited as iron absorption-promoting ligands.     

The effect of pH,salts and sugars on the rheological properties of cress seed (Lepidium sativum) gum

Effect of shear rate (15–600 s^?1), gum concentration (1–2%), pH (3–9), sucrose (10–40%), lactose (5–15%), NaCl (100–300 mm ) and CaCl₂ (5–50 mm ) was evaluated on apparent viscosity (η_a), flow behaviour index (n), consistency coefficient (K) and yield stress (τ₀) indices of cress seed gum (CSG) solutions. Different rheological models were used to fit the experimental data, although the Herschel–Bulkley model was found the best model. An increase in gum concentration led to an increase in τ₀, η_a, and k and a decrease in n values. The addition of salts lowered the k value; however, the n value showed slight significant change. The presence of sugar resulted in the enhancement of n, k, τ₀ and η_ap values. The existence of yield stress and pseudoplastic behaviour of CSG, its stability against salts, wide range of pH and synergic effect of sugar make it a good thickener and stabiliser in food formulations.     

Orange peel fermentation using Lactiplantibacillus plantarum: microbiological analysis and physico-chemical characterisation

To find an innovative use for orange peels discarded in the orange juice-making process, a fermentative process was assessed using a Lactiplantibacillus plantarum strain. Blanched or rinsed peels were submerged in a 5% NaCl–3% inoculated sucrose brine for 10 days. Total soluble solids, pH, sugars and total aerobic and anaerobic counts were determined in the brines to characterise the process. The final products were characterised by instrumental texture, colour and volatile composition. The blanching pretreatment had a significant effect on the whole process and the final product characteristics. Anaerobic bacteria total counts were significantly higher in the blanched samples during the whole fermentation, and pH decreased significantly slower in these samples than in the rinsed ones. Rinsed samples were characterised by higher aerobic total counts, higher sucrose consumption and higher glucose, fructose and polyalcohols production. The texture was softer in the pretreated samples, probably due to the blanching process rather than the fermentation. The volatile composition was quite similar between samples, although it was different from one of the raw orange peels due to a significant decrease in various volatile compounds.     

Fermentation of concentrated skim-milk. Effects of different protein/lactose ratios obtained by ultrafiltration–diafiltration

Defatted milk was ultrafiltered–diafiltered in a 20 kDa polysulphone flat membranes UF unit to obtain concentrated milk with different protein/lactose ratios. Different samples of concentrated–diafiltrated milks with protein concentration between 4·7% and 8·9% and lactose concentration between 2·7 and 0·8% (protein/lactose ratios between 1·7 and 9·5) were fermented (Lactobacillus bulgaricus and Streptococcus thermophilus) to obtain yoghurt-like products. Firmness of the coagulum as well as pH, colony count and organoleptic test were considered in the final products. Concentrated-diafiltrated milk with lactose and protein contents lower than 2% and 6·5%, respectively, lead to a very liquid product. Enriched protein low-lactose fermented milks can be obtained by this technology. © 1998 SCI.     

Ultrathin-layer isoelectric focusing of partially purified peroxidase from tomato fruit

Partially purified peroxidase from tomato fruits was obtained by gel filtration on Ultrogel AcA 34 of a pH 5·5 extract prepared by homogenising the fruits with 0·4m McIlvaine buffer containing 0·2% Triton-X 100, subsequent centrifugation at 2000 × g and ultrafiltration of the supernatant. This fraction, with a molecular weight of 43 000 daltons, showed an optimum activity of pH 5·3 with guaiacol as the hydrogen donor, a K_m for H₂O₂ of 0·8 mm at optimum guaiacol concentration, a K_m of 9·0 mm for guaiacol at optimum H₂O₂ concentration and competitive cyanide inhibition with a K_i of 1·0 μm. Ultrathin-layer isoelectric focusing in 50 μm polyacrylamide gels resulted in the separation of eight peroxidase isozyme bands, detected mainly in the range pH 2 to 4. Parallel experiments carried out with horseradish peroxidase (Boehringer, Mannheim) exhibited the main enzyme activities in the range pH 7 to 9.     

Correlating changes that occur in chemical properties with the generation of antioxidant capacity in different sugar-amino acid Maillard reaction models

Abstract: We investigated the development of antioxidant activity relative to the change of pH, fluorescent intensity, ultraviolet (UV) absorbance (A294), browning (A420), and alpha‐dicarbonyl compounds in sugar‐amino acid Maillard reaction (MR) model systems comprising fructose, glucose, or ribose each with glycine (Fru‐Gly, Glu‐Gly, and Rib‐Gly) or lysine (Fru‐Lys, Glu‐Lys, and Rib‐Lys), respectively, which were heated at 121 °C for 5 to 90 min. For hexose models, the change in pH was shown to fit a second‐order polynomial regression with A294 and A420. Antioxidant activity was significantly and positively correlated with UV absorbance (r = 0.905, P < 0.001) and browning products (r = 0.893, P < 0.001) rather than with fluorescent products or the alpha‐dicarbonyl compounds. Type of sugar was most important in evoking a change in UV absorbance, browning, alpha‐dicarbonyl compounds, and antioxidant activity of MR products (MRPs). In conclusion, the antioxidant activity of MRPs in six model systems was more closely associated with products derived at the intermediate‐to‐late stages of the reaction and influenced mostly by the type of sugar. Practical Application: We report on the different factors and their interactions that are important for understanding the functional attributes of food components that comprise the generation of Maillard browning products and the associated antioxidant activities generated during high‐temperature food processing.