Effect of pH on fluorescence formation related to fish deterioration

 Earlier studies have investigated fluorescence at different excitation/emission maxima during common types of fish processing. A shift towards higher wavelength maxima was observed and measured as the ratio between two of the maxima tested. This fluorescence ratio (δF) correlated with increased fish damage. The present work is focused on the effect that pH can have on the formation of fluorescent compounds in fish muscle systems. Minced hake muscle was homogenised with 0.1 M phosphate buffer of different pH values (5, 6, 7 and 8) and was stored at 30  °C for up to 30 days. Lipid damage, measured as the δF value of the aqueous reaction medium and the fish lipid extract, indicated little difference between the effect of different pH values under the conditions employed in the present experiment. The effetct of formaldehyde (FA) in the same reaction medium was also evaluated. It was observed that FA had a positive effect on the fluorescence shift occurring in the aqueous reaction medium, so that a higher δF value was observed for pH 7 and pH 8, especially for the latter. It is concluded that changes in the δF value during fish storage and/or processing are of special interest as, in addition to FA formation and lipid oxidation, significant pH increases are expected to occur as a result of damage. Received: 2 February 1998 / Revised version: 6 April 1998     

Hydrolysis of soya milk oligosaccharides by Bifidobacterium longum CRL 849

 The reduction of soya milk oligosaccharides by Bifidobacterium longum CRL 849 was studied. The utilization of stachyose was concomitant with the use of sucrose. Maximum hydrolysis of stachyose (49.3%) occurred during the first 7 h of incubation at 37  °C, while a 79.3% decrease in the concentration of sucrose was observed after 9 h. No raffinose was detected after hydrolysis of the stachyose. Cell population decreased after 8 h of incubation because of the low pH attained (pH 4.7). l(+)-Lactate concentration was higher than acetate (molar ratio 6.7 : 1) at 6 h followed by a slow increase in acetate formation. Ethanol was detected in small amounts at the end of the incubation time (24 h). Received: 29 December 1997     

Starch radicals Part III: Semiartificial complexes

 Nine starch varieties were blended and kneaded with d-fructose, d-glucose, sucrose, egg albumin and/or ethyl stearate to form semiartificial complexes. They were then thermolyzed and the numbers of unpaired spins in the solid residues were determined. Individual starch varieties began to generate unpaired spin states within 2 h of heating at 285  °C but some starch blends, particularly those of four-component starch (10 : 1 : 1 : 1) mixtures, generated free radicals within 2 h of heating at 160  °C. However, the free radical counts were minimized by the mutual complexation of the blend components and, to a certain extent, by the free radical scavenging in the blends. Received: 21 October 1997 / Revised version: 15 April 1998     

Freeze- and bake-stable glazing for confectionery products characterized by differential scanning calorimetry

 A freeze- and bake-stable glazing for pastry has been developed based on an optimization procedure in which starches and carrageenans as thickening agents were combined with sugar alcohols and sodium tripolyphosphate. Instrumental gloss measurement on test biscuits showed that glazing consisting of 3% ι-carrageenan and 15% sorbitol (w/w) had maximal gloss. This was confirmed on pastry products. In contrast to glazing based on mannitol, no crystallization problems were encountered for this glazing, for which differential scanning calorimetry showed that some water freezes at –3  °C followed by freezing of supercooled encapsulated water at –20  °C and by a glass transition at –61  °C. During normal frozen storage of preglazed, nonbaked pastry, the glazing is thus in a nonstable rubbery state with a limited resistance to water migration and ice crystal formation, which can be substantially improved by storage at temperatures below –61  °C. Received: 1 March 1999 / Revised version: 19 April 1999     

Contribution to the identification of the pigments responsible for the browning of anthocyanin-flavanol solutions

 The formation of pigments responsible for the browning of solutions of pH 3.2 containing malvidin-3-monoglucoside (Mv3g) and (+)-catechin (cat), stored at 32  °C, was studied by liquid chromatography with double detection by diode array spectrophotometry and mass spectrometry. The progressive accumulation of yellowish pigments, with λ_max in the visible region between 439 nm and 458 nm, was observed throughout the assay period. The results obtained allowed the hypothesis that these pigments result from anthocyanin-flavanol condensation to be discounted, and instead suggested that they derive from cat and involve products from the oxidation of ethanol and tartaric acid. Phloroglucinol, produced in the degradation of Mv3g, was seen to participate in the formation of one of the pigments. A colourless dimer resulting from the direct cat-Mv3g condensation was also found; this product was stable and did not evolve towards the formation of pigments during the period of the assay (90 days). Structures for the principal compounds detected were postulated, taking as a basis the data obtained from their mass spectra. Received: 25 January 1999     

Lipids classes, fatty acids and carotenes of the leaves of six edible wild plants

 α-Linolenic acid and unusual fatty acids of the ω3 and ω6 series play an important role in the modulation of human metabolism. The presence of these acids in the leaves of several edible wild plants has recently been reported. In this study, six edible wild species were selected in order to establish the fatty acid compositions in their leaf lipids. Thus, young leaves from Amaranthus viridis L.(blet), Chenopodium album L. (goosefoot), Crithmum maritimum L. (rock samphire), Plantago major L. (plantain), Portulaca oleracea L. (purslane) and Verbena officinalis L. (vervain) yielded 1.50, 2.20, 3.02, 1.46, 3.81, and 2.28 g of lipids per 100 g dry plant material. Silica gel chromatography yielded 0.64 g (Plantago major) to 2.19 g (Crithmum maritimum) neutral lipids, 0.37 g (Plantago major) to 1.60 g (Portulaca oleracea) glycolipids, and 0.26 g (Crithmum maritimum) to 0.57 g (Verbena officinalis) phospholipids per 100 g (dry weight). Gas chromatography (GC) showed the major fatty acids to be 18 : 3ω3, 18 : 2ω6 and 16 : 0 in all fractions, with high concentrations of 18 : 3ω3 in the glycolipid fraction. GC-mass spectrometric analyses did not reveal the presence of unusual fatty acids. Carotenes were found in high concentrations, ranging from 30.5 mg/100 g (Chenopodium album) to 89.2 mg/100 g (Portulaca oleracea). The analyzed plants are rich sources of essential fatty acids (18 : 2ω6 and 18 : 3ω3) and also of carotenes. Received: 29 October 1998     

Effect of curing and pre-storage dip treatments on the control of storage mould of kola nuts

 The effect of curing kola nuts at 30  °C was evaluated at the Cocoa Research Institute of Nigeria, Ibadan, to determine the effect of curing time and delay between inoculation and initiation of curing on storage rot. For nuts inoculated 24 h prior to curing, 48–72 h curing at 30  °C gave optimal disease control. The incidence of rot was higher when the treatment was delayed for 48 h after inoculation. When artificially wounded and inoculated kola nuts were dipped in a solution of 1.0 g l^–1 of sodium bicarbonate for 2 mins, disease development was significantly reduced compared to the 0.5% sodium hypochorite treatment. Sodium metabisulphite also reduced disease development but the level of disease control achieved was not significantly different from that of the water-dipped controls. The in vivo results were consistent with the in vitro results. Received: 15 April 1998     

Natural fermentation of lentils Influence of time, temperature and flour concentration on the kinetics of thiamin, riboflavin and niacin

 Lentils (Lens culinaris, var. vulgaris cultivar Magda-20) were naturally fermented for 96 h at different lentil flour concentrations (79, 150 and 221 g/l) and temperatures (28, 35 and 42°C). During fermentation, samples were taken at 24-h intervals and the changes in thiamin (vitamin B₁), riboflavin (vitamin B₂) and total and available niacin (vitamin B₃) were investigated. Preparation of the lentil flour suspension to be fermented (i.e. the process of mixing the flour and sterilized tap water) caused an increase of the available niacin content in all batches, while changes in thiamin and riboflavin content were related to the conditions in which the preparation of the suspensions was carried out. The whole natural fermentation process (from the raw state to after 96 h of fermentation), either did not affect or produced a slight decrease in the thiamin content of lentils. In contrast, riboflavin, available niacin and total niacin contents increased throughout the 96 h period, which ended with a 35 – 82% increase of riboflavin, a 24 – 91% increase of available niacin and a 20 – 58% increase of total niacin. The temperature during the fermentation procedure had significant effect on the levels of thiamin and riboflavin in fermented lentils. To obtain lentil flours with an improved amount of riboflavin and available niacin with a minimum loss of thiamin, the natural fermentation of lentils should be carried out for 96 h at 42°C and with a lentil flour concentration of 221 g/l. Received: 21 February 1997     

Effect of curing and pre-storage dip treatments on the control of storage mould of kola nuts

 The effect of curing kola nuts at 30  °C was evaluated at the Cocoa Research Institute of Nigeria, Ibadan, to determine the effect of curing time and delay between inoculation and initiation of curing on storage rot. For nuts inoculated 24 h prior to curing, 48–72 h curing at 30  °C gave optimal disease control. The incidence of rot was higher when the treatment was delayed for 48 h after inoculation. When artificially wounded and inoculated kola nuts were dipped in a solution of 1.0 g l^–1 of sodium bicarbonate for 2 mins, disease development was significantly reduced compared to the 0.5% sodium hypochorite treatment. Sodium metabisulphite also reduced disease development but the level of disease control achieved was not significantly different from that of the water-dipped controls. The in vivo results were consistent with the in vitro results.     

The extraction of yellow gentian root (Gentiana lutea L .)

 Several solvents have been investigated for the preparation of bitter compounds of gentian roots (Gentiana lutea L.) for food applications. The highest concentrations of the bitter compounds, amarogentin and gentiopicroside, were obtained with ethanol : water 55 : 45 (v/v), propylene glycol: water 30 : 70 (v/v) and ethanol: propylene glycol: water 20 : 20 : 60 (v/v/v). Enzyme treatment prior to solvent extraction gave a greater extract yield (3.5%) but the amarogentin and gentiopicroside concentrations remained the same. The volatile fraction was affected by the solvent used through the formation of esters of organic acids from the plant. Received: 22 January 1997 / Revised version: 18 March 1997     

Inactivation of tomato pectic enzymes by manothermosonication

 The resistance of tomato pectic enzymes to manothermosonication (MTS), a combined treatment of heat and ultrasound under moderate pressure, was studied. Pectinmethylesterase (PMF) and polygalacturonases (PG) I and II were inactivated much more efficiently by MTS than by simple heating. In MTS inactivation of these enzymes, the effect of heat and ultrasonic waves was synergistic. D values [the time required for the (original) enzyme activity to decrease by 90%] for PME heat inactivation at 62.5  °C were reduced 52.9-fold by MTS and those for PG I at 86  °C and PG II at 52.5  °C, 85.8-fold and 26.3-fold, respectively. Received: 23 January 1998 / Revised version: 23 March 1998     

Effect of temperature on the cell wall composition of raisins during storage under a modified atmosphere

 Raisins obtained from seedless grapes ("Flame" variety) were kept under a modified atmosphere (MA) composed of CO₂ (60%) and N₂ (40%), and stored at 10  °C (10MA), 20  °C (20MA), 30  °C (30MA) and 40  °C (40MA). An additional sample was stored under air at 20  °C (20A). Colour, and changes in cell wall components were monitored during storage. At the end of the storage period, the 40MA and 20A samples showed a significant decrease (∼18–19%) in the yield of cell wall material (CWM), whereas less than 6% of CWM had been degraded in the 10MA sample. The decrease in CWM was mainly due to pectic polysaccharide degradation, although for 20A and 40MA samples, hemicelluloses were also affected. Throughout storage, 10MA, 20MA and 30MA samples exhibited similar CWM solubility; however, that of the 40MA sample underwent a significant decrease, from 10% to 4.5%, probably due to the formation of new pectic chains of higher molecular weight. In contrast, the CWM solubility of sample 20A increased from 10% to 15%, suggesting that MA may have promoted the inhibition of pectic-polysaccharide-degrading enzymes. In general, the combined use of relatively low temperatures and a MA helped to preserve both the colour of raisins and maintain the levels of their CWMs at values similar to initial concentrations. Received: 5 October 1998     

Inactivation of tomato pectic enzymes by manothermosonication

 The resistance of tomato pectic enzymes to manothermosonication (MTS), a combined treatment of heat and ultrasound under moderate pressure, was studied. Pectinmethylesterase (PMF) and polygalacturonases (PG) I and II were inactivated much more efficiently by MTS than by simple heating. In MTS inactivation of these enzymes, the effect of heat and ultrasonic waves was synergistic. D values [the time required for the (original) enzyme activity to decrease by 90%] for PME heat inactivation at 62.5  °C were reduced 52.9-fold by MTS and those for PG I at 86  °C and PG II at 52.5  °C, 85.8-fold and 26.3-fold, respectively. Received: 23 January 1998 / Revised version: 23 March 1998     

Applicability of lactic acid and nisin to improve the microbiological quality of cold-smoked rainbow trout

 The effect of lactic acid and nisin whey permeate on the microbiological and sensory quality of cold-smoked rainbow trout was studied. Lactic acid and whey permeate were mixed with salt solution and injected into fish fillets in concentrations of up to 2.5 g/kg lactic acid, 30 g/kg whey permeate and 20 g/kg sodium chloride. After smoking at 28  °C for 6 h the fillets were sliced, vacuum packed and stored at 3  °C. The aerobic and anaerobic/facultative anaerobic counts were measured after 1, 8, 15, 22 and 29 days of storage. The influence of the treatment on the sensory characteristics was analysed after 8 and 22 days by sensory profiling. A triangle test was used in order to ascertain at which stage the sensory quality of the samples changed during storage. Bacterial counts of smoked fish stored for 29 days were lowest in those samples containing a combination of lactic acid, sodium chloride and nisin whey permeate. Lactic acid alone did not affect the odour or flavour characteristics of the fish but did affect their texture as determined by sensory evaluation making it less elastic when stored at +3  °C for 8 days. A combination of lactic acid and nisin whey permeate enhanced the characteristic fishy flavour of the product. Differences between treatments in elasticity and fishy flavour were not detected after 22 days of storage. According to the triangle test, the samples from all treatments remained unchanged over the first 15 days. Received: 23 March 1998 / Revised version: 19 June 1998     

Improving the keeping quality of pomegranate fruit by intermittent warming

 Mollar pomegranates (Punica granatum, Punicaceae) were stored at 0  °C or 5  °C and 95% relative humidity (RH) for 80 days. Intermittent warming (IW) cycles of 1 day at 20  °C every 6 days, during which time the fruit had been stored at 0  °C or 5  °C, followed by a shelf-life period of 7 days at 15  °C and 70% RH were applied. IW during storage at 0  °C was the best treatment for maintaining the red skin colour as at harvest. However the red colour of the arils (hue angle) was kept better under warming at 5  °C. Although significant changes in the individual anthocyanins were observed in all treatments, particularly after the shelf-life period, the total anthocyanin content of the juice at harvest was maintained. While storage at 0  °C avoided decay although increased the risk of chilling injuries such us pitting and husk scald, 5  °C-storage reduced these injuries but fungal attacks were not inhibited. After the shelf-life period, IW alleviated chilling injuries without any incidence of decay. Warming treatments gave very good results with respect to storage and the keeping quality of pomegranates, particularly when applied during storage at 0  °C. Received: 10 February 1998 / Revised version: 24 April 1998     

The aminopeptidase C (PepC) from Lactobacillus helveticus CNRZ32. A comparative study of PepC from lactic acid bacteria

 The aminopeptidase C (PepC) of Lactobacillus helveticus CNRZ32 was purified by anion exchange chromatography from cell free extracts of an E. coli DH5α clone overexpressing the Lactobacillus aminopeptidase. PepC was found to have a tetrameric structure in its native form with subunits of 50 kDa each, a pH optimum of 6.5 and maximum activity at 45  °C. Sulfhydryl-blocking reagents inhibited the enzyme activity whereas reducing or metal chelating reagents had an activating effect on the PepC activity. The PepC hydrolyzed a wide range of p-nitroaniline derivatives, dipeptides and several tripeptides which contained basic amino acids (Arg, Lys), Pro residues, or cheese flavour precursor amino acids (Met, Leu, Phe) at the N-terminal position. The substrate specificity and residual activity of PepC from several lactic acid bacteria, including the PepC described above, were compared at conditions of pH and NaCl present in cheese. Received: 25 February 2000     

Chemical analysis of heterocyclic aromatic amines: the results of two European projects compared

 This report describes two studies which compared the results of the analyses of four heterocyclic aromatic amines (HAAs): 2-amino-3-methylinidazo[4,5-f]quinoline (IQ); 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx); 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), first as pure methanolic solutions and, in a second step, in a food matrice (beef extract) spiked with known amounts of these four HAAs. Details are given for the preparation of the methanolic solutions of the four HAAs and for the homogeneity and stability studies. The results of different statistical treatments revealed no significant heterogeneity within or between ampoules. The results of the stability studies clearly indicated that, except for PhIP, no effect of storage period (up to 6 months) or storage temperature (up to 25  °C), existed for the five HAAs methanolic solutions. Each of the eight European participating laboratories, which has leading experience in the analysis of HAAs, received sealed ampoules containing the pure reference solutions of the four HAAs together with a mixture of unknown identity and concentration. For the analysis of the unknwon sample, participants followed, a common work programme, but used different columns, solvent gradients and detection systems (UV, fluorescence, mass spectrometry and electrochemical detection. The analysis of the results of this first comparison revealed a good correlation between the results provided by the participants and high precision regarding the target values, independent of the experimental conditions used. For the second comparison, a common batch of commercial beef extract was prepared and spiked with known amounts of the four HAAs. The long-term stability study at –18  °C, 4  °C, 25  °C, 40  °C and 60  °C revealed high stability of these four HAAs, during up to 6 months of storage. At 40  °C and 60  °C, however, a significant loss was observed, in particular for PhIP. On the other hand, the 1-year stability study revealed that the HAAs content did not change when beef extract was stored at –18  °C. Details of these homogeneity and stability studies are provided. The sealed ampoules containing beef extract, together with the reference methanolic solutions were sent to the participants in refrigerated container. The eight European laboratories, which participated in the first comparision, adopted the work programme of this exercise. They generally followed a previously agreed upon solid-phase extraction procedure, very similar to that described by Gross [8], with analysis by HPLC. Column conditions, solvent elution and detection by UV, fluorescence, mass spectrometry and electrochemical detection varied between laboratories. The objectives of this second phase of the project were to compare and improve usual routine laboratory methods for the determination of IQ, MeIQx, 4,8-DiMeIQx and PhIP in the range of 1–30 ng/g, in a commercial beef extract. The comparision of the results revealed, however, large variations, not only beween but also within laboratories. Major difficulties were encountered by the participants, mainly for the determination of PhIP. Acceptable recovery levels were agreed between participants and different sources of variability in the extraction procedures were identified. In conclusion, whereas the analytical determination of HAAs in beef extract appeared to be satisfactory, the procedures of isolation and purification require further improvement. Received: 23 April 1998     

Chemical analysis of heterocyclic aromatic amines: the results of two European projects compared

 This report describes two studies which compared the results of the analyses of four heterocyclic aromatic amines (HAAs): 2-amino-3-methylinidazo[4,5-f]quinoline (IQ); 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx); 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (4,8-DiMeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), first as pure methanolic solutions and, in a second step, in a food matrice (beef extract) spiked with known amounts of these four HAAs. Details are given for the preparation of the methanolic solutions of the four HAAs and for the homogeneity and stability studies. The results of different statistical treatments revealed no significant heterogeneity within or between ampoules. The results of the stability studies clearly indicated that, except for PhIP, no effect of storage period (up to 6 months) or storage temperature (up to 25  °C), existed for the five HAAs methanolic solutions. Each of the eight European participating laboratories, which has leading experience in the analysis of HAAs, received sealed ampoules containing the pure reference solutions of the four HAAs together with a mixture of unknown identity and concentration. For the analysis of the unknwon sample, participants followed, a common work programme, but used different columns, solvent gradients and detection systems (UV, fluorescence, mass spectrometry and electrochemical detection. The analysis of the results of this first comparison revealed a good correlation between the results provided by the participants and high precision regarding the target values, independent of the experimental conditions used. For the second comparison, a common batch of commercial beef extract was prepared and spiked with known amounts of the four HAAs. The long-term stability study at –18  °C, 4  °C, 25  °C, 40  °C and 60  °C revealed high stability of these four HAAs, during up to 6 months of storage. At 40  °C and 60  °C, however, a significant loss was observed, in particular for PhIP. On the other hand, the 1-year stability study revealed that the HAAs content did not change when beef extract was stored at –18  °C. Details of these homogeneity and stability studies are provided. The sealed ampoules containing beef extract, together with the reference methanolic solutions were sent to the participants in refrigerated container. The eight European laboratories, which participated in the first comparision, adopted the work programme of this exercise. They generally followed a previously agreed upon solid-phase extraction procedure, very similar to that described by Gross [8], with analysis by HPLC. Column conditions, solvent elution and detection by UV, fluorescence, mass spectrometry and electrochemical detection varied between laboratories. The objectives of this second phase of the project were to compare and improve usual routine laboratory methods for the determination of IQ, MeIQx, 4,8-DiMeIQx and PhIP in the range of 1–30 ng/g, in a commercial beef extract. The comparision of the results revealed, however, large variations, not only beween but also within laboratories. Major difficulties were encountered by the participants, mainly for the determination of PhIP. Acceptable recovery levels were agreed between participants and different sources of variability in the extraction procedures were identified. In conclusion, whereas the analytical determination of HAAs in beef extract appeared to be satisfactory, the procedures of isolation and purification require further improvement. Received: 23 April 1998     

Differential lipid damage in various muscle zones of frozen hake (Merluccius merluccius)

 A comparison of the lipid damage produced in different hake zones was carried out during frozen storage at –11 and –18  °C. Three light muscle zones and the dark muscle were considered. Lipid oxidation [conjugated dienes; thiobarbituric acid index (TBA-i); fluorescence formation] and hydrolysis (free fatty acids, FFA) were determined. The most predominant lipid damage in all zones was hydrolysis, at the end of storage reaching values of about 40% (for the light muscle zones) and 12% (for the dark muscle) of the total lipids at –11  °C. Significant (P<0.05) correlation value (r=0.67–0.85) relationships between the frozen storage time and the FFA content were obtained for the four muscle zones at both temperatures. A comparison of the regression lines slopes in the different zones showed that a lower (P<0.05) lipolitic activity was produced in the dark muscle compared to the three light zones at both temperatures. A low lipid oxidation development was produced in the three light muscle parts, so that no significant differences between them could be assessed. However, the dark muscle showed a higher oxidation development (TBA-i and fluorescence formation) as a result of a higher lipid content and the presence of prooxidant constituents. Received: 16 June 1998     

Differential lipid damage in various muscle zones of frozen hake (Merluccius merluccius)

 A comparison of the lipid damage produced in different hake zones was carried out during frozen storage at –11 and –18  °C. Three light muscle zones and the dark muscle were considered. Lipid oxidation [conjugated dienes; thiobarbituric acid index (TBA-i); fluorescence formation] and hydrolysis (free fatty acids, FFA) were determined. The most predominant lipid damage in all zones was hydrolysis, at the end of storage reaching values of about 40% (for the light muscle zones) and 12% (for the dark muscle) of the total lipids at –11  °C. Significant (P<0.05) correlation value (r=0.67–0.85) relationships between the frozen storage time and the FFA content were obtained for the four muscle zones at both temperatures. A comparison of the regression lines slopes in the different zones showed that a lower (P<0.05) lipolitic activity was produced in the dark muscle compared to the three light zones at both temperatures. A low lipid oxidation development was produced in the three light muscle parts, so that no significant differences between them could be assessed. However, the dark muscle showed a higher oxidation development (TBA-i and fluorescence formation) as a result of a higher lipid content and the presence of prooxidant constituents.