INTERACTIONS BETWEEN pH AND MALIC ACID CONCENTRATION ON THE INACTIVATION OF LISTERIA MONOCYTOGENES1

The effects and interactions of malic acid concentration and pH on the inactivation kinetics of a three strain mixture of Listeria monocytogenes was studied in brain heart infusion broth (BHI). The medium was supplemented with malic acid and monosodium malate to achieve pH levels of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5 or 7.0 in conjunction with concentrations of 0.0, 0.1, 0.5, 1.0 and 2.0 M. Duplicate 20-mL portions of each pH/malate level were inoculated (ca. 10⁸ cfu/mL), stored aerobically at 28C, and assayed periodically for viable counts by plating on BHI agar. Survivor curves were generated by fitting data to a linear model that includes a lag term and used to calculate D-values and “times to 4-D inactivation”. Inactivation rates were dependent on both the pH and malic acid concentration. At the higher pH levels, malic acid appeared to provide some degree of protection compared to control cultures where the pH was adjusted with HCl. At lower pH values and at higher malic acid concentrations, a concentration-dependent anion effect was observed. The results indicate that malic acid is a relatively benign organic acid. Its antimicrobial characteristics are similar to those of citric acid and is substantially less bactericidal than lactic or acetic acids.     

Effect of Acid Pretreatment on the Stability of Ascorbic Acid Complexes with Various Iron Sources in a Wheat Flake Cereal

The effect of acid incubation of ascorbic acid with each of five iron sources (ferrous sulfate, ferric chloride, ferric orthophosphate, hydrogen and electrolytically reduced iron) on iron solubilization in a wheat flake cereal was evaluated. Incubation produced more soluble iron at pH 2 but not necessarily at the endogenous pH of the cereal nor at pH 6. At pH 2, Fe⁺² rather than Fe ⁺³ was produced, apparently by a reduction of bound Fe ⁺³ and subsequent release of Fe⁺². At pH 6, the soluble iron was mainly in a complexed form. This indicates that acid incubation with ascorbate might facilitate bioavailability of iron if it were incorporated by fortification techniques.     

Characterization of Solution Properties of Four Iron Sources in Model Systems by Solubility Studies and IR/VIS Reflectance Spectrophotometry

The hydrolysis of iron and the effect of aging on the resolubilization of iron hydroxides under simulated gastric pH conditions were measured and spectroscopy was evaluated as a predictor of these changes. Model systems of ferrous sulfate (FeZSO₄), ferric sulfate (Fe₂(SO₄)₃), ferric chloride (FeCl₃) and hydrogen-reduced elemental iron (HRFe), with and without ascorbic acid, were analyzed at intervals over 10 wk storage for both soluble iron and iron resolubilized from insoluble iron hydroxide polymers. Irreversible hydrolysis, resulting in polymers with ordered structures, occurred in the FeSO₄ and HRFe systems. Spectra of air-dried precipitates correlated with solution behavior of iron providing a possible predictive technique. Added ascorbic acid increased the levels of soluble iron in all systems.     

Malic Acid as a Source of Carbon Dioxide in Cucumber Juice Fermentations

The degradation of malic acid to lactic acid and CO₂ during fermentation of cucumber juice was investigated. This malolactic reaction was the major source of CO₂ when cucumber juice was fermented by Lactobacillus plantarum. It may also be an important CO₂ source in controlled cucumber fermentations. In addition to CO₂ production, the degradation of malate served to buffer the fermentation and increase sugar utilization. The pH after 7 days’fermentation was 2.8 when 13 mM malic acid was present in the juice and 4.1 with 135 mM malic acid. In the same fermentations, 52% of the sugars were degraded with the low malic acid concentration while complete sugar utilization was observed with the highest malic acid level.     

KINETIC STUDIES AND MECHANISM OF CATALYSIS OF MALIC DEHYDROGENASE FROM DIOSCOREA ROTUNDATA TUBER

ABSTRACT The kinetic mechanism of catalysis by malic dehydrogenase (EC 1.1.1.37) isolated from yam (Dioscorea rotundata) tuber has been delineated. Initial velocity studies with the enzyme in the presence and absence of products of the reaction revealed an ordered sequential mechanism. The K_m values obtained from secondary plots were 0.05, 0.08, 0.48 and 2.56 mM for NADH, OAA, and NAD⁺ and L-malic acid, respectively. Product inhibition studies in both the forward and backward reactions support an ordered Bi-Bi sequential mechanism. This begins with an obligatory binding of NAD⁺ to form the first binary complex and a final release of NADH.     

CONTINUOUS PROPIONIC ACID FERMENTATION OF HYDROLYZED COD (GADUS MORHUA) GURRY

Hydrolyzed cod gurry to which 4.0% glucose was added yielded a final pH of 4.5 after 96 h of incubation at 32C following inoculation with a 1.0% (v/v) inoculum (～10⁸ cells/mL) of a propionic acid producing bacterial culture. The use of a 90% (v/v) inoculum still resulted in a prolonged incubation time of 72 h to reach a pH of 4.5. A continuous propionic acid fermentation was successfully achieved for a period of 18 days with the addition of 2.5% to 4.0% glucose and a nutrient feed rate of 30 mL/h using a 500 mL fermentation vessel. A constant pH of 4.5 with an accompanying propionic acid concentration of 1.4% was maintained. Extrapolation of experimental data indicated that a glucose concentration of 2.2% is optimum.     

ACID TOLERANCE OF ESCHERICHIA COLI FOLLOWING COLD SHOCK TREATMENT

The effect of an initial cold shock treatment (2 h at 10C), following an abrupt downshift in temperature from 37 to 10C, on the subsequent growth and survival of Escherichia coli strains O157:H7 and MY20 (Biotype 1) in acidified Trypticase soy broth (TSB) and fruit juices (orange, apple) was investigated. Overall, no difference in growth at 37C was observed between each cold shocked and noncold shocked E. coli strain when cultured in TSB adjusted with either acetic acid (pH 6.0)or malic, citric and tartaric acid (each adjusted to: pH 4.5, 5.0, 5.5, 6.0). However, significant (P ± 0.05) differences in survival were observed between cold shocked and noncold shocked populations in TSB acidified with acetic acid (pH 5.0) or citric, malic and tartaric acid (pH 4.0). For strain MY20, survivor levels for cold shocked cells in TSB acidified with acetic acid citric, malic and tartaric acid at 8C were significantly (P ± 0.05) higher than in noncold shocked populations. Also, at 37C survival levels for cold shocked cells were significantly (P ± 0.05) higher than noncold shocked cells in TSB acidified with either malic or tartaric acid (pH 4.0). For the O157:H7 strain, survivor levels were higher (P ± 0.05) for cold shocked cells when maintained in TSB at 37C regardless of acid type. At 8C, cold shock treatment only increased (P ± 0.05) the survival of the O157:H7 strain in TSB adjusted with acetic acid (pH 6.0). Acid cross protection induced by cold shocking, as evidenced by enhanced survival, was not apparent for either E. coil strain in apple (pH 3.5) or orange juice (pH 3.8) maintained at 8C.     

Preparation and characteristics of yak casein hydrolysate–iron complex

The effect of mass ratio between yak casein hydrolysate and FeSO₄, reaction temperature, pH and holding time on ferrous‐binding capacity of yak casein hydrolysate was investigated. Iron‐releasing percentage and structural characteristics of the formed yak casein hydrolysate–iron complex were also examined. Results showed that casein hydrolysates with different hydrolysis degrees (DH) possessed different ferrous‐binding capacities with the highest of 7‐h hydrolysate. The optimal binding conditions between yak casein hydrolysate and iron are mass ratio of casein hydrolysate to FeSO₄ of 15:1 for 20 min at 40 °C and pH 6.0. The principal binding site for Fe²⁺ in yak casein hydrolysate consists of the carboxylate groups and amide groups. Compared with ferrous sulphate, yak casein hydrolysate–iron complex has higher iron‐releasing percentage at basic conditions, such as pH 7.0, 7.5 and 8.0. Ferrous‐iron enriched casein hydrolysate, i.e. yak casein hydrolysate–iron complex, may have great potential as an effective delivery vehicle of bioavailable iron.     

Microencapsulated Iron for Food Fortification

Lipid microcapsules of FeSO₄, alone or with ascorbic acid, and FeCl₃, were developed to fortify cheese and other high moisture foods with iron. Varying lipid coat composition and amount of core iron solution optimized their stability. A high melting fraction of milk fat (m.p. 43.5°C) was oxidized by iron and was thus unsuitable as coat material. Microcapsules made with cottonseed stearine (m.p. 62.8°C) had good oxidative stability and retained more iron under rapid stirring at 39°C than those made with hydrogenated milk fat (m.p. 49.0°C). Microcapsules having good oxidative stability and low leakage of iron were coated with stearine and had a ratio of 0.10g Fe solution/g lipid coat. Microencapsulation may allow fortification of cheese and other iron sensitive foods.     

Preparation,characterization, bioavailability in vitro and in vivo of tea polysaccharides–iron complex

The task of this study was to prepare a complex of tea polysaccharides (TPS) with ferric iron and research its bioavailability in vitro and in vivo. Optimum condition for preparing tea polysaccharides–iron complex (TPIC) was as follows: TPS and FeCl₃ with a weight ratio of 1:2.4, reacted in a water bath at 60 °C for 3 h, generating an iron content for TPIC of 14.60 %. The digestion in vitro experiment showed that availability of TPIC was sufficient. Then, iron bioavailability in vivo of TPIC was evaluated by the rat hemoglobin-repletion bioassay with ferrous sulfate (FeSO₄) as the positive control. Results indicated that at the end of 21 days’ iron regeneration phase, the values of hemoglobin (Hb) concentration, free erythrocyte protoporphyrin (FEP), serum iron (SI) concentration, and mean cell hemoglobin (MCHC) of rats supplemented with FeSO₄ and TPIC increased quickly to those of normal ones. If the bioavailability of FeSO₄ was given 100 % with Hb concentration, SI concentration and MCHC as the evaluation index, respectively, bioavailability of TPIC were in the range of 101.85–116 %. These results demonstrate that TPIC is a good iron supplement source for increasing uptake and bioavailability in the body.     

CONTROLLED PRODUCTION OF CIDER BY INDUCTION OF ALCOHOLIC FERMENTATION AND MALOLACTIC CONVERSION

Characterization experiments involving lactic bacteria allowed a strain of Leuconostoc oenos to be selected in terms of growth capacity at variable pH, temperature, ethanol and sulphite concentrations, malic to lactic conversion yield, acetic acid uptake and dextran production capacity. Cider was produced under controlled conditions where the effect of Kloeckera apiculata yeasts on the development of Saccharomyces cerevisiae and production of major non-volatile compounds influencing end product quality was studied. The apiculate yeast was found to produce large amounts of acetic acid and use the other organic acids; also, it hindered fermentation to a certain extent. A study of the effect of the inoculation time with L oenos as an inducer of malolactic fermentation revealed sequential inoculation of the lactic bacterium once most major sugars in the must had been depleted to be the most favourable. Using yeast cell walls boosted fermentation development, as well as degradation of malic acid and synthesis of succinic and acetic acid.     

Malolactic fermentation in sea buckthorn (Hippophaë rhamnoides L.) juice processing

Malolactic fermentation (MLF) traditionally used in winemaking was applied to sea buckthorn to reduce the high sourness of the berry juice. Chemical and microbiological aspects, as well as sensory properties of the juice during MLF were studied in order to develop an optimal process. In 1:1 water diluted juice with malic acid content of 15 g/l and pH 2.8, efficient conversion of l-malic acid to l-lactic acid was achieved with direct inoculation of unadapted Oenococcus oeni at a cell density of 10⁹ CFU/ml. The rapid malic acid degradation was performed by non-growing bacterial cells without loss of sugars, vitamin C, or pulp oil. More than 50% of the initial malic acid was metabolized to lactic acid and CO₂ already in 12 h of fermentation and a significant decrease in sourness and astringency was noticed. In 24 h fermentation, pH value was increased to 3.1 and only 3 g/l malic acid remained in the juice. Prolonged fermentation had only minor effect on malolactic reaction, but off-flavor of the juice began to increase.     

Selective Concentration of Bovine Immunoglobulins and α-Lactalbumin from Acid Whey using FeCl3

Immunoglobulins and α-lactalbumin of acid whey were concentrated in supernatant and precipitate when FeCl₃ was added at pH 4.2 and 2.8, respectively. Optimized conditions of pH 4.2 were preferable because of higher retention of immunochemical activity of immunoglobulins. In acid whey treated with 7.5 mM FeCl₃ at pH 4.2 and 4°C, 90% of β-lactoglobulin coprecipitated with serum albumin while 70% of immunoglobulins (92% immunochemically active IgG) and 95% of α-lactalbumin were retained in the supernatant. More than 98% of added iron was subsequently eliminated as precipitate by holding the treated whey at pH 8-9 and 4°C, without losing immunochemical activity of immunoglobulin G, in addition to retained activity of immunoglobulins A and M.     

PRESERVATION OF CABERNET SAUVIGNON GRAPE MUST SAMPLES DESTINED FOR CHEMICAL ANALYSIS: ADDITION OF SODIUM AZIDE, ALLYL ISOTHIOCYANATE, OCTANOIC ACID AND ETHYL BROMOACETATE, AND EFFECT OF PASTEURIZATION

Grape must samples destined for chemical analysis can usually be preserved just for a short period of time. As a consequence of the addition of antifermentative substances, or heat treatment, the analytical parameters of must may change. In this work, five different methods of inhibiting endogenous yeast and bacteria in Cabernet Sauvignon grape must were studied. Sodium azide (NaN₃), allyl isothiocyanate, octanoic acid and ethyl bromoacetate were added to samples at various concentrations, and samples were pasteurized by three different methods. The effects of treatments were monitored with respect to soluble solids content, malic (H₂M) and tartaric (H₂T) acids, pH and the titratable acidity of samples. Allyl isothiocyanate and ethyl bromoacetate (100 mg/L) preserved samples for 6 months, NaN₃ did not prevent the degradation of H₂M and H₂T and pasteurization of sample and octanoic acid did not preserve the samples.     

CINNAMIC ACID AS THE BASIS OF A MEDIUM FOR THE DETECTION OF WILD YEASTS

Cinnamic acid (100 μg ml^?1) incorporated in a solid medium was found to inhibit the growth of brewing strains (Pof^?) of yeast while permitting the growth of Pof+ wild yeast contaminants. Typically, colonies of Saccharomyces cerevisiae var. diastaticus (Pof+) mixed with brewing yeast (S. cerevisiae NCYC 240) were visible after 5d incubation at 25°C. The incubation time required to detect a selection of brewery wild yeast isolates was found to vary from 3–12 d.     

Effect of tea phenolics on iron uptake from different fortificants by Caco-2 cells

The in vitro effects of tea phenolics on Fe uptake from different fortificants (FeSO₄, FeCl₃, FeEDTA) by Caco-2 cells were compared. Cell cultures were exposed to catechin, tannic acid, green or black tea solutions, added within Fe-containing solution, or used to pre-treat cell cultures before Fe-exposure. Cell ferritin formation was used as a measure of Fe uptake. Reverse phase chromatography was used to identify specific phenolics in tea solutions, and the Fe-binding catechol and galloyl groups were determined spectrophotometrically. The results showed a positive effect of catechin on Fe uptake only from dissociable Fe sources, and a marked inhibitory effect of tannic acid regardless of the Fe source. Tea phenolics exhibit similar inhibitory patterns on Fe uptake from FeCl₃ and FeEDTA solutions; however, the Fe uptake from FeSO₄ solutions was significantly less affected. These data improve the understanding of interactions by which tea phenolics affect Fe uptake at the intestinal level.     

Evaluation of iron‐fortified Feta cheese for physicochemical and sensory properties

Standardised cow's milk (fat 3 g/100 g) was used to manufacture Feta cheese fortified with 40, 60 and 80 mg of iron/kg cheese using ferrous sulphate (FeSO₄), ferric chloride (FeCl₃), ferric pyrophosphate (Fe₄ (P₂O₇)₃) and microencapsulated ferrous sulphate. Chemical composition and sensory characteristics of fortified cheeses were determined after 60 days of ripening, during which the iron content and thiobarbituric acid (TBA) values were measured. The metallic taste, colour, flavour, overall score and TBA values were statistically (P < 0.05) affected by the source and concentration of iron. The best quality was found in cheeses fortified with 40 mg/kg of microencapsulated ferrous sulphate.     

INFLUENCE OF EXTRACTANT ON L-ASCORBIC ACID RECOVERY FROM SELECTED FOODS AND BEVERAGES

Recovery of L-ascorbic acid from selected vegetables, beverages and cereal was determined using up to 3 metaphosphoric acid-containing reagents, 3% trichloroacetic acid (TCA), and cold 0.05 Mperchloric acid (HCIO₄). Stability of ascorbic acid in these extractants was monitored over a 4 h holding period. All extractants yielded similar results for the beverages, however, TCA extracts of spinach and HCIO, extracts of tomatoes and cereal produced lower results than the other extractants. L-ascorbic acid content of green beans extracted with HClO₄, TCA, and 3% HPO₃, - 8% HOAC diffed from their initial values at 2, 4, and 4 h, respectively.     

Oxidative stability of oil-in-water emulsions containing iron chelates: Transfer of iron from chelates to milk proteins at interface

Iron supplementation can promote oxidation of food matrices as well as cell lipids. The oxidative stability of oil/water emulsions stabilised by β-lactoglobulin (BLG) or sodium caseinate (SC) was studied in the presence of Fe-bisglycinate, NaFe-EDTA or FeSO₄. Lipid oxidation was evaluated by following the peroxide value (PV) and the thiobarbituric acid reactive substances (TBARS) over 7 days. At pH 6.5, for Fe-bisglycinate iron complement, the oxidation kinetics was more reduced with BLG than with SC. Contrarily to BLG, SC possesses phosphate groups that have more affinity for iron ions than carboxylate residues. Both BLG and SC stabilised emulsions were more oxidised with Fe-bisglycinate or FeSO₄ than with NaFe-EDTA. At pH 3.5, lipid oxidation was higher compared to pH 6.5. These results indicate that the competition for iron complexation between functional groups of protein and salt counter-ions (glycinate, sulphate or EDTA) appear as a key factor in oxidation.     

IRON SOLUBILITY FROM SEAFOODS WITH ADDED IRON AND ORGANIC ACIDS UNDER SIMULATED GASTROINTESTINAL CONDITIONS

Iron absorption from foods is dependent on the type of food consumed and other factors such as ligands which may have enhancing or inhibiting effects on iron absorption. In this study, iron (Fe²⁺ or Fe³⁺) and two organic acids (ascorbate and citrate) were added to three seafood surimis (scallops, prawns, and cod) and then subjected to simulated gastrointestinal conditions (pH 2, 4, and 6) to evaluate iron solubility. Soluble iron decreased with increasing pH in all samples and control. In cod, a combination of ascorbic and citric acid (1:2) was most effective to increase soluble iron; however, in scallops and prawns citric acid alone was best. After sequential pH and enzyme treatments, it was found that the effectiveness of iron solubilization depended primarily upon the type of seafood present and to a lesser extent the oxidation state of the iron salt.