Immobilization of β-galactosidase by bioaffinity adsorption on concanavalin A layered calcium alginate–starch hybrid beads for the hydrolysis of lactose from whey/milk

Aspergillus oryzae β-galactosidase was immobilized on the surface of a novel bioaffinity support: concanavalin A layered calcium alginate–starch beads. The maximum activity of the immobilized β-galactosidase was obtained at 60 °C, approximately 10 degrees higher than that of the free enzyme. The immobilized β-galactosidase exhibited significantly higher stability to heat, urea, MgCl₂, and CaCl₂ than the free enzyme. An enhancement of the activity of immobilized β-galactosidase by up to 5.0% MgCl₂ was seen, whereas the activity of the free enzyme decreased above 3.0% MgCl₂. Immobilized β-galactosidase retained 61%, 50% and 43% activity in the presence of 5% CaCl₂, 5% galactose and 4 m urea, respectively, when incubated for 1 h at 37 °C. The immobilized β-galactosidase had a much higher K_iapp value than the free enzyme, which indicated less susceptibility to product inhibition by galactose. The immobilized β-galactosidase preparation was superior to the free enzyme in hydrolysing lactose in whey or milk in a batch process: it hydrolyzed 89% of the lactose in whey in 3 h and 79% of the lactose in milk in 4 h. The immobilized β-galactosidase retained 61% of its original activity after 2 months storage at 4 °C, while the soluble enzyme showed only 37% of the initial activity under identical conditions.     

Purification and characterization of the 3-phosphoglycerate kinase from the thermophile Lactobacillus delbrueckii subsp. lactis

The 3-phosphoglycerate kinase (PGK) of the thermophile Lactobacillus delbrueckii subsp. lactis was purified to homogeneity and found to be a monomeric enzyme with a molecular weight of 45 kDa. PGK is a Michaelis–Menten type enzyme with a K_m=0.7 mm for ATP and a K_m=2.6 mm for 3-phosphoglycerate. pH tolerance of PGK was found to be oriented towards acidic conditions with an optimum at pH 6.8 and a plateau of 90% of the total activity between pH 6.0 and 7.4. The optimum temperature for PGK activity is 45°C, which is consistent with the value expected for a thermophilic bacterium with an optimal growth temperature of 45°C.     

High-pressure homogenisation of raw bovine milk. Effects on fat globule size distribution and microbial inactivation

Whole raw milk was processed using a 15 L h⁻¹ homogeniser with a high-pressure (HP) valve immediately followed by a cooling heat exchanger. The influence of homogenisation pressure (100–300 MPa) and milk inlet temperature T_in (4°C, 14°C or 24°C) on milk temperature T₂ at the HP valve outlet, on fat globule size distribution and on the reduction of the endogenous flora were investigated. The T_in values of 4–24°C led to milk temperatures of 14–33°C before the HP valve, mainly because of compression heating. High T_in and/or homogenisation pressure decreased the fat globule size. At 200 MPa, the d_4.3 diameter of fat globules decreased from 3.8±0.2 (control milk) to 0.80±0.08 μm, 0.65±0.10 or 0.37±0.07 μm at T_in=4, 14°C or 24°C, respectively. A second homogenisation pass at 200 MPa (T_in=4°C, 14°C or 24°C) further decreased d_4.3 diameters to about 0.2 μm and narrowed the size distribution. At all T_in tested, an homogenisation pressure of 300 MPa induced clusters of fat globules, easily dissociated with SDS, and probably formed by sharing protein constituents adsorbed at the fat globule surface. The total endogenous flora of raw milk was reduced by more than 1 log cycle, provided homogenisation pressure was ⩾200 MPa at T_in=24°C (T₂∼60°C), 250 MPa at T_in=14°C (T₂∼62°C), or 300 MPa at T_in=4°C (T₂∼65°C). At all T_in tested, a second pass through the HP valve (200 MPa) doubled the inactivation ratio of the total flora. Microbial patterns of raw milk were also affected; Gram-negative bacteria were less resistant than Gram-positive bacteria.     

Influence of ohmic heating/osmotic dehydration treatments on polyphenoloxidase inactivation,physical properties and microbial stability of apples (cv. Granny Smith)

The combined effect of ohmic heating (OH) and osmotic dehydration (OD) with vacuum impregnation (VI), on the polyphenoloxidase (PPO) inactivation, physical properties and microbial stability of apples stored at 5 °C or 10 °C was analyzed. The treatments were performed using a 65% (w/w) sucrose solution and with ohmic heating at 13 V/cm at 30 °C, 40 °C or 50 °C for 90 min. Examination of the dehydrated samples showed that the water loss and the solid gain were greater with the OD/OH and VI/OH treatments at 50 °C. PPO was completely inactivated by the OD/OH and VI/OH treatments at 50 °C. There was a correlation between the PPO activity, the color change and the browning index of the treated and stored samples; the values for these parameters were stable when PPO was inactivated. The lowest loss of firmness and color was obtained with the VI/OH treatment at 50 °C. The shelf-life of the apples treated with VI/OH at 50 °C and stored at 5 °C was extended to more than 4 weeks. Therefore, the VI/OH treatment at 50 °C was determined to be the best process for dehydrating apples.Industrial relevanceThe aim of this research was 1 to study the combined effect of ohmic heating (OH) and osmotic dehydration (OD) with vacuum impregnation (VI) on the polyphenoloxidase inactivation and microbial stability of osmotically dehydrated apples stored at either 5 °C or 10 °C. Two technologies, OH and OD were performed at 30, 40 or 50 °C with an electric field intensity of 13 V/cm and conventional heating for 90 min. The results showed a correlation between the PPO activity, the color change and the browning index of the treated and stored samples; the values were stable when PPO was inactivated. PPO was completely inactivated by the OD/OH and VI/OH treatments at 50 °C. The shelf-life of the apples treated was extended to more than 4 weeks. Under the investigated conditions, VI/OH treatment at 50 °C and stored at 5 °C may be considered the better minimal processing that preserves the fresh-like properties.     

Growth of Listeria Monocytogenes in Traditional Austrian Meat Jelly Products

Individual components of a traditional meat jelly (cooked meat chunks, gelatin and preboiled vegetable) with differences in pH and a_w can constitute a niche for the multiplication of Listeria. Listeria monocytogenes counts remained stable in jelly over 21 days at 2 and 8 °C, whereas in meat and vegetables, a >1 log₁₀ unit increase was observed after 7 days at 2 °C (or >5 log₁₀ at 8 °C). In the composed product, Listeria numbers remained stable at 2 °C (21 days), but increased more than 1 log₁₀ during 7 days at 8 °C. Improving safety of jellied meat by lowering pH is discussed.     

Chymotrypsin from the hepatopancreas of cuttlefish (Sepia officinalis) with high activity in the hydrolysis of long chain peptide substrates: Purification and biochemical characterisation

Chymotrypsin from the hepatopancreas of cuttlefish (Sepia officinalis) was purified to homogeneity, with a 120-fold increase in specific activity and 23% recovery. The molecular weight of the purified chymotrypsin was estimated to be 28 kDa by sodium dodecyl sulphate–polyacrylamide gel electrophoresis. The optimum pH and temperature for the chymotrypsin activity were pH 8.5 and 55 °C, respectively, using succinyl-l-ala-ala-pro-l-phenylalanine-p-nitroanilide (SAAPFpNA) as a substrate. The enzyme was extremely stable in the pH range of 7.0–10.0 and highly stable up to 50 °C after 1 h incubation. This proteinase was strongly inhibited by chymostatin, soybean trypsin inhibitor, diisopropylfluorophosphate and phenylmethylsulfonyl fluoride, but was not inhibited by tosyl-l-phenylalanine chloromethyl ketone, N-carbobenzoxy-phenylalanine chloromethyl ketone or N_α-tosyl-l-lysine chloromethyl ketone. The enzyme hydrolysed long chymotrypsin peptide substrates SAAPFpNA, SAAPLpNA and ZAALpNA and did not hydrolyse short chymotrypsin substrates. Kinetic parameters of the enzymatic reaction demonstrated that the best substrate was SAAPFpNA, with k_cat 18 s^?1 and K_m 22 μM. However, the enzyme had a lower K_m for SAAPLpNA, 54 μM.The N-terminal amino acid sequence of the first 20 amino acids of the purified chymotrypsin was IVGGQEATIGEYPWQAALQV.     

Effect of modified atmosphere packaging,storage period,and storage temperature on the residual nitrate of sliced-pastırma,dry meat product,produced from fresh meat and frozen/thawed meat

The amount of nitrite in sliced-pastirma made, from fresh or frozen (which was stored at −18 °C for 240 days and then thawed at 10 °C for 24 h) M. Longissimus dorsi muscle was determined. Sliced-pastirma samples were stored in modified atmosphere packages (50% N₂ + 50% CO₂) at 4 and 10 °C for 150 days, and the amount of residual nitrite was measured after 0, 30, 60, 90, and 150 days of storage. The residual nitrite of pastirma samples made with frozen/thawed meat was higher than that of the pastirma made from fresh meat at both 0 day and at the end of the storage (150 days). The storage temperature (p < 0.01), storage period (p < 0.01) and the storage period × the storage temperature interaction (p < 0.01) had significant effects on the amount of the residual nitrite.     

X-ray diffraction analysis of lactose crystallization in freeze-dried lactose–whey protein systems

Water sorption, time-dependent crystallization and XRD patterns of lactose and lactose–WPI mixtures were studied with glass transition data. The results indicated that the sorbed water of lactose–WPI mixtures was fractional and water content of individual amorphous components in lactose–WPI mixtures at each a_w from 25 °C to 45 °C could be calculated. Crystallization occurred in pure lactose whereas partial crystallization was typical of lactose–WPI mixtures (protein content ≤ 50%) at intermediate and high a_w (> 0.44 a_w) from 25 °C to 45 °C. The extents of crystallization were significantly delayed by WPI. The T_g values of lactose–WPI systems showed the composition-dependent property in systems and might indicate the occurrence of phase separation phenomena during 240 h storage. XRD showed no anhydrous β-lactose and mixed α-/β-lactose with molar ratios of 4:1 crystals in crystallized lactose–WPI systems (70:30 and 50:50 solids ratios). Reduced crystallization in the presence of WPI was more pronounced possibly because of reduced nucleation and diffusion during crystal-growth. The present study showed that WPI could present an important role in preventing sugar crystallization.     

Effects of high pressure treatment on indigenous enzymes in bovine milk: Reaction kinetics,inactivation and potential application

High pressure-induced inactivation of the indigenous milk enzymes alkaline phosphatase (ALP), γ-glutamyltransferase (GGT) and phosphohexoseisomerase (PHI) was studied in the pressure range 400–800 MPa at temperatures between 5 and 40 °C. With respect to pressure stability the following ranking was observed: ALP>GGT>PHI. PHI was inactivated after pressure treatment at 500 MPa and 20 °C for 10 min. In terms of reaction kinetics, inactivation of GGT followed first-order reaction kinetics in the range of 400–800 MPa whereas a reaction order of 1.5 was found for ALP. Reactivation of pressure-treated ALP was observed at low enzyme activity resulting from severe pressure treatment and 2 h storage at 35 °C. The influence of process temperature on the pressure-induced inactivation of GGT and ALP was limited in the range 5–40 °C.     

Biochemical characterisation of an aminopeptidase with highest preference for lysine from Japanese flounder skeletal muscle

An aminopeptidase was purified from Japanese flounder skeletal muscle to homogeneity by ammonium sulphate fractionation and three chromatographies. The enzyme was approximately 100 kDa with isoelectric point of 5.7 as estimated by two-dimensional polyacrylamide gel electrophoresis. Its optimum temperature and pH were 45 °C and 7.5, respectively. According to peptide mass fingerprinting study, the enzyme revealed high identity to a puromycin-sensitive aminopeptidase. It had a broad specificity toward aminopeptidase substrates and preferred to hydrolyse Lys-MCA with k_cat/K_m of 8.1 × 10⁶ M^?1 s^?1, and the activation energy (E_a) of 72.5 kJ M^?1. Metal-chelating agents effectively inhibited the enzyme activity, and Zn²⁺, Mn²⁺ and Co²⁺ significantly restored the apoenzymatic activity dialysed by EDTA, whilst inhibitors to other proteinases did not show much effect. Furthermore, bestatin strongly inhibited its activity. These results indicate that the purified enzyme is a metalloaminopeptidase which would possibly contribute to free amino acids increase in fish muscle.     

Inactivation of Escherichia coli O157:H7 and Listeria monocytogenes in milk by caprylic acid and monocaprylin

Listeria monocytogenes and Escherichia coli O157:H7 are major foodborne pathogens implicated in various outbreaks involving pasteurized or unpasteurized milk, and various dairy products. The objective of this study was to determine the antibacterial effect of caprylic acid (CA, C_8:0) and its monoglyceride, monocaprylin (MC) on L. monocytogenes and E. coli O157:H7 in whole milk. A five-strain mixture of E. coli O157:H7 or L. monocytogenes was inoculated in autoclaved milk (10⁶ CFU/ml) containing 0, 25, or 50 mM of CA or MC. At 37°C, all the treatments, excepting 25 mm CA, reduced the population of both pathogens by approximately 5.0 log CFU/ml in 6 h. At 24 h of storage at 8°C, MC at both levels and CA at 50 mM decreased L. monocytogenes and E. coli O157:H7, respectively by >5.0 log CFU/ml. At 48 h of 4°C storage, populations of L. monocytogenes and E. coli O157:H7 were decreased to below detection level (enrichment negative) by 50 mm of MC and CA, respectively. Results indicate that MC could potentially be used to inhibit L. monocytogenes and E. coli O157:H7 in milk and dairy products, but sensory studies need to be conducted before recommending their use.     

Effect of heat shock on thermal tolerance and susceptibility of Listeria monocytogenes to other environmental stresses

In the present study, Listeria monocytogenes Scott A, V7 and CCRC 14930 were first subjected to heat shock at 45°C for 1 h or at 48°C for 10 min. Thermal tolerance at 55°C and survival of the heat shocked as well as non-heat shocked cells of L. monocytogenes in the presence of 25% NaCl, 0.01% crystal violet, 0.1% H₂O₂, and 18% ethanol were examined.It was found that heat shock response of L. monocytogenes varied with strains, the condition of heat shock treatment and type of subsequent stress. Compared with the non-heat shocked cells, the 45°C-1 h heat shocked cells of strains Scott A and V7 showed an increased survival after exposure to 55°C for 60 min. Meanwhile, survival of the 48°C-10 min heat shocked L. monocytogenes cells, regardless of strain, exhibited no significant difference (p>0.05) with their respective control cells. Generally, heat shocking at 45°C for 1 h increased the tolerance of L. monocytogenes to NaCl, ethanol and crystal violet. On the other hand, heat shocking at 48°C for 10 min although increased the resistance of L. monocytogenes to NaCl reduced its resistance to H₂O₂ and crystal violet.     

Enzymatic synthesis of banana flavour (isoamyl acetate) by Bacillus licheniformis S-86 esterase

New carboxylesterase from organic-solvent-tolerant Bacillus licheniformis S-86 strain was characterized. The enzyme named as type II esterase showed an optimal activity in the temperature range 60–65 °C and pH 8.0. The enzyme was moderatly thermostable (half-life of 1 h at 50 °C), but remarkable stable at extremely alkaline pH, retaining 100% of its activity at pH 10.0–11.0. Furthermore, the esterase showed high stability in detergents (86% residual activity in 10% SDS), and also 0.1% ionic and non-ionic detergents are inducers of enzyme activity. PMSF, a serine protease inhibitor, did not show any effect on the activity. The immobilized type II esterase was able to synthesize isoamyl acetate from isoamyl alcohol and p-nitrophenyl acetate (acyl donor) in n-hexane. The resulting ester yield (42.8%), obtained at a low temperature (28 °C) and with a very low amount of enzyme (4.6 × 10^?5 mg ml^?1), indicates a high potential for type II esterase in isoamyl acetate synthesis for production purposes.     

Destruction of Salmonella Enteriditis inoculated onto raw almonds by high hydrostatic pressure

The effects of continuous (50,000, 60,000 and 70,000 psi with holding times of 5 and 10 min) and discontinuous (oscillatory) (six cycles at 60,000 psi with a holding time of 20 s) high hydrostatic pressure (HHP) treatments on the viability of two Salmonella Enteriditis strains (FDA and PT30) inoculated onto raw almonds were evaluated at 25, 50, and 55 °C. Complete inactivation of the S. Enteriditis was achieved in 0.1% peptone water after continuous pressurization at 60,000 psi and 25 °C for 5 min. Continuous pressurization of raw almonds inoculated with S. Enteriditis at 60,000 psi and 50 °C for 5 min resulted in less than a log reduction (log₁₀ 0.83) of vegetative cells. The decimal reduction time using the continuous pressurization parameters was determined to be 9.78 min. A discontinuous process consisting of six cycles of pressurization at 60,000 psi and 50 °C for 20 s provided greater than a one log reduction (log₁₀ 1.27 for FDA and log₁₀ 1.16 for PT30) of the S. Enteriditis concentration. The low water activity (a_w) of the almonds was found to impart baroprotective attributes on the S. Enteriditis cells. When the almonds were directly suspended in water and then pressurized, a log₁₀ reduction of 3.37 was achieved. HHP of certain dry foods appears to be feasible if the food is directly suspended in the pressurizing medium (water).     

Effect of preheating temperature and calcium ions on the properties of cold-set soybean protein gel

To elucidate the effect of preheating temperature and calcium ions on the properties of cold-set soybean protein gel, Ca²⁺ induced gelation of soybean protein were investigated by rheological approaches, electrophoresis analysis, confocal scanning laser microscopy (CSLM) and surface hydrophobicity (S₀). The results showed that, both CaCl₂ concentration (20–40 mM) and preheating temperature (80–120 °C) took significant influence on the dynamic viscoelasticity of the gel samples. The bands distribution of samples preheated at 120 °C were different from the bands distribution of samples preheated below 100 °C in denature and native electrophoresis. The CSLM analysis showed that the gel became coarser as the Ca²⁺ increased. On the other hand, two different kinds of gel were shown under the same Ca²⁺ concentration: a bead-like structure (below 100 °C) and a filamentous structure (above 100 °C). According to fractal theory, weak-linked gel (α was >0.8) was formed when preheating below 100 °C, while a transition gel (α = 0.52–0.62) was obtained when preheating over 100 °C. All these results suggested that the preheating temperature influence the type of cold-set gel.     

Mechanical and thermal characteristics of amaranth starch isolated by acid wet-milling procedure

Effects of soaking conditions during acid wet-milling of amaranth grain on mechanical and thermal properties of amaranth starch were investigated. A factorial design based on temperature (40–60 °C) and SO₂ concentration (0.1–1.0 g/L) was used. Dynamic oscillatory tests involved heating and cooling cycles (25–90 °C) with tempering at 90 °C followed by a frequency sweep (0.1–20 Hz) at constant temperature. Thermal properties of starch suspensions were based on DSC tests. Viscoelastic modulus and thermal properties were affected by both factors, being significant the interaction effect. Maximum values of viscoelastic modulus at the end of heating and cooling steps were determined for samples corresponding to soaking conditions of 50 °C and 1.0 g/L SO₂. Based on Ross-Murphy index from frequency sweep analysis it was found that paste behavior occurs at 60 °C and 0.1 g/L, while at 47.4 °C and 0.72 g/L starch suspension gave a strong gel. Onset (61.7 °C) and peak (66.2 °C) temperatures showed a minimum at 50 °C and 1.0 g/L. As SO₂ concentration decreased, the end of gelatinization took place at lower temperature. Maximum gelatinization enthalpy (12.7 J/g) was found at 40 °C and 0.1 g/L SO₂.     

Thermal and high-pressure stability of purified polygalacturonase and pectinmethylesterase from four different tomato processing varieties

Polygalacturonase (PG) and pectinmethylesterase (PME) were extracted and purified from four tomato varieties (Galeón, Malpica, Perfectpeel and Soto) used in the processing industry. The processing stability (thermal and high pressure) of PG and PME from the four varieties was analyzed, and they all showed the same behavior. PG was present in two isoforms, PG1 (inactivated at 90 °C, 5 min) and PG2 (inactivated at 65 °C, 5 min). In contrast, PG1s and PG2s showed the same pressure stability, both can be inactivated at room temperature in the pressure range of 300–500 MPa. On the other hand, purified PMEs could be thermally inactivated (5 min, 70 °C) but 50% of its activity remained after high-pressure treatment (850 MPa, 15 min, 25 °C). High pressure processing can thus be used for selective inactivation of PG in tomato processing (while keeping PME intact). This fact could open prospectives for improving texture/rheology of processed tomato based products; however further research in the texture/rheology area is needed.     

Extraction of boldo (Peumus boldus M.) leaves with supercritical CO2 and hot pressurized water

High-activity fractions in boldo leaves were extracted with supercritical CO₂ (SC-CO₂) and hot pressurized water (HPW). Total yield after 3 h of extraction (0.6–3.5%) in low-pressure SC-CO₂ experiments increased with process pressure (60–150 bar) and decreased with temperature (30–60 °C), as expected. The extract obtained with SC-CO₂ at 50 °C and 90 bar contained approximately 50% of essential oil components. In higher pressure experiments with solvent mixtures, the yield increased with pressure (300–450 bar) and modifier concentration (2–10% ethanol), ranging 0.14–1.95 ppm for the alkaloid boldine and 1.8–4.8% for total solids following 1.5 h treatment at 50 °C. Boldine recovery was solubility-controlled, reaching 7.4 ppm after 7-h extraction at 450 bar and 50 °C using an ethanol–SC-CO₂ mixture (5% co-solvent). Boldine solubility and yield decreased when using pure CO₂ at higher pressure (600 bar, 50 °C). The extract yield after 3 h batch-wise HPW extraction increased from 36.9% at 100 °C to 53.2% at 125 °C, and then decreased as temperature was increased to 175 °C. Boldine yield decreased from 26.8 ppm at 100 °C to 0.7 ppm at 125 °C, and was negligible at ⩾150 °C. The essential oil yield increased to a maximum at 110 °C and was negligible at ⩾150 °C also. The ranking of antioxidant potency of various extracts was as follows: HPW at 110 °C > methanol (soxhlet extraction) ≫ high-pressure SC-CO₂ with or without polar co-solvent > low-pressure SC-CO₂.     

High pressure carbon dioxide combined with high power ultrasound processing of dry cured ham spiked with Listeria monocytogenes

Traditionally, thermal treatments for the inactivation of Listeria monocytogenes in meat products involve undesirable changes of the product quality. In recent years, efforts have been carried out to develop alternative methods to inactivate L. monocytogenes without affecting the product quality attributes. In this context, the feasibility of combined high pressure carbon dioxide and high power ultrasound (HPCO₂ + HPU) treatment to inactivate L. monocytogenes inoculated on the surface of dry cured ham was investigated. Inactivation data were determined at 6, 8 and 12 MPa, as a function of temperature (22, 35, 45 °C) and treatment time (0.5 to 30 min), and compared to those obtained after thermal and HPCO₂ treatments.Color, pH and acidity changes of the samples after both thermal and HPCO₂ + HPU treatments were measured and compared, sensorial profile of the treated samples was evaluated by a sensory panel and shelf-life was determined by a storage study for 4 weeks at 4 °C.The results clearly revealed that HPU alone was not able to induce any microbial inactivation while HPCO₂ + HPU treatment always assured a certain level of inactivation, variable with the process temperature used: the inactivation efficiency was demonstrated higher at 35 °C rather than 22 °C and no enhancement was observed at 45 °C compared to 35 °C. Process conditions of 12 MPa, 35 °C, at 10 W for 5 min assured inactivation to undetectable level of L. monocytogenes spiked on the surface of the product with an initial concentration of about 10⁹ CFU/g. No differences were detected between acidity, pH, color and sensory attributes of the untreated and HPCO₂ + HPU treated dry cured ham surface, while slight differences were measured between the values obtained for the untreated and thermally treated samples. Additionally, the storage study demonstrated that a full microbial and quality shelf-life was assured for 4 weeks at 4 °C. The results obtained may open the doors to the application of such an innovative process at industrial level, in particular to treat ham-type or meat products.     

Effect of mild heat pre-treatment with alkaline electrolyzed water on the efficacy of acidic electrolyzed water against Escherichia coli O157:H7 and Salmonella on Lettuce

Cut lettuce dip-inoculated with Escherichia coli O157:H7 and Salmonella was treated with alkaline electrolyzed water (AlEW) at 20°C for 5 min, and subsequently washed with acidic electrolyzed water (AcEW) at 20°C for 5 min. Pre-treatment with AlEW resulted in an approximate 1.8 log₁₀ cfu/g reduction of microbial populations, which was significantly (p⩽0.05) greater than microbial reductions resulting from other pre-treatment solutions, including distilled water and AcEW. Repeated AcEW treatment did not show a significant bacterial reduction. Mildly heated (50°C) sanitizers were compared with normal (20°C) or chilled (4°C) sanitizers for their bactericidal effect. Mildly heated AcEW and chlorinated water (200 ppm free available chlorine) with a treatment period of 1 or 5 min produced equal reductions of pathogenic bacteria of 3 log₁₀ and 4 log₁₀ cfu/g, respectively. The procedure of treating with mildly heated AlEW for 5 min, and subsequent washing with chilled (4°C) AcEW for period of 1 or 5 min resulted in 3–4 log₁₀ cfu/g reductions of both the pathogenic bacterial counts on lettuce. Extending the mild heat pre-treatment time increased the bactericidal effect more than that observed from the subsequent washing time with chilled AcEW. The appearance of the mildly heated lettuce was not deteriorated after the treatment. In this study, we have illustrated the efficacious application of AlEW as a pre-wash agent, and the effective combined use of AlEW and AcEW.