Effect of water activity on the thermal stability of Thermomyces lanuginosus xylanases for process time–temperature integration

Three strains of the thermophilic fungus Thermomyces lanuginosus were used to produce β-xylanases. The thermal stability of these xylanases at low levels of water activity was studied. Isothermal inactivation experiments were performed in the temperature range of 100–130 °C. Reduction of water activity to 0.63 and as low as 0.13 had a drastic effect on the observed D and z-values. At water activity of 0.13 the D_120 °C and z-values of the three xylanases ranged from 20.4 to 37.6 min and from 23.3 to 28.9 °C, respectively. The applicability of the developed kinetic models was tested under time–temperature profiles representative of typical thermal processes. The developed systems can be applied as time–temperature integrators (TTI) at this high thermal processing range. Calculations demonstrated that the use of a triple xylanase TTI system could provide acceptable F-values prediction for z-values lower than the achieved range.     

Thermal inactivation of Salmonella spp. during conching

Although chocolate is a microbiologically stable product it has been described as a vehicle for Salmonella spp. Because of the low water activity (a_w) and the high fat content of chocolate Salmonella spp. shows an increased heat resistance, even during the thermal process of chocolate making. The aim of this study was to evaluate the thermal inactivation of Salmonella spp. during conching in various masses of chocolate and cocoa butter at different temperatures (50-90 °C). The effect of thermal treatment on Salmonella spp. was determined with the MPN (Most-Probable-Number) method. Results of thermal treatment showed approximate D-values for cocoa butter from D_50°C = 245 min to D_60°C = 306 min, for cocoa liquor from D_50°C = 999 min to D_90°C = 26 min and for dark chocolate of D_50°C = 1574 min. z-values were found to be z = 20 °C in cocoa liquor and z = 14 °C in dark chocolate. This study demonstrates that the conching process alone does not ensure the inactivation of Salmonella spp. in different chocolate masses and that an additional decontamination step at the beginning of the process as well as an HACCP concept is necessary during chocolate production to guarantee the absence of Salmonella spp. in chocolates and related products.     

Inactivation kinetics of Bacillus spores in batch- and continuous-heating systems

Kinetic parameters for the thermal inactivation of Geobacillus stearothermophilus ATCC 7953 and Bacillus flexus 1316 spores were determined for temperatures ranging from 100 to 130 °C and 100 to 125 °C, respectively. Ringer's solution (pH = 7.1) was used as the heating medium. A batch-heating system, in which the samples are kept in small tubes that are heated with steam, and a continuous-heating system, which is based upon a heat exchanger and enables high temperature short time heating, were used. Experiments were conducted in both systems and the heat resistances of the two species were determined. Additionally, a comparison of the two heating systems was carried out. The reaction rate constant at the reference temperature, k_ref, the activation energy, E_a, the D-value and the z-value were calculated for the two species. The D-values at 121 °C for G. stearothermophilus and for B. flexus in the batch-heating system were found to be 42 and 4.2 s, respectively. The z-values were calculated as 13 K for G. stearothermophilus and 16 K for B. flexus in the batch-heating system. The results from both systems differed significantly, wherein the continuous-heating system had been more lethal than the batch-heating system.     

Heat resistance of Listeria species to liquid whole egg ultrapasteurization treatment

The lethality of ultrapasteurization treatments (70 °C/1.5 min.) applied at constant temperature (isothermal condition) and at a constantly raising temperature of 2 °C/min (non-isothermal condition) in liquid whole egg (LWE) against two strains of Listeria monocytogenes (STCC 5672 and 4032) and one of Listeria innocua has been investigated. Isothermal survival curves up to 71 °C were obtained, which followed first-order inactivation kinetics. The obtained D_t values indicated that L. innocua was significantly (p < 0.05) more heat resistant than L. monocytogenes strains. Non-significant (p > 0.05) differences were observed among z values (12.4 ± 0.4 °C, 13.1 ± 0.4 °C and 12.2 ± 0.7 °C for L. innocua and L. monocytogenes 5672 and 4032, respectively). Based on obtained D_t and z values, isothermal ultrapasteurization treatment (70 °C/1.5 min.) would provide 3.5-, 5.0-, and 6.5-Log₁₀ cycles of L. innocua and L. monocytogenes 5672 and 4032, respectively. Non-isothermal heating lag phase increased the thermotolerance of Listeria species in LWE. The simulated industrial pasteurization treatment for LWE (heating-up phase from 25 to 70 °C followed by 1.5 min. at 70 °C) would attain 5-Log₁₀ reductions of L. monocytogenes 5672 and 4032, and 3.7-Log₁₀ reductions of L. innocua. Therefore, the safety level of industrial ultrapasteurization concerning L. monocytogenes could be lower than that estimated with data obtained under isothermal conditions.     

Thermal inactivation of lactoperoxidase in goat,sheep and bovine milk – A comparative kinetic and thermodynamic study

Using isothermal heating, inactivation of lactoperoxidase (LPO) in goat, sheep and cow milk was studied in the temperature range of 70–77 °C. Kinetic and thermodynamics studies were carried out at different time–temperature combination in order to evaluate the suitability of LPO as marker for the heat-treatment of milk and dairy products from different species. The thermal inactivation of LPO followed the first-order kinetics. D- and k-values decreased and increased, respectively with increasing temperature, indicating a more rapid LPO inactivation at higher temperatures. The influence of temperature on the inactivation rate constant was quantified using the Arrhenius and thermal death time models. The corresponding z-values were 3.38 ± 0.013, 4.11 ± 0.24 and 3.58 ± 0.004 °C in goat, sheep and cow milk, respectively. Activation energy values varied between milk species with 678.96 ± 21.43 kJ mol⁻¹ in goat milk, 560.87 ± 28.18 kJ mol⁻¹ in sheep milk and 641.56 ± 13.12 kJ mol⁻¹ in cow milk, respectively.     

Kinetics of thermal inactivation of tomato lipoxygenase

The kinetics of thermal inactivation of tomato lipoxygenase were studied. Tomato dices and a non-purified enzymatic extract were treated at different time–temperature combinations in the range of 0–150 min and 80–98 °C and the thermodynamic (activation energy) and kinetic (D_T and z) parameters for lipoxygenase inactivation were calculated. The kinetic behaviour of lipoxygenase from the tomato samples indicated the presence of two different isoenzymes having different thermal stabilities. Furthermore, the results showed that the isoenzymes were more heat-resistant than expected on the basis of literature reports and that the determined D_T and z values for lipoxygenase were orders-of-magnitude larger than those for microrganisms. This suggested that high temperature-short time treatments cannot be sufficient to achieve complete inactivation of tomato lipoxygenase.     

High-pressure destruction kinetics of Clostridium sporogenes ATCC 11437 spores in milk at elevated quasi-isothermal conditions

The high-pressure sterilization establishment requires data on isobaric and isothermal destruction kinetics of baro-resistant pathogenic and spoilage bacterial spores. In this study, Clostridium sporogenes 11437 spores (10⁷ CFU/ml) inoculated in milk were subjected to different pressure, temperature and time (P, T, t) combination treatments (700–900 MPa; 80–100 °C; 0–32 min). An insulated chamber was used to enclose the test samples during the treatment for maintaining isobaric and quasi-isothermal processing conditions. Decimal reduction times (D values) and pressure and temperature sensitivity parameters, Z_T (pressure constant) and Z_P (temperature constant) were evaluated using a 3 × 3 full factorial experimental design. HP treatments generally demonstrated a minor pressure pulse effect (PE) (no holding time) and the pressure hold time effect was well described by the first order model (R² > 0.90). Higher pressures and higher temperatures resulted in a higher destruction rate and a higher microbial count reduction. At 900 MPa, the temperature corrected D values were 9.1, 3.8, 0.73 min at 80, 90, 100 °C, respectively. The thermal treatment at 0.1 MPa resulted in D values 833, 65.8, 26.3, 6.0 min at 80, 90, 95, 100 °C respectively. By comparison, HP processing resulted in a strong enhancement of spore destruction at all temperatures. Temperature corrected Z_T values were 16.5, 16.9, 18.2 °C at 700, 800, 900 MPa, respectively, which were higher than the thermal z value 9.6 °C. Hence, the spores had lower temperature sensitivity at elevated pressures. Similarly, corrected Z_P values were 714, 588, 1250 MPa at 80, 90, 100 °C, respectively, which illustrated lower pressure sensitivity at higher temperatures. By general comparison, it was concluded that within the range operating conditions employed, the spores were relatively more sensitive to temperature than to pressure.     

A mathematical model of inactivation kinetics for a four-strain composite of Salmonella Enteritidis and Oranienburg in commercial liquid egg yolk

The goal of this study was to develop a general model of inactivation of salmonellae in commercial liquid egg yolk for temperatures ranging from 58 °C to 66 °C by studying the inactivation kinetics of Salmonella in liquid egg yolk. Heat-resistant salmonellae (three serovars of Enteritidis [two of phage type 8 and one PT 13] and one Oranienburg) were grown to stationary phase in Tryptic Soy Broth and concentrated 10-fold by centrifugation. Each inoculum was added to liquid egg yolk and mixed thoroughly, resulting in a final population of ca. 7 log CFU/ml egg yolk. Inoculated yolk was injected into sterile glass capillary tubes, flame-sealed and heated in a water bath at 58, 60, 62, 64, and 66 °C. Capillary tubes were ethanol sanitized, rinsed, and contents were extracted. Yolk was diluted, surface plated onto Tryptic Soy Agar + 0.1% sodium pyruvate and 50 μg/ml nalidixic acid and incubated at 37 °C for 24 h before colonies were enumerated. Decimal reduction values were calculated from survivor curves with a minimum inactivation of 6 log CFU/ml at each temperature. Survival curves (except for 66 °C) featured initial lag periods before first order linear inactivation. Estimated asymptotic D-values were 1.83 min at 58 °C, 0.69 min at 60 °C, 0.26 min at 62 °C, 0.096 min at 64 °C and 0.036 min at 66 °C. The estimate of the asymptotic z-value was ca. 4.7 °C with standard error of 0.07 °C. A linear relationship between the log₁₀ of the lag times and temperature was observed. A general kinetic model of inactivation was developed. The results of the study provide information that can be used by processors to aid in producing safe pasteurized egg yolk products and for satisfying pasteurization performance standards and developing industry guidance.     

Effects of light,temperature and water activity on the kinetics of lipoxidation in almond-based products

Lipoxidation in almond-derived products was investigated using the chemiluminescence (CL) and thiobarbituric acid-reactive substances (TBARS) methods to detect the first and later reaction products, respectively. The effects of light during storage at 5 °C, 22 °C and 40 °C were studied, as well as the effects of combined heat/water activity treatments in the 60–120 °C and 0.38–0.72 range. During storage, light was found to enhance the CL and TBARS values, and specific responses were observed in almond paste and the final Calisson product. During the heating of almond paste, as the initial water activity (a_w) increased, the CL rate constants increased during heating to 60 °C and 80 °C, but interestingly, these values decreased during further heating to 120 °C, whereas the maximum TBARS rate constants occurred at a_w 0.57 at all the heating temperatures tested. The activation energies, based on the CL and TBARS values, decreased specifically when the a_w increased from 0.38 to 0.72, giving overall values ranging from110 kJ mol⁻¹ to 60 kJ mol⁻¹. Likewise, in the same water activity range, the temperature-dependent rate constant enhancing factor (Q₁₀) decreased from 3.3 to 1.6.     

Enhancing the thermal destruction of Escherichia coli O157:H7 in ground beef patties by trans-cinnamaldehyde

The effect of trans-cinnamaldehyde (TC) on the inactivation of Escherichia coli O157:H7 in undercooked ground beef patties was investigated. A five-strain mixture of E. coli O157:H7 was inoculated into ground beef (7.0 log CFU/g), followed by addition of TC (0, 0.15, and 0.3%). The meat was formed into patties and stored at 4 °C for 5 days or at −18 °C for 7 days. The patties were cooked to an internal temperature of 60 or 65 °C, and E. coli O157:H7 was enumerated. The numbers of E. coli O157:H7 did not decline during storage of patties. However, cooking of patties containing TC significantly reduced (P < 0.05) E. coli O157:H7 counts, by >5.0 log CFU/g, relative to the reduction in controls cooked to the same temperatures. The D-values at 60 and 65 °C of E. coli O157:H7 in TC-treated patties (1.85 and 0.08 min, respectively) were significantly lower (P < 0.05) than the corresponding D-values for the organism in control patties (2.70 and 0.29 min, respectively). TC-treated patties were more color stable and showed significantly lower lipid oxidation (P < 0.05) than control samples. TC enhanced the heat sensitivity of E. coli O157:H7 and could potentially be used as an antimicrobial for ensuring pathogen inactivation in undercooked patties. However detailed sensory studies will be necessary to determine the acceptability to consumers of TC in ground beef patties.     

Inactivation of Cronobacter sakazakii by ultrasonic waves under pressure in buffer and foods

The objective of this research was to characterize the resistance of Cronobacter sakazakii to ultrasonic waves under pressure (manosonication, MS). The D_MS value (decimal reduction time value) of C. sakazakii in standard conditions (35 °C, 117 μm, 200 kPa, citrate-phosphate buffer pH 7.0) was 0.41 min. This value was higher than that of Yersinia enterocolitica (D_MS = 0.19 min) and lower than those of Salmonella enterica serovar Enteritidis (D_MS = 0.61 min), Listeria monocytogenes (D_MS = 0.86 min), and Enterococcus faecium (D_MS = 1.2 min). Strain studied (ATCC 29544, NCTC 8155, 9238, and 9529), growth temperature (10, 20, 30, and 37 °C), and pH of the treatment media (4.0, 5.0, 6.0, and 7.0) did not significantly change C. sakazakii MS resistance. Conversely, entry into stationary growth phase, decreasing water activity of the treatment media (0.98, 0.96, and 0.94), and treatment in food products (apple and orange juices, chicken and vegetable soups, and rehydrated powdered milk) resulted in up to a 1.6-, 3.9-, and 2.5-fold maximum change in D_MS values, respectively. Whereas an exponential relationship between the amplitude of ultrasonic waves and D_MS values was found, the relationship between static pressure and D_MS values was better described by a quadratic equation. The energy transferred into the medium determined the lethality of the ultrasonic waves regardless of the combination of pressure (0, 50, 100, 200 and 300 kPa) and amplitude (34, 62, 90, 117 and 145 μm) applied. There was an exponential relationship between D_MS values and the power input: an increase of 134 W increased the inactivation rate ten times regardless of the treatment medium. No C. sakazakii cells with sublethally injured cytoplasmic membrane or with sublethal oxidative damage occurred after MS treatments, but the results indicated that damage to the outer membrane preceded microbial death.     

Characterization of polyphenol oxidase from butter lettuce (Lactuca sativa var. capitata L.)

Polyphenol oxidase (PPO) was isolated from butter lettuce (Lactuca sativa var. capitata L.) grown in Poland and its biochemical characteristic were studied. PPO from butter lettuce showed a higher affinity to 4-methylcatechol than to catechol. The K_M and V_max values were: 3.20 ± 0.01 mM and 4081 ± 8 U/ml min⁻¹ for catechol and 1.00 ± 0.09 mM and 5405 ± 3 U/ml min⁻¹ for 4-methylcatechol. The optimum pHs of the enzyme were found to be 5.5 using catechol and 6.8 using 4-methylcatechol as substrate. The enzyme had a temperature optimum of 35 °C. The enzyme was relatively stable at 30 °C and 40 °C. The times required for 50% inactivation of activity at 50 °C, 60 °C and 70 °C were found to be about 30, 20 and 5 min, respectively. Inhibitors used for investigation in this study were placed in relative order of inhibition: p-hydroxybenzoic acid > glutathione ≈ ascorbic acid > l-cysteine > EDTA > citric acid. The enzyme eluted in the chromatographic separations was analyzed electrophoretically under denaturating conditions. The analysis revealed a single band on the SDS–PAGE which corresponded to a molecular weight of 60 kDa.     

Characterisation of thermostable trypsin and determination of trypsin isozymes from intestine of Nile tilapia (Oreochromis niloticus L.)

Trypsin from intestinal extracts of Nile tilapia (Oreochromis niloticus L.) was characterised. Three-step purification – by ammonium sulphate precipitation, Sephadex G-100, and Q Sepharose – was applied to isolate trypsin, and resulted in 3.77% recovery with a 5.34-fold increase in specific activity. At least 6 isoforms of trypsin were found in different ages. Only one major trypsin isozyme was isolated with high purity, as assessed by SDS-PAGE and native-PAGE zymogram, appearing as a single band of approximately 22.39 kDa protein. The purified trypsin was stable, with activity over a wide pH range of 6.0–11.0 and an optimal temperature of approximately 55–60 °C. The relative activity of the purified enzyme was dramatically increased in the presence of commercially used detergents, alkylbenzene sulphonate or alcohol ethoxylate, at 1% (v/v). The observed Michaelis–Menten constant (K_m) and catalytic constant (K_cat) of the purified trypsin for BAPNA were 0.16 mM and 23.8 s⁻¹, respectively. The catalytic efficiency (K_cat/K_m) was 238 s⁻¹ mM⁻¹.     

Decimal reduction times of Salmonella Typhimurium in guinataang kuhol: An indigenous Filipino dish

This study established the thermal inactivation of Salmonella Typhimurium (E1366) in a mixture of golden apple snail (Pomacea conaliculata Linn.) meat and coconut cream medium—a dish native to the Philippines locally known as guinataang kuhol. Artificially inoculated S. Typhimurium were heated to 60, 75 and 90 °C for 0-1.5 min. Survivor cells were enumerated using a selective and a nonselective enumerating medium, bismuth sulphite agar (BSA) and nutrient agar (NA), respectively. The thermal inactivation rates of Salmonella Typhimurium were expressed in terms of decimal reduction times (D-values). Results showed that in both enumerating media and in all heating temperatures, the number of surviving cells significantly (P<0.05) decreased with increasing treatment times. Survivor cell populations enumerated on NA were always significantly greater than those enumerated on BSA. The D-values calculated from the survivor counts on both media were however not significantly different. The calculated D-values ranged from 0.22 to 0.48 min.     

The effects of packaging method (vacuum pouch vs. plastic tray) on spoilage in a cook-chill pork-based dish kept under refrigeration

The effects of two packaging methods on the spoilage of a cook-chill pork-based dish kept under refrigeration were studied. Raw pork cuts and pre-cooked tomato sauce were packed under vacuum “sous vide” in polyamide–polypropylene pouches (SV) or into translucent polypropylene trays under modified atmosphere (80% N₂ + 20% CO₂) and sealed with a top film (PT). Samples were cooked inside the pack at an oven temperature/time of 70 °C/7 h, chilled at 3 °C and stored at 2 °C for up to 90 days. Microbial (psychrotrophs, lactic-acid bacteria, Enterobacteriaceae, moulds and yeasts), physical–chemical (pH, water activity and total acidity) and sensory (colour, odour, flavour, texture and acceptance) parameters were determined. Heat penetration was faster in SV (2 °C/min) than in PT (1 °C/min) (core temperature). Both packaging methods were equally effective in protecting against microbial spoilage for 90 day at 2 °C. Minor counts were only detected for lactic-acid bacteria and anaerobic psychrotrophs in SV. No Enterobacteriaceae growth was found. Slight differences between SV and PT in pH and total acidity were observed. SV and PT had similar effects on the sensory preservation of the dishes. A gradual loss of acceptance of the cooked pork and tomato sauce was observed. Rancid flavour in PT and warmed-over-flavour in SV were noted in the final stages of storage. According to acceptance scores, the shelf-life of both SV and PT was 56 days at 2 °C. Both packaging methods can be used to manufacture sous vide meat-based dishes subsequently stored under refrigeration for catering use.     

Sensory,physical and chemical characteristics of cooked ham manufactured from rapidly chilled and earlier deboned M. semimembranosus

Effects of rapid chilling of carcasses (at − 31 °C in the first 3 h of chilling, and then at 2–4 °C) and earlier deboning (8 h post-mortem), compared to rapid (till 24 h post-mortem) and conventional chilling (at 2–4 °C, till 24 h post-mortem), on quality characteristics of pork M. semimebranosus and cooked ham were investigated. Quality measurements included pH value, colour (CIEL*a*b* values) and total aerobic count of M. semimebranosus, as well as sensory (colour, juiciness, texture, and flavour), physical (pH value, colour – CIEL*a*b* values and texture – Warner–Bratzler shear and penetration forces) and chemical (protein, total fat, and moisture content) characteristics of cooked ham. The cooked ham was manufactured from pieces of M. semimebranosus with ultimate lightness (CIEL* value) lower than 50. Rapid chilling and earlier deboning significantly increased quantity of M. semimebranosus desirable for cooked ham manufacturing. Earlier start of pork fabrication did not affect important quality characteristics of cooked ham.     

Characterisation of a β-N-acetylhexosaminidase from a commercial papaya latex preparation

A β-N-acetylhexosaminidase (β-NAHA) (EC 3.2.1.52) with molecular mass of 64.1 kDa and isoelectric point of 5.5 was purified from a commercial papaya latex preparation. The optimum pH for p-nitrophenyl-N-acetyl-β-d-glucosaminide (pNP-β-GlcNAc) hydrolysis was five; the optimum temperature was 50 °C; the K_m was 0.18 mM, V_max was 37.6 μmol min⁻¹ mg⁻¹ and activation energy (E_a) was 10.3 kcal/mol. The enzyme was thermally stable after holding at 30–45 °C for 40 min, but its activity decreased significantly when the temperature exceeded 50 °C. Heavy metal ions, Ag⁺ and Hg²⁺, at a concentration of 0.25 mM and Zn²⁺ and Cu²⁺, at a concentration of 0.5 mM, significantly inhibited enzyme activity. The β-NAHA had only one active site for binding both pNP-β-GlcNAc and p-nitrophenyl-N-acetyl-β-d-galactosaminide (pNP-β-GalNAc). A prototropic group with pKa value of about five on the enzyme may be involved in substrate binding and transformation, as examined by Dixon–Webb plots.     

Detection and prevalence of pathogenic Yersinia enterocolitica in refrigerated and frozen dairy products by duplex PCR and dot hybridization targeting the virF and ail genes

Pathogenic Yersinia enterocolitica is involved in yersiniosis through expression of chromosome-borne or plasmid-borne virulence factors. Yersinia enterocolitica is a cold-tolerant pathogen frequently isolated from refrigerated or frozen foods. However, little attention has been focused on the prevalence of pathogenic Y. enterocolitica in refrigerated or frozen dairy samples in China. In this study, we developed a new duplex PCR targeting the plasmid-borne virF gene and chromosome-borne ail gene for detection of pathogenic Y. enterocolitica isolates. We established a detection limit for the duplex PCR of 6.5 × 10² cfu/mL in artificially contaminated dairy samples. In addition, the duplex PCR could detect directly 4.5 to 5.7 cfu of Y. enterocolitica in 5 mL of brain heart infusion broth after 6 h of enrichment at 28°C. A newly developed dot hybridization assay further confirmed specificity of the duplex PCR for detection of virulent Y. enterocolitica. Furthermore, 13 Y. enterocolitica and 5 pathogenic strains, from 88 commercial frozen or refrigerated dairy products, were detected successfully by the China National Standard method (GB/T4789.8-2008) and the duplex PCR, respectively. Finally, biotypes and serotypes of pathogenic Y. enterocolitica strains were further characterized. The duplex PCR developed here is reliable for large-scale screening, routine monitoring, and risk assessment of pathogenic Y. enterocolitica in refrigerated or frozen dairy products.     

Artificial intelligence in predicting extraction of anti-cancer compounds

Saponins from the particulate Saponaria vaccaria L. seeds (0.29–0.84 mm, 15.35–61.40 g H₂O/100 g dry mass) were extracted for methanol concentrations (MeOH) of 30, 50, 70, and 90 mL/100 mL H₂O and temperatures (T) of, 30, 45, and 60 °C at ten extraction intervals (t) between 1 and 180 min. A calibration equation was developed from the liquid-chromatogram–mass spectroscopy peaks to quantify the extract yields (mg mL⁻¹) for various types of saponins. An artificial neural network (ANN) with three inputs, MeOH, T, and t predicted the extraction kinetics and the yields with less than ca. 12% error. The ANN model not only slightly outperformed the numerical diffusional model, but it also made the prediction simple and faster eliminating the use of the partition coefficient and the effective diffusivity. Therefore an ANN model can be a right approach to predict the yields of saponins and similar products.     

A β-galactosidase from chick pea (Cicer arietinum) seeds: Its purification,biochemical properties and industrial applications

A β-galactosidase from Cicer arietinum seeds has been purified to apparent electrophoretic homogeneity using a combination of various fractionation and chromatographic techniques, giving a final specific activity of 220 units mg⁻¹, with approximately 1840 fold purification. Analysis of the protein by SDS–PAGE revealed two subunits with molecular masses of 48 and 38 kDa, respectively. These bands were further confirmed with LC–MS/MS, indicating that Chick pea β-galactosidase (CpGAL) is a heterodimer. Molecular mass was determined to be 85 kDa by Superose-12 FPLC column, which is in agreement with the molecular mass suggested by mass spectroscopy to be 83 kDa. The optimum pH of the enzyme was 2.8 and it hydrolysed o-nitrophenyl β-d galactopyranoside (ONPG) with a K_m value of 1.73 mM at 37 °C. The energy of activation (E_a) calculated in the range of 35 to 60 °C, using Arrhenius equation, was determined to be 11.32 kcal mol⁻¹. The enzyme could also hydrolyse lactose, with an optimum pH of 4.0 at 40 °C. K_m and E_a for lactose hydrolysis was found to be 10 mM and 10.57 kcal mol⁻¹, respectively. The enzyme was found to be comparatively thermostable showing maximum activity at 60 °C for both ONPG and lactose. Galactose was found to be the competitive inhibitor. β-Galactosidase also exhibited glycoproteineous properties when applied on Con-A Sepharose column. The enzyme was localised in germinated seeds with X-gal activity staining and shown to be expressed prominently at grown radical tip and seed coat. Sequence alignment of CpGAL with other known plant β-galactosidase showed high amino acid sequence homology.