Immobilization of Aspergillus niger Mycelium on Seeds for Glucoamylase Production

Mycelium of the glucoamylolytic mutant Aspergillus niger C-58-III was immobilized on wheat, rye, barley, pea, buckwheat and mustard seeds in repeated-batch flasks. After every 24th it was possible to obtain culture broth rich in glucoamylase. The highest yield of enzyme (66.4 U × ml⁻¹) was obtained on the mustard carrier. Immobilized cells were successfully reused with high level of enzyme formation being mantained for longer period (192h). Some of the variables influencing the enzymatic activity have been standardized. Enzyme productivity reached in immobilized cells of A. niger was 1.6-times higher in comparison with free cells.     

Production,Purification, and Characterization of Thermoanaerobacterium thermosaccharolyticum Glucoamylase

Glucoamylase from Thermoanaerobacterium thermosaccharolyticum ATCC 7956 (DSM 571) was produced in extracellular form. It was purified to homogeneity by two separate methods, one with two chromatographic steps and the other with three. This glucoamylase is closely related to glucoamylases from Clostridium sp. G0005 and T. thermosaccharolyticum DSM 572. Activities and K_M values with maltose substrate are less than one‐tenth and about fourfold, respectively, those of Aspergillus niger glucoamylase. T. thermosaccharolyticum glucoamylase is about twenty times as thermostable as A. niger glucoamylase and its optimal pH is somewhat higher at 4.9; however, it is produced in much lower activities. Sorbitol strongly stabilizes A. niger glucoamylase.     

In-milk inactivation of Escherichia coli O157:H7 by the environmental lytic bacteriophage ECPS-6

Shiga toxin-producing Escherichia coli is a common foodborne pathogen which transmission includes dairy products. In the search for novel biocontrol methods, bacteriophages have become important candidates for the eradication of foodborne pathogens. The aim of this study was to evaluate the bacteriophage-mediated reduction of E. coli O157:H7 in raw and filtered milk. Laboratory-scale tests showed that the bacteriophage ECPS-6 efficiently adsorbed to E. coli O157: H7 cells. Furthermore, ECPS-6 remained stable when heated at 70°C for 20 min and in a wide pH range from 3.0 to 11.0. The trials on contaminated milk were performed using filtered and unfiltered raw milk contaminated with 1 × 10⁵ CFU × ml⁻¹ of E. coli O157: H7. Bacteriophage was added at multiplicity of infection (MOI) 5 and 50. The ECPS-6 reached the highest lytic activity at MOI = 5 (25°C) which resulted in 4.74 Log₁₀ CFU × ml⁻¹ and 7.3 Log₁₀ CFU × ml⁻¹ reduction after 10 days for both tested strains, respectively. Under refrigerated conditions (4°C) the quantity of E. coli decreased to 1.5 Log₁₀ CFU × ml⁻¹ and 3.04 Log₁₀ CFU × ml⁻¹ for these strains, respectively. Usage of MOI = 50 for the treatment unfiltered milk led to the reduction of E. coli O157:H7 A-2 below the detection limit after 6 hr.     

Reduction of raffinose oligosaccharides in chickpea (Cicer arietinum) flour by crude extracellular fungal α-galactosidase

The efficiency of crude extracelluar α-galactosidases from Cladosporium cladosporides, Aspergillus oryzae and A niger in reducing the raffinose and stachyose content in chickpea flours was studied and compared with other traditional treatments. The optimum pH for α-galactosidase activity was found to be 4·5 for A oryzae and 5·0 for Cl cladosporides and A niger, while the optimum temperature of enzyme activity was 40°C for Cl cladosporides and 50°C for A oryzae and A niger. The specific activities of α-galactosidase from Cl cladosporides, A oryzae and A niger were 3·35, 3·94 and 5·94 units μg⁻¹ protein, respectively. The enzyme activity was stable between pH 4·0 and 7·0 for A oryzae and A niger and between pH 5·0 and 7·0 for Cl cladosporides. The enzymes were thermostable when incubated at temperature ranges of 40–60°C for Cl cladosporides and 40–50°C for A oryzae and A niger. The optimum conditions for removing the raffinose and stachyose were obtained by incubating chickpea flours with 30 ml of crude fungal α-galactosidase extract (290, 210 and 130 units ml⁻¹ for Cl cladosporides, A oryzae and A niger, respectively) for 3 h at the optimum conditions of each strain. Crude fungal α-galactosidases reduced the raffinose oligosaccharides content in chickpea flours by 100%, while germination reduced the raffinose content by 69% and stachyose content by 75%. Other traditional techniques reduced the raffinose content by 13–49% and stachyose content by 10–32%. © 1998 Society of Chemical Industry     

Simulation and modelling of the effect of small inoculum size on time to spoilage by Bacillus stearothermophilus

Mathematical models developed in predictive food microbiology typically use large (approx. 10⁵cfu ml⁻¹) inoculum sizes. Real food systems may contain low microbial loads (1–10 cfu ml⁻¹) introducing an additional uncertainty unaccounted for by model predictions. This research investigated the effects of very low inoculum sizes on the time to spoilage of Bacillus stearothermophilus ATCC 12980 spores. Microtiter plates (96-well) were filled with tryptic soy broth and inoculated with various concentrations of B. stearothermophilus spores. Time to spoilage was defined as colour change of the medium containing bromcresol purple (corresponding to c. 10⁸cfu ml⁻¹) from purple to yellow. A Poisson distribution best described the number of spores in a well. Spoilage times showed maximum variability (7·25–17 h) at 1 spore well⁻¹, and negligible variability (c. 6·5 h) at 500 spores well⁻¹. A simplified Gompertz function described spoilage kinetics. Mathematical modelling and simulation approaches were used to study spoilage times. The modelling approach had a fail-safe bias in its predictions and provided greater accuracy than the simulation, which was fail-dangerous. The simulation approach provided potentially greater mechanistic insight into the causes of spoilage time variability, and supported the notion that the effects of biovariability and interactions among individual spores manifest at very low inoculum sizes.     

Purification and Characterization of a Pullulan-Hydrolyzing Glucoamylase from Sclerotium rolfsii

The pullulan-hydrolyzing enzyme from the culture filtrates of Sclerotium rolfsii grown on soluble starch as a carbon source has been purified by ultrafiltration (Amicon, PM-10), ion-exchange chromatography (DEAE-Cellulose DE-52) and gel filtration chromatography (Bio-Gel P-150). The enzyme moved as a single band in non-denaturing polyacrylamide gel electrophoresis carried out at pH 2.9 and 7.5. The relative molecular mass of the enzyme was estimated to be 64.000 D by SDS-PAGE and 66.070 D by gel filtration on Bio-Gel P150. The enzyme hydrolyzed pullulan optimally at 50°C between pH 4.0–4.5, whereas, soluble starch was optimally hydrolyzed at a pH of between 4.0–4.5 and at 65°C. The Michaelis constant (Km) for pullulan was 5.13mg·ml⁻¹ (V_max 1.0U · mg⁻¹) and for soluble starch, it was 0.6mg · ml⁻¹ (V_max 8.33 U · mg⁻¹). The enzyme was observed to be a glycoprotein (12–13% carbohydrate by weight) and had a strong affinity for Concanavalin A. The enzyme hydrolyzed α-D-glucans in an exo-manner, which resulted in the release of glucose as the sole product of hydrolysis. Acarbose, a maltotetraose analog, was found to be a potent inhibitor of both pullulan and starch hydrolysis (100% inhibition at 0.06 μM). The enzyme has been characterized as a glucoamylase (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3) showing a significant action on pullulan.     

Biochemical and morphological characterization of a new fungal contaminant in balsamic and cider vinegars

In this study we have determined the morphological, some physiological and biochemical characteristics of a fungal strain isolated from balsamic and cider vinegars. The examination of fungal contamination of 100 balsamic and cider vinegar samples showed that 90% contained more than 10⁴ colony forming units (CFU ml^?1), but in most samples (65%) values of more than 10⁵ CFU ml^?1 were obtained. The most frequent contamination was with Monascus sp. (80% of the samples examined showed 2 × 10³ to 6 × 10⁵ CFU ml^?1). This mould can produce secondary metabolites such as citrinin and monacolin K_L (lactone form), and monacolin K_A (acid form). Using high-performance liquid chromatography (HPLC) with fluorescence detection, citrinin was found in 68% of the samples. The concentration of these mycotoxins varied between 1.6 × 10^?2 µg ml^?1 and 7.2 × 10^?2 µg ml^?1. The concentrations of citrinin were very low, and we can anticipate that this compound at these concentrations has no toxic effects on renal cells. Monacolin was not detected in any sample studied.     

Fumonisin B2 production by Aspergillus niger from grapes and natural occurrence in must

Aspergillus niger has been recently found to produce fumonisin B₂ (FB₂). Thirty-one strains belonging to four Aspergillus species isolated from grape were evaluated for FB₂ production on agar plates. Four out of eight strains of A. niger produced FB₂ (29–293 µg g^?1). None of the strains of A. uvarum (n = 7), A. tubingensis (8) and A. carbonarius (8) produced detectable amounts of toxin. The capability to produce FB₂ was also confirmed by some A. niger strains artificially inoculated on grape berries. Natural occurrence of FB₂, at levels of 0.01 and 0.4 µg ml^?1, was found in two samples of must collected in Apulian cellars in 2007. This is the first report of FB₂ contamination in must. These findings suggest that there is a potential risk of exposure to FB₂ in the grape–wine chain for consumers and that A. niger may represent the major fumonisin-producing species among black Aspergilli occurring on grapes.     

Pungency threshold of capsaicin produced by in vitro culture of placental tissues of capsicum frutescens mill.

Production of capsaicin in free and immobilized placenta was compared in order to assess their capsaicin production ability. In immobilized placenta, maximum accumulation of capsaicin was on the 14th day of culture with a production of 2045 ug/culture, whereas in free placenta, the accumulation reached maximum on the 7th day (2050ug/culture). It was found that there was no significant difference between free and immobilized placenta in terms of capsaicin production, suggesting that immobilization is not necessary to enhance capsaicin accumulation in piacentai tissue. Pungency threshold of capsaicin produced in in vitro grown free piacentai tissues of Capsicum frutescens was done by Scoville heat units (SHU) method. Pungency analysis of capsaicin produced in free piacentai tissue showed that on day zero, pungency of tissue culture produced capsaicin (0.2267x10⁵ SHU) was comparable to standard natural capsaicin (0.222×10⁵ SHU), while on the 3rd day pungency of tissue culture produced capsaicin (0.3639×10⁵ SHU) was slightly lower than standard natural capsaicin (0.4415×10⁵ SHU).     

Hardaliye: fermented grape juice as a traditional Turkish beverage

Twenty-six aged hardaliye samples, collected from different spots of the Kirklareli province of Turkey and hardaliye produced in laboratory conditions using the traditional method were investigated in this study. The pH ranged from 3·21 to 3·97. Red colour (Hunter Lab a_Lvalue) of samples ranged from 1·33 to 9·66. The total bacterial count ranged from 3·5×10²to 8×10⁵cfu ml⁻¹. The lactic acid bacteria counts of the samples were found to be between 1·0×10²and 4·0×10⁴cfu ml⁻¹. Yeasts and moulds, which were found in 21 samples out of 26, ranged from 1·0×10²to 8·1×10⁴cfu ml⁻¹. Coliforms and Escherichia coli were found in none of the samples. The changes of some microbiological and chemical properties of hardaliye during fermentation were investigated. The pH of hardaliye dropped from 3·86 to 3·39. The ethanol content of the end product was determined as 595·50 mg dl⁻¹. During the fermentation process, the total bacteria count, lactic acid bacteria count and yeast count changed from 2·1×10⁵, 6·0×10⁴and 1·2×10⁵cfu ml⁻¹to 1·3×10², 1·2×10³and 1·1×10³cfu ml⁻¹, respectively.Lactobacilli isolated from hardaliye samples were characterized by API 50 CH and other phenotypic criteria. A succession of Lactobacillus species, dominated by L. paracasei subsp. paracasei and L. casei subsp. pseudoplantarum were found during the fermentation process.     

Use of nisin for inhibition of Alicyclobacillus acidoterrestris in acidic drinks

The inhibitory effects of nisin on the growth of the thermoacidophilic spoilage bacteriumAlicyclobacillus acidoterrestris were investigated for the purpose of preventing flat-sour-type spoilage in acidic drinks. Minimum inhibitory concentration values of nisin against the spores were from less than 0·78 to 12·5 IU ml⁻¹and from 25 to 100 IU ml⁻¹on mYPGA plates at pH 3·4 and 4·2, respectively. The levels of nisin inhibition against the vegetative cells were, however, higher than those of the spores. In determining the effects of nisin on the thermal resistance of A. acidoterrestris spores, the addition of nisin contributed to the reduction of the thermal resistance of A. acidoterrestris spores in acidic drinks. Furthermore, the outgrowth of A. acidoterrestris spores was inhibited by the addition of 25–50 IU ml⁻¹nisin in both orange and fruit-mixed drinks, but was not inhibited by the higher level (600 IU ml⁻¹) addition in a clear-apple drink. From these findings, we conclude that it would be useful to add nisin for preventing the spoilage caused by A. acidoterrestris in all but clear-apple acidic drinks.     

In vitro effects of steroidal saponins from Yucca schidigera extract on rumen microbial protein synthesis and ruminal fermentation

In a first experiment, ground alfalfa hay and rolled barley grain were incubated in buffered ruminal fluid with and without Yucca schidigera extract (YE, 0 or 10 mg ml⁻¹). Gas and total VFA production from barley grain were increased (P < 0.05) by YE during the first 10 h of incubation; from alfalfa hay, these were reduced (P < 0.001) throughout the 24 h. Yucca extract reduced (P < 0.001) acetate/propionate ratios and ammonia concentrations, irrespective of substrate. In a second experiment, ground barley grain was incubated in a buffered suspension of mixed ruminal microbes obtained by low‐speed centrifugation of ruminal fluid. Steroidal saponins (SAP) isolated from YE were included at 0, 15, 75 or 225 µg ml⁻¹. Microbial incorporation of ¹⁵N was increased (P < 0.05) by 15 µg SAP ml⁻¹ but decreased (P < 0.05) by 225 µg SAP ml⁻¹. Gas and VFA production peaked with 75 µg SAP ml⁻¹ and were elevated (P < 0.05) relative to control in the presence of 75 µg SAP ml⁻¹. Microbial protein synthesis was increased (P < 0.05) by SAP at 15 µg ml⁻¹ and reduced (P < 0.05) by the higher concentrations. Acetate/propionate ratios were linearly reduced by SAP from 8 to 24 h incubation (P < 0.01). The effects of SAP on digestive microbes were less pronounced (P < 0.05) with barley grain digestion than with alfalfa. A YE × diet interaction was recorded. The biological activity of YE was shown to be attributable to its SAP and to be diet‐dependent. © 2000 Society of Chemical Industry. Contributions of Y Wang, T A McAllister, L J Yanke and Z Xu © Minister of Public Works and Government Services Canada 2000     

Breaking of structural dormancy in seeds of Tetrapleura tetraptera by Aspergillus niger

Aspergillus niger (strain UUF9202) significantly reduced the dormancy period and boosted aggregate germination percentage (AGP) in seeds of Tetrapleura tetraptera. A net reduction in mean germination time (MGT) to 12 days and an increase from 0 to 42% AGP were obtained in 32 days. The optimal spore inoculum of the fungus was 8.5 × 10≥10 ml^?1 per 100 seeds in 96 h pregermination incubation. The potential of some physical factors in combination with this fungal concentration in enhancing AGP was evaluated. The treatments were as follows: (i) hot water scarification at 30°C, 50°C, 80°C and 100°C plus A niger; and (ii) nicking by decoating at either the micropylar or chalazal ends plus A niger. Micropylar scarification plus A niger gave the highest AGP (94%) followed by chalazal scarification plus A niger (86%) and hot water dip at 80°C + A niger (82%). The control seeds which were soaked in presterilised (by autoclaving at 121°C for 15 min) A niger spore suspension at 30°C gave zero germination.     

Detoxification of essential oil components (citral and menthol) by Aspergillus niger and Rhizopus stolonifer

The antifungal activity of two essential oil components (citral and menthol) towards Aspergillus niger and Rhizopus stolonifer was studied in shake culture. The activity was high with lower inoculum density (measured in terms of number of spores ml^?1), and it decreased as the inoculum density increased. Citral at 200 μg ml^?1 and menthol at 400 μg mh^?1 were lethal to the spores of A. niger and R. stolonifer, respectively, after a 48-h treatment. At lower concentrations both the compounds were only fungistatic: the growth of the fungi resumed after an initial delay or upon transfer to fresh medium. Periodic estimations of the essential oil components by gas-liquid chromatography showed a continuous decrease in their concentrations in the culture medium, indicating their removal tending to detoxification. The difference in the activity of the two compounds was related to the capacity of the fungi to detoxify the compounds.     

The effect of EDTA and trisodium phosphate,alone and in combination with nisin,on the growth of Arcobacter butzleri in culture

Arcobacter butzleri is a common contaminant of foods of animal origin and there is increasing evidence linking the organism to human illness. This study investigated the effectiveness of ethylene diamine tetra-acetic acid (EDTA) and trisodium phosphate (TSP) alone and in combination with nisin, on the growth of A. butzleri in culture. At 1 mM, 5 mM, 10 mM and 20 mM, EDTA inhibited growth, both alone and combined with 500 IU ml⁻¹nisin, but only at 20 mM did the addition of nisin produce a statistically significant difference compared with EDTA alone (4·3 log₁₀and 5·6 log₁₀reduction respectively). Short-term simultaneous exposure with EDTA and 500 IU ml⁻¹nisin was more effective than sequential treatment on both logarithmically growing and stationary phase cells.Growth of A. butzleri was inhibited by 0·5 mM TSP with simultaneous treatment being more effective than sequential treatment. After 10 min simultaneous treatment, only at 40 mM TSP (but not at lower concentrations) was there a statistically significant difference between TSP treatment alone and TSP plus 500 IU ml⁻¹nisin (5·2 log₁₀compared with 7·7 log₁₀reduction).Thirty-minute treatment with 20 mM EDTA or 10 min with 10% TSP at 4°C, followed by incubation in the presence of 500 IU ml⁻¹nisin, resulted in no viable cells being recovered after 24 h (>99·9% cell kill) indicating that a multiple hurdle approach is the most effective method of reducing growth and survival of A. butzleri in culture.     

An inhibitory synergistic effect of a nisin–monolaurin combination on Bacillus sp. vegetative cells in milk

The inhibitory activities of nisin and monolaurin, used alone or in combination, were investigated against four Bacillus species vegetative cells in milk at 37°C for 5 days. In the absence of inhibitors, the four strains grew and sporulated at the end of the exponential growth step and throughout the stationary phase. Nisin (100 IU ml⁻¹) induced an immediate reduction in the population level but transient because regrowth appeared and was strain-dependent; cell concentrations reached the control culture level, e.g. 6–7 log₍₁₀₎as well as the spore load (4–5 log₍₁₀₎). On the other hand, monolaurin (250 μ g ml⁻¹) had a durable bacteriostatic effect followed by a regrowth level constantly lower than that of the control culture; sporulation was low (between 13 and 7×10³spl ml⁻¹) and did not occur in the case of B. coagulans. The use of these inhibitors in combination, induced a synergistic bactericidal effect leading to a total inhibition (0 cfu ml⁻¹) until day 5, except in the case of B. cereus where a concentration of 500 cfu ml⁻¹was constant till the end of the experiment; consequently, sporulation was absent.     

The effects on vegetative cells and spores of three bacteriocins from lactic acid bacteria

The sensitivities of vegetative cells of strains ofListeria, Clostridium, Staphylococcus, Lactococcus, Lactobacillus, MicrococcusandPediococcus, and of spores ofClostridiumandBacillusto three broad spectrum bacteriocins (nisin A, nisin Z and pediocin) from lactic acid bacteria were determined by a critical dilution micro-assay. The minimal inhibitory concentrations (MIC) of partially purified bacteriocins, prepared by a pH-dependent adsorption/desorption process, were determined and expressed in arbitrary units ml⁻¹and in μ g ml⁻¹of pure bacteriocin. The MICs of bacteriocins varied considerably between species and even between strains of the same species, as clearly shown for nine strains ofListeria monocytogenes. When bacteriocin activity was expressed in μ g ml⁻¹, pediocin was more effective againstListeria monocytogenesthan nisin A or nisin Z. The latter bacteriocins, in concentrations between 23 and 69 μ g ml⁻¹, prevented outgrowth ofClostridiumandBacillusspores for at least 10 days. Although pediocin at 17 μ g ml⁻¹prevented outgrowth ofB. stearothermophilusandC. butyricumspores for up to 7 days, it apparently activated the germination ofB. subtilisspores.     

Optimization of Aspergillus niger Fermentation for the Production of Glucose Oxidase

A number of nutritional factors influencing glucose oxidase (EC 1.1.3.4) production by Aspergillus niger NCIM 545 were studied. The synthesis of glucose oxidase by A. niger was investigated in two steps using submerged fermentation at 30 ± 2 °C and 180 rpm for 96 h. Primarily, nutritional components were selected by one-factor-at-a-time method, and the significance of each component with respect to glucose oxidase production was identified by Plackett–Burman design (seven variables including six nutritional viz. sucrose, sodium nitrate, peptone, calcium carbonate, magnesium sulfate, and potassium dihydrogen phosphate, and one dummy or unassigned variable were studied with eight experiments). In the second step, concentration of most significant factors and their interaction were studied with response surface methodology (central composite design). Each variable in the design was studied at five different levels, with all variables taken at a central coded value of zero. Considerable amount of glucose oxidase was produced from A. niger species with sucrose as the carbon source, sodium nitrate as the inorganic nitrogen source, and peptone as the organic nitrogen source. Glucose oxidase activity increased remarkably by 28.93 fold (from 0.00993 to 0.29 U ml⁻¹) with CaCO₃-supplemented media. The outcome of Plackett–Burman design showed CaCO₃, peptone, and MgSO₄ as significant parameters. Further optimization using a three-factor central composite design with 20 experiments increased yield of glucose oxidase from 0.29 to 2.05 U ml⁻¹ (sevenfold) with a decrease in cultivation time from 96 to 72 h.     

Purification and immobilisation of inulinase from Aspergillus candidus for producing fructose

A high‐inulinase‐producing strain of Aspergillus candidus (10 units cm⁻³ of medium) for producing fructose from inulin has been identified. The extracellular inulinase from this fungi was purified 56‐fold by ammonium sulphate fractionation, DEAE cellulose and Sephadex G‐150 column chromatography. Invertase to inulinase ratio of 1.8 in culture filtrate was reduced to 0.14 in the purified preparation. The pH and temperature optima were 5.5 and 45°C, respectively. The molecular weight of inulinase was determined as 54±4 kDa. K_m of inulinase with inulin as a substrate was 3.8 mmole dm ⁻³. The purified preparation produced only fructose as the product of inulin, indicating that inulinase has primarily exo‐inulinase activity. Inulinase was immobilised on chitin and casein using glutaraldehyde as a linking agent and on cellulose using FeCl₃‐HCl as a metal chelation agent. Maximum immobilisation of 45.8% was achieved on cellulose. All three immobilised preparations had a higher temperature optima of 55°C. The inulinases immobilised on cellulose and casein were stable at pH 5–7. The cellulose‐immobilised preparation was more stable than the other two preparations after heating for 1 h at 55°C. © 1999 Society of Chemical Industry     

Covalent Immobilization of a α-Amylase onto Poly(methyl methacrylate-2-hydroxyethyl methacrylate) Microspheres and the Effect of Ca2+ Ions on the Enzyme Activity

α-Amylase was covalently immobilized onto poly(methyl methacrylate-2-hydroxyethyl methacrylate) microspheres, which were activated by using either epichlorohydrin (ECH) or cyanuric chloride (C₃N₃Cl₃). The properties of the immobilized enzyme were investigated and compared with those of the free enzyme. For the assays carried out at 25 °C and pH 6.9, the relative activities were found to be 73.0% and 90.8% for epichlorohydrin and cyanuric chloride bound enzymes, respectively. Upon immobilization, the maximum activities were obtained at lower pH values and higher temperatures as compared with the free enzyme. Kinetic parameters were calculated as 2.51 g/L, 28.54 g/L and 15.50 g/L for K_m and 1.67 × 10⁻³ gL⁻¹ min⁻¹ 2.89 × 10⁻⁴ gL⁻¹ min⁻¹ and 1.89 × 10⁻³ gL⁻¹ min⁻¹ for V_max for free, epichlorohydrin and cyanuric chloride bound enzymes, respectively. Enzyme activities were found to be ca. 32.7% for ECH and 41.1% for C₃N₃Cl₃ activated matrices after storage for one month. On the other hand the free enzyme lost its activity completely within 20 days. Immobilization, storage stability and repeated use capability experiments carried out in the presence of Ca²⁺ ions demonstrated higher stability in the presence of these ions. The enzymes immobilized in the presence of Ca²⁺ ions retained 90.6% and 90.8% of the original activities even after 30 days in the case of ECH and C₃N₃Cl₃ activations, respectively. In repeated batch experiments, i.e., 20 uses of the enzyme in 3 days; in the absence of Ca²⁺ ions retentions of 79.2% and 77.1% of the original enzyme activities were observed for ECH and C₃N₃Cl₃ immobilized enzymes, respectively, whereas, in the case of addition of Ca²⁺ ions to the assay medium, these values were enhanced to 95.3% and 92.2%.