Calcium Alginate Immobilization Research Articles

Accurate and non-destructive monitoring of mold contamination in foodstuffs based on whole-cell biosensor array coupling with machine-learning prediction models

Mold contamination in foodstuffs causes huge economic losses, quality deterioration and mycotoxin production. Thus, non-destructive and accurate monitoring of mold occurrence in foodstuffs is highly required. We proposed a novel whole-cell biosensor array to monitor pre-mold events in foodstuffs. Firstly, 3 volatile markers ethyl propionate, 1-methyl-1 H-pyrrole and 2,3-butanediol were identified from pre-mold peanuts using gas chromatography-mass spectrometry. Together with other 3 frequently-reported volatiles from Aspergillus flavus infection, the volatiles at subinhibitory concentrations induced significant but differential response patterns from 14 stress-responsive Escherichia coli promoters. Subsequently, a whole-cell biosensor array based on the 14 promoters was constructed after whole-cell immobilization in calcium alginate. To discriminate the response patterns of the whole-cell biosensor array to mold-contaminated foodstuffs, optimal classifiers were determined by comparing 6 machine-learning algorithms. 100 % accuracy was achieved to discriminate healthy from moldy peanuts and maize, and 95 % and 98 % accuracy in discriminating pre-mold stages for infected peanuts and maize, based on random forest classifiers. 83 % accuracy was obtained to separate moldy peanuts from moldy maize by sparse partial least square determination analysis. The results demonstrated high accuracy and practicality of our method based on a whole-cell biosensor array coupling with machine-learning classifiers for mold monitoring in foodstuffs.

Jet cutter technique as a tool to achieve high lipase hydrolytic activity

Lipase immobilization has been widely studied because it allows for enzyme reuse and provides more assertive control over the catalytic process. This study aimed to evaluate the effect of bead size on the performance of entrapped lipase. Eversa® Transform 2.0 was immobilized on calcium alginate beads by jet cutting and dripping. Beads produced by jet cutting were small (D[3,4] = 803.36 ± 16.9 µm) and had a relatively narrow size distribution (span of 0.79). Beads obtained by extrusion dripping measured 2459.98 ± 15.6 µm and had a span of 0.45. Infrared spectroscopy and microscopic analysis confirmed the presence of lipase in both types of beads. Lipase showed high hydrolytic activity in its free form (15,000 U g−1). Immobilization in calcium alginate was effective but decreased recovered enzyme activity. The porosity of loaded beads varied with size. The high surface area (5.46 vs 3.13 m2 g−1) and porosity (76.33% vs 21.65%) of beads produced by jet cutting, as compared with those produced by dripping, favored enzyme activity (3000 vs 1500 U g−1 protein). The results indicate that facilitated mass transfer is an important factor in the development of immobilized enzymes.

Increased metal tolerance and bioaccumulation of zinc and cadmium in Chlamydomonas reinhardtii expressing a AtHMA4 C-terminal domain protein.

The use of microalgal biomass for metal pollutant bioremediation might be improved by genetic engineering to modify the selectivity or capacity of metal biosorption. A plant cadmium (Cd) and zinc (Zn) transporter (AtHMA4) was used as a transgene to increase the ability of Chlamydomonas reinhardtii to tolerate 0.2 mM Cd and 0.3 mM Zn exposure. The transgenic cells showed increased accumulation and internalization of both metals compared to wild-type. AtHMA4 was expressed either as the full-length (FL) protein or just the C-terminal (CT) tail, which is known to have metal-binding sites. Similar Cd and Zn tolerance and accumulation was observed with expression of either the FL protein or CT domain, suggesting that enhanced metal tolerance was mainly due to increased metal binding rather than metal transport. The effectiveness of the transgenic cells was further examined by immobilization in calcium alginate to generate microalgal beads that could be added to a metal contaminated solution. Immobilization maintained metal tolerance, while AtHMA4-expressing cells in alginate showed a concentration-dependent increase in metal biosorption that was significantly greater than alginate beads composed of wild-type cells. This demonstrates that expressing AtHMA4 FL or CT has great potential as a strategy for bioremediation using microalgal biomass.

Efficient and long-term vanillin production from 4-vinylguaiacol using immobilized whole cells expressing Cso2 protein

Vanillin is a well-known fragrant, flavoring compound. Previously, we established a method of coenzyme-independent vanillin production via an oxygenase from Caulobacter segnis ATCC 21756, called Cso2, that converts 4-vinylguaiacol to vanillin and formaldehyde using oxygen. In this study, we found that reactive oxygen species inhibited the catalytic activity of Cso2, and the addition of catalase increased vanillin production. Since Escherichia coli harbors catalases, we used E.coli cells expressing Cso2 to produce vanillin. Cell immobilization in calcium alginate enabled the long-term use of the E.coli cells for vanillin production. Thus, we demonstrate the possibility of using immobilized E.coli cells for both continuous and repeated batch vanillin production without any coenzymes.

Production of 2-keto-gluconic acid from glucose by immobilized Pseudomonas plecoglossicida resting cells.

2-Keto-d-gluconic acid (2KGA) is an important organic acid derived from d-glucose and is used to produce the food antioxidant erythorbic acid. To improve the 2KGA production performance and cell reusability, various carriers such as calcium alginate, k-carrageenan, chitosan, and poly(vinyl alcohol)-alginate were evaluated to immobilize Pseudomonas plecoglossicida JUIM01 resting cells. Calcium alginate was shown to be a suitable carrier since the immobilized cells had the highest number of reuse times and produced the highest 2KGA concentration of 171.77g/L, with a productivity of 3.58g/L·h and conversion ratio of 98.38%. The cell concentration, cultivation temperature, aeration rate and initial glucose concentration were further optimized in a 5-L airlift bioreactor to obtain the best 2KGA production performance by calcium alginate-immobilized P. plecoglossicida cells. Under the optimal conditions including a cell concentration of 4.0g/L, glucose concentration of 126.0g/L, temperature of 34°C and aeration rate of 2.8 L/min, 134.45g/L 2KGA was produced by alginate-immobilized P. plecoglossicida cells within 30h, with a total productivity of 4.48g/L·h and yield of 1.07g/g (conversion ratio of over 99.0%). The immobilized cells maintained a stable conversion capacity after nine reuses and 25days of storage at 4°C, which indicated that calcium alginate immobilization of P. plecoglossicida cells had industrial practicability for 2KGA production.

Mucin immobilization in calcium alginate: A possible mucus mimetic tool for evaluating mucoadhesion and retention of flavour

To reduce animal testing, there is a need to develop novel in-vitro models for evaluating the retention of bioactive compounds in food and pharmaceutical products. Here, a mucus-mimetic platform was developed through a one-step approach based on encapsulating mucin within alginate gel beads. We found that mucins form micron sized aggregates distributed across the surface of the calcium-alginate bead, as shown by environmental scanning electron microscopy (ESEM). Retention of bioactive compounds on the mucin-functionalised surface was tested using a commercial orange drink formulation. To aid flavour retention, different mucoadhesive polymers with varying charge, including anionic, neutral and strongly cationic, were tested for their ability to interact with mucin and aid retaining flavour compounds within the mucin-alginate bead. The alginate-mucin mucus mimic was validated using an ex-vivo bovine tongue, with the flavour retention results showing qualitative agreement. The developed method proved to be a convenient, efficient tool for providing information on the effectiveness of mucoadhesive polymers without variability, safety and sustainability issues associated with an ex-vivo or in-vivo system. We propose that by encapsulating other relevant oral proteins, alongside mucins, current gaps between in-vitro and the ex-vivo systems may be narrowed.

Immobilized and Free Cells of Geotrichum candidum for Asymmetric Reduction of Ketones: Stability and Recyclability.

Marine-derived fungus Geotrichum candidum AS 2.361 was previously reported by our group as an active strain for the enantioselective reduction of ketones. Although some other Geotrichum strains were also found from the terrestrial sources, information on their stability and reusability is scarce. Herein, the stabilities—in terms of pH tolerance, thermostability, and storage stability, and reusability—of G. candidum AS 2.361 were described for the asymmetric reduction of a series of aromatic ketones. Two differently immobilized cells (agar immobilization and calcium alginate immobilization) as well as free cells were prepared. For three substrates (1-(3-bromophenyl) ethan-1-one (1b), 1-(2-chlorophenyl) ethan-1-one (1d), and acetophenone (1g)) immobilized cells on agar showed a great improvement in the bioreduction activities compared to the free cells, increasing yields up to 97% with ee values of 99%. Cells immobilized on agar/calcium alginate could maintain more than 90% of the original activities within the assayed pH ranges of 3.5–11, while free cells were highly sensitive to alkaline and acidic conditions. Concerning thermostability, immobilized cells on agar kept 99% of their original activities after incubation at 60 °C for 1 h, while almost no activity was detected for the free cells under the same condition. Immobilized cells were stable at 4 °C for 80 days without any activity loss, while free cells started to decrease the activity after storage at 4 °C for six days. The immobilized cells retained almost 99% activity after four reuse cycles, while free cells lost almost all the activities at on the third cycle.

KINETIC AND ENZYME RECYCLING STUDIES OF IMMOBILIZED b-GLUCOSIDASE FOR LIGNOCELLULOSIC BIOMASS HYDROLYSIS

b-glucosidases are very important in the improvement of cellulose degradation rates by diminishing the inhibition of end products. Although, cellulase cost has reduced significantly in last decade but still the high costs of biomass saccharification is one of the main challenges for the commercialization of ethanol from LC biomass. In this work, -glucosidase immobilization in calcium alginate was optimized using response surface methodology. 89% immobilization efficiency was achieved under optimum conditions (i.e., calcium alginate 4.19% (w/v), calcium chloride 0.14 M and -glucosidase 4.42% (v/v) or 57.46 U/mL of reaction mixture). pH and temperature optima of both free and immobilized -glucosidase was found to be 5 and 50 C, respectively. Enzyme kinetics of free and immobilized -glucosidase was done to evaluate Michaelis constant (Km) and Vmax values, using pnitrophenyl- -D-glucopyranoside (pNPG) as the substrate and glucose as inhibitor. Increase in Km value (0.995 mM) for immobilized -glucosidase as compared to the free -glucosidase (0.617 mM) was observed. A decrease in Vmax from (1191.74 mol min-1 for free -glucosidase) to (736.65 mol min-1 immobilized -glucosidase) was observed after immobilization. Glucose was found to inhibit -glucosidase by competitive mode of inhibition (Ki= 3.01 mM). Studies were carried out in a batch reactor using 2% cellobiose, 0.1 M sodium acetate buffer, 5 pH and 50 C. 84% cellobiose to glucose conversion was found in first cycle which was about 67% in fifth cycle. This study suggests that enzyme immobilization improved enzyme stability, substrate inhibition and permits enzyme reusability.

A potential tissue culture approach for the phytoremediation of dyes in aquaculture industry

Brilliant green has been used as an effective compound to control external fungal and protozoan infections of fish though is carcinogenic and teratogenic. Present study focuses on the efficient degradation of brilliant green using callus cultures of the plant, Tecoma stans var. angustata, showing a peroxidase activity of 3.07 IUg−1. Callus cultures retained 86% activity after immobilization in calcium alginate. Dye degradation parameters were initially optimized using batch cultures. A packed bed reactor was constructed using the immobilized beads and different concentrations of the dye from 8.5 to 45mgl−1 along with H2O2 were given in an up-flow mode. Km and Vmax for the dye degradation were 0.01924gl−1 and 0.035gl−1h−1. The bioreactor could degrade 94% of the dye (concentration–35mgl−1) at a residence time of 42.0×10−3h. Percentage of degradation varied depending on the flow rate, residence time and dilution rate. TLC and reverse phase HPLC analysis showed that the dye was completely degraded to minor non toxic metabolites via complete degradation of the aromatic rings and by cleavage of functional groups. The current study is a preliminary work that can be used for application in Aquaculture and allied Industries where biodegradation of brilliant green is required.

Enhanced dibenzothiophene biodesulfurization by immobilized cells of Brevibacterium lutescens in n-octane-water biphasic system.

In this study, it was the first report that the Brevibacterium lutescens CCZU12-1 was employed as a sulfur removing bacteria. Using dibenzothiophene (DBT) as the sole sulfur source, B. lutescens could selectively degrade DBT into 2-hydroxybiphenyl (2-HBP) via the "4S" pathway. In the basal salt medium (BSM) supplemented with 0.25 mM DBT and 0.5 g/L Tween-80, high desulfurization rate (100 %) was obtained by growth cells after 60 h. Furthermore, the n-octane-water (10:90, v/v) biphasic system was built for the biodesulfurization by resting cells. Moreover, a combination of magnetic nano Fe3O4 particles with calcium alginate immobilization was used for enhancing biodesulfurization. In this n-octane-water biphasic system, immobilized B. lutescens cells could be reused for not less than four times. Therefore, B. lutescens CCZU12-1 shows high potential in the biodesulfurization.

Reusability of ADH and GDH producing Arxula adeninivorans cells and cell extract for the production of 1-(S)-phenylethanol

Abstract An Arxula adeninivorans strain co-expressing ADH of Rhodococcus ruber and GDH of Bacillus megaterium , coding for (S)-specific alcohol dehydrogenase (ADH) and glucose dehydrogenase (GDH) respectively, was used for the synthesis of enantiomerically pure 1-(S)-phenylethanol as permeabilized yeast cells (PC), permeabilized immobilized cells (PIC) or immobilized crude extract (IE). Permeabilization was achieved with Triton X-100 and resulted in cells that had the same activity as crude extract. Calcium alginate immobilization allowed the entrapment of enzymes and PIC, which resulted in a gain of 60 and 82% activity respectively compared to crude extract. All enzyme preparations were used in successive reaction cycles. PC and IE lost activity after 10 reactions whereas PIC were active with over 80% activity for 28 cycles. After 5 PIC catalyzed reactions, 94.8% chemically pure 1-(S)-phenylethanol with ee of ≥99% was isolated, which is a conversion of 79.6% of the substrate, acetophenone. Furthermore the reaction medium containing BIS-TRIS buffer could be regenerated by the addition of Ca(OH) 2 , which precipitated calcium gluconate and restored full buffering capacity. Stability is a key factor for the cost-effective use of an enzyme system in industry and these results indicate that A. adeninivorans PIC will be useful for the synthesis enantiomerically pure alcohols.

Highly enantioselective oxidation of racemic phenyl-1,2-ethanediol to optically pure (R)-(−)-mandelic acid by a newly isolated Brevibacterium lutescens CCZU12-1

Enantioselective oxidation of racemic phenyl-1,2-ethanediol into (R)-(-)-mandelic acid by a newly isolated Brevibacterium lutescens CCZU12-1 was demonstrated. It was found that optically active (R)-(-)-mandelic acid (e.e.p > 99.9 %) is produced leaving the other enantiomer (S)-(+)-phenyl-1,2-ethanediol intact. Using fed-batch method, a total of 172.9 mM (R)-(-)-mandelic acid accumulated in the reaction mixture after the seventh feed. Moreover, oxidation of phenyl-1,2-ethanediol using calcium alginate-entrapped resting cells was carried out in the aqueous system, and efficient biocatalyst recycling was achieved as a result of cell immobilization in calcium alginate, with a product-to-biocatalyst ratio of 27.94 g (R)-(-)-mandelic acid g⁻¹ dry cell weight cell after 16 cycles of repeated use.

Cyanide degradation by immobilized cells of Rhodococcus UKMP-5M

Anthropogenic sources contribute to the bulk presence of cyanide, which causes substantial health and environmental concerns. A petroleum-contaminated soil isolate, Rhodococcus UKMP-5M has been verified to efficiently degrade high concentration of cyanide in the form of KCN in our previous study. In order to enhance the cyanide-degrading ability of this bacterium, different encapsulation matrices were screened to immobilize cells of Rhodococcus UKMP-5M for degradation of cyanide. It was revealed that the biocatalyst activity and bead mechanical strength improved significantly when calcium alginate encapsulation technique was employed as compared to free cells. The results also indicated that the immobilized cell system could tolerate a higher level of KCN concentration and were able to support a higher biomass density. In addition, the embedded cells retained almost 96% of their initial cyanide removal efficiency during the first five batches and the entrapped cell system maintained 64% of its initial activity after eight successive batches. The encapsulated beads could be easily recovered from the production medium and reused for up to five batches without significant losses of cyanide-degrading ability, which proved to be advantageous from an economic point of view. From this study, it could be inferred that the novel Rhodococcus UKMP-5M strain demonstrated high cyanide-degrading ability and the optimized calcium alginate immobilization technique provided a promising alternative for practical application of large scale remediation of cyanide-bearing wastewaters.

Biosynthesis of benzoylformic acid from benzoyl cyanide by a newly isolated Rhodococcus sp. CCZU10-1 in toluene–water biphasic system

Benzoylformic acid was synthesized from the hydrolysis of benzoyl cyanide by a newly isolated Rhodococcus sp. CCZU10-1. In this study, an aqueous–toluene biphasic system was developed for highly efficient production of benzoylformic acid from the hydrolysis of benzoyl cyanide. In the aqueous–toluene biphasic system, the phase volume ratio, buffer pH and reaction temperature were optimized. Using fed-batch method, a total of 932mM benzoylformic acid accumulated in the reaction mixture after the 10th feed. Moreover, enzymatic hydrolysis of benzoyl cyanide using calcium alginate entrapped resting cells was carried out in the aqueous–toluene biphasic system, and efficient biocatalyst recycling was achieved as a result of cell immobilization in calcium alginate, with a product-to-biocatalyst ratio of 14.26g benzoylformic acid g−1 dry cell weight (DCW) cell after 20 cycles of repeated use.

Producción y Caracterización de Xilanasas Por Inmovilización en un Sistema de Cultivo Continuo de la Bacteria Termófila Cepa FT3 en Alginato de Calcio

Las xilanasas son exoglucanasas capaces de degradar xilano. Estas enzimas se pueden aplicar en varios procesos industriales como es la degradación de residuos agrícolas, producción de azúcares fermentables, producción de etanol, industria papelera y en distintos procesos relacionados a la industria alimenticia. La aplicación industrial sin embargo, requiere estabilidad principalmente a elevadas temperaturas, por ello existe gran interés en el estudio de microorganismos termófilos. En este estudio se optimizó la producción de xilanasas, en una primera fase en cultivo en batch y posteriormente en cultivo continuo en un sistema de inmovilización con alginato de calcio de la cepa bacteriana FT3 anaeróbica termòfila, aislada de la región altiplánica de Bolivia. Los resultados muestran que usando un medio basai, la actividad es de 3 [UI/mL]. Esta actividad fue incrementada a 4.5 [UI/mL], en cultivo en batch con alginato calcico de alta viscosidad al 2 %. Cuando el sistema inmovilizado fue ensayado en un cultivo continuo que permitiera la recuperación de la enzima, se logró incrementar la actividad hasta 13 [UI/mL]. El cambio de NH4C1 por extracto de levadura como fuente de nitrógeno y residuos de soya como fuente de carbono en el medio permitió mayor incremento de la actividad enzimàtica en el biorreactor hasta 17 [UI/mL],

Biocatalytic synthesis of (R)-(−)-mandelic acid from racemic mandelonitrile by cetyltrimethylammonium bromide-permeabilized cells of Alcaligenes faecalis ECU0401

The nitrilase from Alcaligenes faecalis ECU0401 belongs to the category of arylacetonitrilase, which could hydrolyze 2-chloromandelonitrile, 3,4-dimethoxyphenylacetonitrile, mandelonitrile, and phenylacetonitrile into the corresponding arylacetic acids. To overcome the permeability barrier and prepare whole cell biocatalysts with high activities, permeabilization of Alcaligenes faecalis ECU0401 in relation to nitrilase activity was optimized by using cetyltrimethylammonium bromide (CTAB) as permeabilizing agent. The nitrilase activity from Alcaligenes faecalis ECU0401 increased 4.5-fold when the cells were permeabilized with 0.3% (w/v) CTAB for 20 min at 25 degrees C and pH 6.5. Consequently, almost all the mandelonitrile was consumed and converted to (R)-(-)-mandelic acid with greater than 99.9% enantiomeric excess (e.e.) by the CTAB-permeabilized cells. The permeability barrier has been significantly reduced in the hydrolysis of mandelonitrile by using CTAB-permeabilized cells and a dynamic resolution was successfully achieved, giving a 100% theoretical yield of (R)-(-)-mandelic acid. Efficient biocatalyst recycling was achieved as a result of cell immobilization in calcium alginate, with a product-to-biocatalyst ratio of 3.82 g (R)-(-)-mandelic acid g(-1) dry cell weight (dcw) cell after 20 cycles of repeated use.

A Comparative Study of Immobilized Nitrifying and Co-Immobilized Nitrifying and Denitrifying Bacteria for Ammonia Removal from Sludge Digester Supernatant

Bench scale kinetic experiments were conducted to examine the use of cell immobilization in calcium alginate to remove ammonia in anaerobic sludge digester supernatant. Two systems, immobilized nitrifiers and co-immobilized nitrifiers and denitrifiers, were studied with and without the addition of methanol. Results indicated that partial nitrification (to nitrite) was achieved in both systems. The co-immobilized reactors did not exhibit the extent of nitrite accumulation observed in the solely nitrifying reactors. The nitrifying reactors were unable to buffer the hydrogen ion production, during the nitrification process, to the level the co-immobilized cell reactors achieved. Both of these differences suggested the occurrence of denitrification in the co-immobilized reactors. Scanning electron microscopic images of bacteria immobilized in the alginate spherical beads support the results of the kinetic experiments. Nitrifiers colonized in the 100–200 μm peripheral layer of the beads. Large voids caused by nitrogen gas due to denitrification were found in a number of co-immobilized bead samples.

Evaluation of various parameters of calcium-alginate immobilization method for enhanced alkaline protease production by Bacillus licheniformis NCIM-2042 using statistical methods

Calcium-alginate immobilization method for the production of alkaline protease by Bacillus licheniformis NCIM-2042 was optimized statistically. Four variables, such as sodium-alginate concentration, calcium chloride concentration, inoculum size and agitation speed were optimized by 2 4 full factorial central composite design and subsequent analysis and model validation by a second-order regression equation. Eleven carbon, 11 organic nitrogen and seven inorganic nitrogen sources were screened by two-level Plackett–Burman design for maximum alkaline protease production by using optimized immobilized conditions. The levels of four variables, such as Na-alginate 2.78%; CaCl 2, 2.15%; inoculum size, 8.10% and agitation, 139 rpm were found to be optimum for maximal production of protease. Glucose, soybean meal and ammonium sulfate were resulted in maximum protease production at 644 U/ml, 720 U/ml, and 806 U/ml when screened for carbon, organic nitrogen and inorganic nitrogen sources, respectively, using optimized immobilization conditions. Repeated fed batch mode of operation, using optimized immobilized conditions, resulted in continuous operation for 12 cycles without disintegration of beads. Cross-sectional scanning electron microscope images have shown the growth pattern of B. licheniformis in Ca-alginate immobilized beads.

Influence of phenol on the biodegradation of pyridine by freely suspended and immobilized Pseudomonas putida MK1

To study the effect of co-contaminants (phenol) on the biodegradation of pyridine by freely suspended and calcium alginate immobilized bacteria. Varying concentrations of phenol were added to free and calcium alginate immobilized Pseudomonas putida MK1 (KCTC 12283) to examine the effect of this pollutant on pyridine degradation. When the concentration of phenol reached 0.38 g l(-1), pyridine degradation by freely suspended bacteria was inhibited. The increased inhibition with the higher phenol levels was apparent in increased lag times. Pyridine degradation was essentially completely inhibited at 0.5 g l(-1) phenol. However, immobilized cells showed tolerance against 0.5 g l(-1) phenol and pyridine degradation by immobilized cell could be achieved. This works shows that calcium alginate immobilization of microbial cells can effectively increase the tolerance of P. putida MK1 to phenol and results in increased degradation of pyridine. Treatment of wastewater stream can be negatively affected by the presence of co-pollutants. This work demonstrates the potential of calcium alginate immobilization of microbes to protect cells against compound toxicity resulting in an increase in pollutant degradation.

Growth patterns of Escherichia coli O157:H7 in ground beef treated with nisin, chelators, organic acids and their combinations immobilized in calcium alginate gels

The effects of antimicrobial substances including nisin, acetic acid, lactic acid, potassium sorbate and chelators (disodium ethylenediamine tetraacetic acid [EDTA] and sodium hexametaphosphate [HMP]), alone or in combination and, with or without immobilization in calcium alginate gels, on the growth of Escherichia coli O157:H7 in ground beef were investigated. Results showed that acetic acid and potassium sorbate could inhibit the growth of E. coli O157:H7 effectively at 10°C and at 30°C. Both EDTA and HMP did not halt the growth of E. coli O157:H7. In an antimicrobial system immobilized with calcium alginate, most of the antimicrobials could not inhibit the growth of E. coli O157:H7 in ground beef at 10°C and at 30°C, with the exception of acetic acid and lactic acid. Immobilization did not enhance the effectiveness of acetic acid against E. coli O157:H7 in ground beef at 10°C and at 30°C ( P>0.05) but it did enhance the effectiveness of lactic acid at 10°C. In a system combining different antimicrobials, treatment with nisin /EDTA or nisin/potassium sorbate at 10°C revealed a significantly lower population change of E. coli O157:H7 compared to samples treated with nisin, EDTA or potassium sorbate alone. The use of calcium alginate immobilization further enhanced the effectiveness of the combination system of nisin/EDTA, nisin/acetic acid and nisin/potassium sorbate on the growth of E. coli O157:H7 in ground beef at 10°C but it was not effective at 30°C.