Biological control of foodborne pathogens by lacticacid bacteria: A focus on juice processing industries

Abstract The use of lactic acid bacteria (LAB) in foods as biocontrol agents against foodborne pathogens has become increasingly known. Under the premise that controlling the adhesion of microorganisms to food contact surfaces is an essential step for meeting the goals of food processing, the aim of this work was to investigate the inhibitory and anti-biofilm effectiveness of Lactobacillus rhamnosus GG (ATCC 53103) and Lactobacillus casei (ATCC 393) against Escherichia coli O157:H7, Salmonella enterica and Listeria monocytogenes. Lactobacillus strains (108UFCCFU/ml) and pathogens (104UFCCFU/ml) were evaluated to monitor LAB anti-adhesive and antibiofilm effect, in two main scenarios: (i) co-adhesion and (ii) pathogen incorporation to stainless steel surfaces with a protective biofilm of Lactobacillus cells. In (i) the predominant effect was observed in L. rhamnosus against S. enterica and L. monocytogenes, whereas in (ii) both LAB significantly reduced the number of pathogenic adherent cells. The effect of pre-established LAB biofilms was more successful in displacing the three pathogens than when they were evaluated under co-adhesion. These findings show that both LAB can be considered good candidates to prevent or inhibit the adhesion and colonization of L. monocytogenes, S. enterica and E. coli O157:H7 on surfaces and conditions of relevance for juice processing industries, offering alternatives for improving the safety and quality of fruit-based products.

Resumen Existe un creciente interés en el uso de bacterias ácido lácticas (BAL) como agentes de biocontrol frente a patógenos de transmisión alimentaria. Bajo la premisa de que el control de la adhesión de microorganismos a superficies de contacto con alimentos es el paso esencial para evitar su contaminación, el objetivo de este trabajo fue investigar la efectividad inhibitoria y antibiofilm de Lactobacillus rhamnosus GG (ATCC 53103) y Lactobacillus casei (ATCC 393) frente a Escherichia coli O157:H7, Salmonella enterica y Listeria monocytogenes. A fin de cumplir con el objetivo propuesto, las cepas de Lactobacillus (108UFCUFC/ml) y los patógenos (104UFCUFC/ml) se ensayaron en 2 escenarios: (1) coadhesión, y (2) incorporación de los patógenos a las superficies de acero inoxidable con un biofilm preformado de Lactobacillus. En (1), el efecto predominante se observó con L. rhamnosus frente a S. enterica y L. monocytogenes, mientras que en (2), ambas BAL redujeron significativamente el número de células patógenas adheridas. En función de estos resultados, concluimos que el efecto de un biofilm preformado de ambas BAL fue más exitoso en el desplazamiento de los 3 patógenos que en coadhesión. Ambas BAL pueden considerarse buenas candidatas para mitigar la adhesión y colonización de L. monocytogenes, S. enterica y E. coli O157:H7 en superficies en condiciones de relevancia para la industria procesadora de jugos, y, de esta manera, ofrecer alternativas para mejorar la seguridad y calidad de los alimentos a base de frutas.

Inactivation of Alicyclobacillus acidoterrestris ATCC 49025 spores in apple juice by pulsed light: Influence of initial contamination and required reduction levels / Inactivación de esporas de Alicyclobacillus acidoterrestris ATCC 49025 por luz pulsada en jugo de manzana: Influencia de la contaminación inicial y de las reducciones requeridas

The purpose of this study was to analyze the response of different initial contamination levels of Alicydobadllus acidoterrestris ATCC 49025 spores in apple juice as affected by pulsed light treatment (PL, batch mode, xenon lamp, 3pulses/s, 0-71.6 J/cm²). Biphasic and Weibull frequency distribution models were used to characterize the relationship between inoculum size and treatment time with the reductions achieved after PL exposure. Additionally, a second order polynomial model was computed to relate required PL processing time to inoculum size and requested log reductions. PL treatment caused up to 3.0-3.5 log reductions, depending on the initial inoculum size. Inactivation curves corresponding to PL-treated samples were adequately characterized by both Weibull and biphasic models (R²d j 94-96%), and revealed that lower initial inoculum sizes were associated with higher inactivation rates. According to the polynomial model, the predicted time for PL treatment increased exponentially with inoculum size.

El objetivo del presente trabajo fue evaluar la influencia de la concentración de esporas de Alicyclobacillus acidoterrestris ATCC 49025 en la respuesta de inactivación por acción de la luz pulsada (modo estanco, lámpara de xenón, 3 pulsos/s, 0-71,6 J/cm²) en jugo de manzana comercial. Para caracterizar la relación existente entre la concentración de esporas y el tiempo de tratamiento con las reducciones logarítmicas alcanzadas luego de la exposición a la luz pulsada (LP), se aplicaron 2 modelos: el de Weibull y el bifásico. Adicionalmente, se estimó la relación entre el tiempo de tratamiento con LP y la concentración inicial de inoculo en el jugo con las reducciones logarítmicas logradas mediante regresión múltiple y la metodología de superficie de respuesta (MSR). La inactivación por LP provocó entre 3 y 3,5 reducciones logarítmicas, según la concentración inicial de esporas. Las curvas de inactivación fueron adecuadamente caracterizadas por los modelos matemáticos propuestos (Restado = 94-96%). El análisis por MSR permitió predecir un aumento exponencial del tiempo de tratamiento requerido conforme se incrementa el nivel de contaminación inicial.

A Novel Raw Starch Hydrolyzing Thermostable α-Amylase Produced by Newly Isolated Bacillus mojavensis SO-10: Purification, Characterization and Usage in Starch Industries

ABSTRACT The aim of this study is the production, purification, and characterisation of thermostable raw starch hydrolyzing α-amylase produced by Bacillus mojavensis SO-10. The maximum production conditions of α-amylase were found at 36th hour, 35 °C and pH 7.0. We utilized three steps to purify the thermostable α-amylase and as a result, 34-fold and 18% yield were obtained. The molecular weight of purified α-amylase was determined as 73 kD. The Km and Vmax rates were detected as 0.010 mM and 3.38 µmol min−1, respectively. This purified α-amylase exhibited the highest activity at pH 5.0-6.0 and 70 ºC and showed stability over a wide variety of pH and temperature at 4.0-8.0, and 40-50 ºC, respectively. The thermostable purified α-amylase exhibited stability in the presence of denaturing agents and heavy metal ions. The purified enzyme hydrolyzed the raw starches of corn and wheat grains in the ratio of 36.7% and 39.2% respectively. The end-yields of soluble starch hydrolysis were analyzed by thin-layer chromatography (TLC). In addition, the usage of purified α-amylase in clarification of apple juice and domestic washing detergent industries were evaluated.

Antibacterial activity of lime (Citrus x aurantifolia) essential oil against Listeria monocytogenes in tyndallised apple juice / Actividad antimicrobiana del aceite de lima (Citrus x aurantifolia) contra Listeria monocytogenes en jugo de manzana tindalizado

The antibacterial activity of lime (Citrus x aurantifolia) essential oil (EO) against the foodborne pathogen Listeria monocytogenes in tyndallised apple juice was studied at two temperatures. The EO concentration required to produce a significant increase in the lag phase of bacterial growth was determined. The addition of 200 µL of lime EO per 100 mL of apple juice completely inhibited the growth of L. monocytogenes at 5 ºC and at 37 ºC. This concentration of EO extended the lag time at least 292.7% compared to juice without EO. This is especially important considering that L. monocytogenes was able to grow in the juice at low temperatures in the absence of EOs.

En este trabajo se estudió la actividad antibacteriana del aceite esencial de lima (Citrus x aurantifolia) contra Listeria monocytogenes, patógeno alimentario, cultivado en jugo de manzana tindalizado a dos temperaturas. Se determinó la concentración necesaria del aceite esencial para producir una extensión significativa de la fase de retraso. La adición de 200 µL de aceite esencial de lima por 100 mL de jugo de manzana tindalizado, a 5 ºC produjo la inhibición total del crecimiento de L. monocytogenes, en tanto que con el mismo volumen a 37 ºC la fase de retraso se extendió a 24,7 h (292,7%). Esto es importante debido a que L. monocytogenes fue capaz de crecer en este sustrato a temperaturas bajas en ausencia de aceite esencial.

Application of decolourized and partially purified polygalacturonase and α-amylase in apple juice clarification

Polygalacturonase and α-amylase play vital role in fruit juice industry. In the present study, polygalacturonase was produced by Aspergillus awamori Nakazawa MTCC 6652 utilizing apple pomace and mosambi orange (Citrus sinensis var mosambi) peels as solid substrate whereas, α-amylase was produced from A. oryzae (IFO-30103) using wheat bran by solid state fermentation (SSF) process. These carbohydrases were decolourized and purified 8.6-fold, 34.8-fold and 3.5-fold, respectively by activated charcoal powder in a single step with 65.1%, 69.8% and 60% recoveries, respectively. Apple juice was clarified by these decolourized and partially purified enzymes. In presence of 1% polygalacturonase from mosambi peels (9.87 U/mL) and 0.4% α-amylase (899 U/mL), maximum clarity (%T660nm = 97.0%) of juice was attained after 2 h of incubation at 50 ºC in presence of 10 mM CaCl2. Total phenolic content of juice was reduced by 19.8% after clarification, yet with slightly higher %DPPH radical scavenging property.

Survey of molds, yeast and Alicyclobacillus spp. from a concentrated apple juice productive process

Bacteria and molds may spoil and/or contaminate apple juice either by direct microbial action or indirectly by the uptake of metabolites as off-flavours and toxins. Some of these microorganisms and/or metabolites may remain in the food even after extensive procedures. This study aim to identify the presence of molds (including heat resistant species) and Alicyclobacillus spp., during concentrated apple juice processing. Molds were isolated at different steps and then identified by their macroscopic and microscopic characteristics after cultivation on standard media at 5, 25 and 37ºC, during 7 days. Among the 19 isolated found, 63% were identified as Penicillium with 50% belonging to the P. expansum specie. With regards to heat resistant molds, the species Neosartorya fischeri, Byssochlamys fulva and also the genus Eupenicillium sp., Talaromyces sp. and Eurotium sp. were isolated. The thermoacidophilic spore-forming bacteria were identified as A. acidoterrestris by a further investigation based on 16S rRNA sequence similarity. The large contamination found indicates the need for methods to eliminate or prevent the presence of these microorganisms in the processing plants in order to avoid both spoilage of apple juice and toxin production.

Green Synthesis of Fluorescent Carbon Quantum Dots for Detection of Hg2+ / 分析化学

Fluorescent carbon quantum dots ( CQDs) were synthesized by one-step hydrothermal treatment of apple juice. Experiments showed that Hg2+could quench the fluorescence of the CQDs with specificity. Based on this phenomenon, a selective and sensitive sensor was constructed for Hg2+ detection. In a NaH2 PO4-Na2HPO4 buffer solution (pH 7. 0), their fluorescence intensity showed good linear relationship with the concentrations of Hg2+ from 5 to 100 nmol/L and 1 to 50 μmol/L, respectively, with the detection limit of 2. 3 nmol/L (S/N=3). Its practical application was further demonstrated by the detection of Hg2+ in real water samples.

Cashew apple juice as microbial cultivation medium for non-immunogenic hyaluronic acid production

In this work, natural cashew apple juice was used as cultivation medium as an alternative to substitute brain heart infusion medium. The effect of aeration and juice supplementation with yeast extract on the production of hyaluronic acid in batch fermentation was also investigated. Similar levels of cell mass were obtained in inoculum using cashew apple juice supplemented with yeast extract or the conventional brain heart infusion medium. Fermentation in Erlenmeyer flasks produced low biomass and hyaluronic acid concentrations. The hyaluronic acid concentration and viscosity increased from 0.15 g/L and 3.87 cP (no aeration or medium supplementation) to 1.76 g/L and 107 cP, when aeration (2 vvm) and 60 g/L of yeast extract were used. The results suggest the production of low-molecular weight hyaluronic acid oligomers instead of the high molecular weight polymer.

Apple wine processing with different nitrogen contents

The aim of this work was to evaluate the nitrogen content in different varieties of apple musts and to study the effect of different nitrogen concentrations in apple wine fermentation. The average total nitrogen content in 51 different apples juices was 155.81 mg/L, with 86.28 percent of the values above 100 mg/L. The apple must with 59.0, 122.0 and 163.0 mg/L of total nitrogen content showed the maximum population of 2.05x 10(7); 4.42 x 10(7) and 8.66 x 10(7) cell/mL, respectively. Therefore, the maximum fermentation rates were dependent on the initial nitrogen level, corresponding to 1.4, 5.1 and 9.2 g/L.day, respectively. The nitrogen content in the apple musts was an important factor of growth and fermentation velocity.

Isolation and Identification of Thermoacidiphilic Bacteria from Apple Juice Concentrate / 微生物学通报

The thermotolerant bacteria which was isolated from the Apple Juice Concentrate(AJC) was investigated by comparison with the standard strain of Aliyclobacillus acidoterrestris DSM3922 using Kirin-medium acidified with malic acid.The results shows the two polluted bacteria isolated from AJC can grow under the temperature of 21?C～55?C and pH of 2.4～6.2,which was corresponded with the characteristics of the thermo-acidiphilic Alicyclobacillus spp..Further more,the cell morphology,colony morphology,cultural characteristics and physiological characteristics tests indicated the two isolated strains of thermotolerant bacteria have obviously similar characteristics with the standard strain of Aliyclobacillus acidoterrestris DSM3922.

Ocorrência de patulina em suco de maçä por cromatografia líquida de alta eficiência / Ocurrence of patulin in apple juice by high-performance liquid chromatography

Patulina é uma micotoxina produzida por várias espécies de fungos (Penicillium, Aspergillus e Biossochlamys), principalmente pelo Peniccillium expansum, fungo presente nas maçäs podres. É um composto tóxico, que em experimentos com animais tem demonstrado ter atividade mutagênica, carcinogênica. O conteúdo de patulina em suco de maçä é um bom indicador da qualidade das maçäs utlizadas no processamento. Treze marcas comercializadas em Belo Horizonte/Minas Gerais foram coletadas entre março e setembro de 1999. A metodologia utilizada foi a cromatografia líquida de alta eficiência (CLAE) em fase reversa. Patulina foi extraída com acetato de etila e o extrato foi purificado por participaçäo com soluçäo de carbonato de sódio. A separaçäo e quantificaçäo foi realizada em um cromatógrafo líquido por meio de uma coluna de fase reversa C18(3um) e detetor de ultravioleta a 278nm. Patulina e 5-hidroximetilfurfural (5-HMF) foram completamente separados utilizando como fase móvel 0,8(por cento) tetrahidrofurano em água contendo 0,02(por cento) de azida de sódio em uma vazäo de 0,5mL/minuto. O limite de detecçäo foi <5ug/L. Foram obtidos recuperaçöes média de patulina superiores a 73(por cento) e coeficientes de variaçäo menores que 7(por cento). Patulina foi detectada em apenas uma amostra (10ug/L). Apesar da baixa incidência de patulina em suco de maçä neste estudo, autoridades brasileiras de saúde devem continuar a monitorar estes produtos. (AU)

Patulin is a mycotoxin produced by several species of moulds (Penicillium, Aspergillus andBissochlamys), but mainly by the apple-rotting fungus Penicillium expansum. Patulin is a highly toxiccompound that has been shown to be mutagenic, carcinogenic and teratogenic in experiments with animals.The patulin content of apple juice is a good indicator of the quality of the apples used in the process.Thirteen brands were collected from retail markets in the city of Belo Horizonte, state of Minas Gerais,Brazil, between March and September 1999. A rapid reversed-phase liquid chromatographic determinationof patulin was used. Patulin was extracted with ethyl acetate and the extract was cleaned up by partitionwith sodium carbonate solution. The identification and quantification of patulin was determined by C18reversed phase column (3 µm) and a variable-wavelength UV-VIS detector set at 278 nm. Patulin and 5-hydroxymethylfurfural were completely resolved by using 0,8% tetrahydrofuran in water containing 0,02%sodium azide as the mobile phase at a flow rate of 0,5 mL/min. The detection limit was < 5 µg/L. Averagerecoveries greater than 73% of patulin and coefficients of variation of less than 7% were obtained. Patulinwas detected in only one sample (10 µg/L). Although the incidence of patulin in apple juice in the presentstudy was low, Brazilian health authorities should continue monitoring these products. (AU)

Study on isolation and detection of thermo-acidophilic bacteria in apple juice concentrate / 西安交通大学学报(医学版)

Objective To determine the best method to detect thermo-acidophilic bacteria in apple juice concentrate. Methods L 16 (4 5) orthogonal test was applied to analyse the four factors, including culture medium, ways of heat shock, dilution degrees of AJC and pH value. The results were comprehensively scored according to the number of colonies. Results The isolated bacteria were polymorphous. A better method was as follows: after being diluted to 1∶10, the AJC was submitted to a heat shock at 80 ℃ for 10 min, and then spread on k agar with a pH value of 3.5. Samples were incubated at 45 ℃ for 3 days, and then enumerated. Conclusion This method can considerably improve the sensitivity of detecting thermo-acidophilic bacteria in apple juice concentrate.