Chitosan inhibits growth of spoilage micro-organisms in chilled pork products

The aim of this study was to develop novel preservation systems for chilled, comminuted pork products that are sold raw using the natural compound chitosan (polymeric ß -1,4- N -acetylglucosamine). In vitro testing showed that viable numbers of Saccharomycodes ludwigii were reduced by up to 4 log cfu ml⁻¹ on exposure to 0·05% chitosan in 0·9% saline at pH 6·2. Higher concentrations of chitosan (0·25 and 0·5%) were required to achieve similar levels of inactivation withLactobacillus viridescens, Lac. sake and Listeria innocua. Torulaspora delbrueckii and Salmonella enteritidis PT4 were resistant to chitosan at the concentrations tested in this study (up to 0·5%). Trials in real foods showed that dipping of standard and skinless pork sausages in chitosan solutions (1·0%) reduced the native microflora (total viable counts, yeasts and moulds, and lactic acid bacteria) by approximately 1–3 log cfu g⁻¹ for 18 days at 7°C. Chitosan treatment increased the shelf-life of chilled skinless sausages from 7 to 15 days. Addition of 0·3 and 0·6% chitosan to an unseasoned minced pork mixture reduced total viable counts, yeasts and moulds, and lactic acid bacteria by up to 3 log cfu g⁻¹ for 18 days at 4°C compared with the untreated control. The results indicated that chitosan was an effective inhibitor of microbial growth in chilled comminuted pork products.     

The effect of a competitive microflora,pH and temperature on the growth kinetics of Escherichia coli O157:H7

Growth curves were generated for Escherichia coli O157:H7 in brain–heart infusion broth incubated at 37 or 15°C in the presence of individual and combinations of competing microflora. Broths were inoculated withE. coli O157:H7 (log₁₀3·00 cfu ml⁻¹) and competitors (log₁₀4·00 cfu ml⁻¹) and the initial pH of the broth was either neutral (7·0) or adjusted to 5·8 and then sequentially reduced to 4·8 over 10 h to simulate fermentation conditions. Growth curves were also generated for the competitors in these cultures, including Pseudomonas fragi, Hafnia alvei, Pediococcus acidilactici (pepperoni starter culture) and Brochothrix thermosphacta . Gompertz equations were fitted to the data and growth kinetics including lag phase duration, exponential growth rates and maximum population densities (MPD) calculated. In pure culture, the growth parameters for E. coli O157:H7 in neutral pH broths were significantly different from those recorded in simulated fermentation broths (P<0·05). The presence of competitors in the broth also had a significant effect on the growth kinetics of the pathogen. H. alvei significantly inhibited the growth (lag phase, growth rate and MPD) of E. coli O157:H7 at 37°C, neutral pH and outgrew the pathogen under these conditions. In neutral pH cultures, two other competitors, B. thermosphacta and P. acidilactici also inhibited the lag phase of the pathogen but had no effect on the other growth parameters. In simulated fermentation broths, the growth rate of E. coli O157:H7 was consistently slower and the MPD lower in the presence of a competitive microflora than when grown individually. At 15°C, only one competitor, P. fragi significantly inhibited the lag phase of the pathogen. The implications of these findings for food safety are discussed.     

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.     

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.     

Spore-forming bacteria in commercial cooked,pasteurised and chilled vegetable purées

In commercial purées of broccoli, carrot, courgette, leek, potato and split pea, pasteurized in their final packaging and analysed at two periods, Bacillus spp. were the dominant aerobic mesophilic bacteria (AMB). Initial numbers were generally lower than 2 log cfu g⁻¹. They increased up to 6–8 log cfu g⁻¹after about 20 days of storage at 10°C. At 4°C, numbers of AMB after 20 days were lower than 3 log cfu g⁻¹in potato purée, lower than 4 log cfu g⁻¹in leek purée, and between 3 and 6 log cfu g⁻¹in other products. Strict anaerobes were in markedly lower numbers than AMB. At all storage temperatures tested courgette purée usually showed the most rapid bacterial growth and spoilage. On this product, an increase in storage temperature from 4°C to 10°C resulted in a threefold reduction in time to 5 log cfu g⁻¹, and time to spoilage. Growth kinetics of AMB in courgette purée at 20°C, 15°C, 10°C, 6·5°C and 4°C were determined using a mathematical model. Three hundred and forty eight isolates were identified using the API system. Bacillus circulans, B. macerans and B. polymyxa were among the main species isolated from products stored at 4°C and 10°C, while B. subtilis and B. licheniformis were the dominant species in product stored at abuse temperature. Bacillus cereus was isolated from all storage conditions, but mostly from products stored at abuse temperature.     

Microbial competition: effect of Pseudomonas fluorescens on the growth of Listeria monocytogenes

Listeria monocytogenes Scott A was cultured alone and in coculture with Pseudomonas fluorescens ATCC 33231 to characterize quantitatively the effects of microbial competition on the growth of this psychrotrophic pathogen. The bacteria were cultured in brain–heart infusion broth (BHI), using a 3×3×3×2 complete factorial design to assess the impact of temperature (4, 12, 19°C), initial pH (5·0, 6·0, 7·0), and sodium chloride content (5, 25, 45 gl⁻¹) on the interaction between the two micro-organisms. Samples were periodically plated on BHI agar and Vogel Johnson agar to obtain total counts and L. monocytogenes counts, respectively. Growth curves were generated by fitting the data to the Gompertz equation, and the derived growth kinetics were compared. WhenP. fluorescens did influence the growth of L. monocytogenes, the primary effect was a suppression of the maximum population density (MPD) reached by the pathogen. Suppression of L. monocytogenes was generally associated with low incubation temperatures (4°C) and sodium chloride levels (5 and 25 gl⁻¹). Slight increases (<1·0 log cfu ml⁻¹) in the MPD attained by L. monocytogenes were observed when grown in the presence of P. fluorescens at higher temperatures (12 and 19°C) and sodium chloride levels (25 and 45 gl⁻¹) when the pH was 5·0. The current study supports earlier work that indicates that reliance on microbial competition as a barrier to control L. monocytogenes in refrigerated foods will require detailed knowledge of how the interaction between the pathogen and the microflora is affected by environmental and food characteristics such as storage temperature, pH, and water activity.     

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.     

Temperature abuse initiating yeast growth in yoghurt

The occurrence of yeasts in dairy products is significant because they can cause spoilage, effect desirable biochemical changes and they may adversely affect public health. While fermentative and spoilage activities of yeasts at elevated temperatures are well known in many food and beverage commodities, little attention has been given to the specific occurrence and significance of yeasts in dairy products at these temperatures. Since yeasts play a substantial role in the spoilage of commercial fruit yoghurts, especially when cold storage practices are neglected, the deterioration of yoghurt samples obtained from the manufacturers were evaluated at different temperatures for a period of 30 days during this study. Total yeast were enumerated, isolated and identified from the yoghurt samples. The highest number of yeast populations, up to 10⁵ and 10⁶ cfu/g, was found when yoghurts were exposed to elevated temperatures in the range of 25 °C, while lower yeast counts (10³ and 10⁴ cfu/g) were obtained from samples kept refrigerated at a temperature of 5 °C. Based on the results obtained, the interaction between the yeasts and lactic acid bacteria resulted in a decline in pH values and the stabilization of viable lactic acid bacterial loads. The most prevalent yeast species isolated, included strains of Kluyveromyces marxianus, Saccharomyces cerevisiae, Saccharomyces exiguus andYarrowia lipolytica.     

Microbial growth in vacuum packaged frankfurters produced in Spain

The effects of storage temperature and time on the microflora of vacuum packed frankfurters were analysed. Total aerobic counts, lactic acid bacteria, Brochothrix thermosphacta and yeast and moulds, were determined as functions of time. Statistically significant differences were observed (P ≤0·001) amongst the counts obtained at 2 and 7°C. At the end of the controlled storage period (42 days) the sausages kept at 7°C showed signs of spoilage. The appearance of superficial slime occurred after 28 days in samples with lactic acid bacteria at counts higher than 10⁸ cfu g⁻¹. The swelling of the pack showed after 42 days in those samples which had a level higher than 5 × 10⁸ cfu g⁻¹. On the contrary, the samples kept at 2°C did not show the aforesaid spoilage signs during the period of controlled storage. Basing our conclusions on the results obtained, a period of shelf life of 28 days was established at 7°C, whilst at 2°C a minimum shelf life of 42 days was obtained.     

Influence of storage temperature on the quality of beef liver; pH as a reliable indicator of beef liver spoilage

The development of the microbial flora specifically involved in the spoilage of sliced beef livers packaged and stored under aerobic conditions at 0 and 3 °C for 14 days was studied. Changes in the pH value of the product were also determined. The possibility that pH value could be considered as a quick and reliable indicator of incipient spoilage was particularly considered. All microbial groups (except micrococci) showed differences in their rates of growth between 0 and 3 °C. Pseudomonads and lactic acid bacteria were the main components of the spoilage flora. When the 37 °C aerobic plate counts (APCs) reached 10⁵–10⁶ CFU g⁻¹ and the 20 °C APCs and pseudomonad counts reached 10⁶–10⁷ CFU g⁻¹, visible surface colonies (VSCs) were observed. The presence of VSCs is the most important criterion to determine organoleptic beef liver spoilage and has hence enabled us to establish a shelf-life of up to 8–10 and 5.5–6.5 days for samples stored at 0 and 3 °C respectively. Our study shows that the determination of pH, which is simple, economical and rapid, is capable of giving a reliable estimate of the spoilage status of beef livers. pH values lower than 6.15 may be considered as indicative of beef liver spoilage. © 1999 Society of Chemical Industry     

A medium for the impedimetric detection of yeasts in foods

A mycological medium, CBAS, was developed for the detection of yeasts in foods using polarization capacitance (Cpol) measurements. The signal obtained due to the growth of yeasts in CBAS provided earlier detections and stronger responses than the signals produced by these yeasts in other common media. Of several impedance components evaluated, Cpol provided the most useful signal for yeasts in CBAS. Further investigation showed that pH change was responsible for approximately 50% of the Cpol signal. Cpol detection times correlated highly with plate counts of a yeast isolated from orange juice when tested in CBAS (r = ?0·99) and in a 1:10 dilution of orange juice in CBAS (r = ?0.94). A concentration of 10² yeast cfu ml⁺¹ in a 1:10 dilution of orange juice in CBAS was detected within 26 h by Cpol in contrast with a 3 to 5 day plate count.     

The fate of Escherichia coli O157 : H7 in Myzithra,Anthotyros, and Manouri whey cheeses during storage at 2 and 12°C

Myzithra, Anthotyros and Manouri whey cheeses were inoculated the day after production withEscherichia coli O157 : H7 at concentrations of approx. 1·8×10⁶cfu g⁻¹, and stored at 2 and 12°C for 30 and 20 days, respectively. The pH of the whey cheeses decreased from an initial value of approx. 6·20 to 5·83 or 5·60 (Myzithra) 5·75 or 5·20 (Anthotyros) and 5·80 or 5·30 (Manouri) by the end of the corresponding storage periods at 2 and 12°C, respectively. Escherichia coli O157 : H7 populations in the whey cheeses at the end of the 12°C storage period, had grown with an increase of approx. 1·3 log₁₀cfu g⁻¹. E. coli O157 : H7 populations in whey cheeses at the end of the 2°C storage period did not grow and decreased, with an approx. 2·5 log₁₀cfu g⁻¹reduction. Results showed that E. coli O157 : H7 can grow at 12°C and survive at 2°C storage in Myzithra, Anthotyros and Manouri whey cheeses, and therefore post-manufacturing contamination with this pathogen must be avoided by employing hygienic control programmes such as HACCP.     

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.     

The potential application of plant essential oils as natural food preservatives in soft cheese

Investigations were carried out to assess the efficiency of four plant essential oils; bay, clove, cinnamon and thyme as natural food preservatives. The effect of the plant essential oils at concentrations of 0·1, 0·5 and 1% was studied in low-fat and full-fat soft cheese against Listeria monocytogenes and Salmonella enteritidis at 4° and 10°C respectively, over a 14-day period. The composition of the cheese was shown to be an important factor in determining the effectiveness of the plant essential oils. In the low-fat cheese, all four oils at 1% reduced L. monocytogenes to ≤1·0 log₁₀cfu ml⁻¹. In contrast, in the full-fat cheese, oil of clove was the only oil to achieve this reduction. Oil of thyme proved ineffective against S. enteritidis in the full-fat cheese, yet was equally as effective as the other three oils in the low-fat cheese, reducing S. enteritidis to ≤1·0 log₁₀cfu ml⁻¹from day 4 onwards. It is concluded that selected plant essential oils can act as potent inhibitors of L. monocytogenes and S. enteritidis in a food product.     

Incorporation of bacteriocin in plastic retains activity and inhibits surface growth of bacteria on meat

The bacteriocin, nisin, was incorporated into a polyethylene based plastic film and retained activity against the indicator bacteria Lactobacillus helveticus and Brochothrix thermosphacta . Beef carcass surface tissue sections (BCT) topically inoculated with the psychrotrophic spoilage bacterium B. thermosphacta were vacuum-packaged both with and without wrapping with the nisin impregnated plastic and held at 4° C. An initial reduction of 2 log₁₀cycles of B. thermosphacta was observed with nisin-impregnated wrapped BCT within the first 2 days of storage. After 20 days of refrigerated storage, B. thermosphacta populations from nisin impregnated plastic wrapped samples were significantly less than (P<0·05) control vacuum-packaged samples; log₁₀5·8 vs 7·2 cfu cm⁻²respectively. Temperature abuse was simulated by shifting inoculated packs from 4° C (after 2 days) to 12° C. Again, by 20 days, the B. thermosphacta populations of treated samples wrapped with nisin impregnated plastic were significantly less than (P<0·05) control vacuum-packaged samples; log₁₀3·6 vs 6·3 cfu cm⁻²respectively. This work highlights the potential for incorporating antimicrobial peptides with a wider and different range of inhibitory activity directly into plastics of different properties for use in controlling food spoilage as well as preservation to enhance product microbial safety.     

Properties of polyphenol oxidase in mango (Mangifera indica) kernel

Polyphenol oxidase (PPO) activity of filtered extract of ground mango kernel suspension (400 g litre⁻¹) was studied spectrophotometrically at 420 nm using catechol as substrate. The enzyme was most active at pH 6·0 and 25°C. Activity was reduced by 50% at pH values of 5·0 and 7·1, and also at temperatures of 14°C and 30°C. The calculated activation energy and the Michaelis constant (K_m) were 21·4 kcal mol⁻¹ °C⁻¹ and 24·6 mM , respectively. The V_max value was 2·14 units g⁻¹ mango kernel. The time to heat inactivate PPO decreased rapidly to < 10 min with increasing temperature of ⩾ 70°C at 50% activity. © 1998 SCI.     

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.     

The population change of yeasts in commercial salami

The survival of yeast during a pilot scale production of commercial salami was investigated. A total of 108 distinctive yeast strains were isolated and identified during the processing of the salami. Initially, the number of yeasts remained below 10³cfu g⁻¹but their numbers increased after the 12^thday of maturation, reaching a maximum of 2·0×10⁵cfu g⁻¹at day 20. During maturation, the pH declined from 5·72 to 4·36, water content from 58% to 43%, while the salt content increased by 1%. The number of lactic acid bacteria remained above 10⁵cfu g⁻¹throughout processing and maturation. Of the 108 yeast strains isolated, 22 strains were identified as members of the species Debaryomyces hansenii, being present in all samples taken. In descending numbers, Rhodotorula mucilaginosa, Yarrowia lipolytica and Cryptococcus albidus, were also frequently isolated during processing and maturation.     

Effect of pH and Ionic Strength on the Heat Stability of Rapeseed 12S Globulin (Cruciferin) by the ANS Fluorescence Method

The heat stability of rapeseed 12S globulin (cruciferin) was examined using 8-anilinonaphthalene-1-sulphonic acid (ANS) as a fluorescence probe. Heating cruciferin (0·06–0·3 mg ml⁻¹ in 10 mM glycyl–glycyl piperizine buffer, pH 7·0, with 0·1–1·0 M NaCl) for 20 min increased its hydrophobicity as monitored by ANS fluorescence measurements. The mid-point temperature for the heat effect (T_m) increased linearly with increasing solvent pH (T_m (°C)=4·16 pH+41 (μ=0.1)) or sodium chloride concentration (T_m (°C)=14·7 [NaCl]+71 (pH=7·0)). The range of T_m values for cruciferin was 45–96°C. At 20°C cruciferin was unstable at pH<3·0 but relatively stable under alkaline conditions (pH 8–10). Though possessing an oligomeric structure, cruciferin appears to heat denature in accordance with the two-stage deactivation model for simple globular proteins.     

Influence of the adherent population level on biofilm population,structure and resistance to chlorination

The aim of this work was to study the influence of adherent population level on biofilm development, either on biofilm population, or on its structure. Our results, obtained from 13 strains isolated from dairy industries environments, demonstrated that, on the whole, Gram-negative bacteria were, although not significantly, globally more adherent to glass than Gram-positive ones (total mean of 6·1 and 5·7 log cfu cm⁻²respectively for an inoculum of 8 log cfu ml⁻¹). Gram-negative bacteria also exhibited a significantly higher biofilm population after 2 days of culture than Gram-positives (7·0 and 6·3 log cfu cm⁻²respectively) and a significant correlation between adhesion ability and biofilm population was seen. Elsewhere, we demonstrated that the level of adherent population did not influence biofilm biomass after 2 days of culture, although it strikingly influenced the biofilm structure. Biofilm resistance to chlorine was significantly increased with age of biofilm (2 log order after 1 day of culture and less than 1 log order after 3 days), but not by its structure.