Occurrence of Alicyclobacillus in the fruit processing environment--a review

Concentrated fruit products have a significant place in modern consumption markets and are valuable semi-prepared food components to the bakery, dairy, confectionary, canning, baby food, frozen food, distilling and beverage industries. There is continuous pressure on the beverage industry to improve the quality of concentrated fruit products in order for reconstituted fruit beverages to compete with beverages that are made from fresh fruits. In recent years, Alicyclobacillus spp. have become a major concern to the beverage industry worldwide as many high-acid, concentrated fruit products have been found to be contaminated with these spoilage microbes. The thermo-acidophilic nature of alicyclobacilli and highly resistant endospores allows for their survival during the production of concentrated fruit products. Under favourable conditions, endospores can germinate and multiply to numbers high enough to cause spoilage and product deterioration through the production of chemical taint compounds. It is imperative to understand the nature of Alicyclobacillus within the fruit concentrate processing environment so as to develop effective control strategies and to prevent spoilage in juice and beverage products that are reconstituted from fruit concentrates. This paper reviews the occurrence of alicyclobacilli in the fruit processing environment, control measures, as well as detection, identification and standardised test methods that are currently used for Alicyclobacillus in concentrated fruit products.     

The growth of Propionibacterium cyclohexanicum in fruit juices and its survival following elevated temperature treatments

This study investigated the growth of Propionibacterium cyclohexanicum in orange juice over a temperature range from 4 to 40 degrees C and its ability to multiply in tomato, grapefruit, apple, pineapple and cranberry juices at 30 and 35 degrees C. Survival after 10 min exposure to 50, 60, 70, 80, 85, 90 and 95 degrees C in culture medium and in orange juice was also assessed. In orange juice the organism was able to multiply by 2 logs at temperatures from 4 to 35 degrees C and survived for up to 52 days. However, at 40 degrees C viable counts were reduced after 6 days and no viable cells isolated after 17 days. The optimum growth temperature in orange juice over 6 days was 25 degrees C but over 4 days it was 35 degrees C. The growth of P. cyclohexanicum was monitored in tomato, grapefruit, cranberry, pineapple and apple juices at 30 and 35 degrees C over 29 days. Cranberry, grapefruit and apple juice did not support the growth of P. cyclohexanicum. At 30 degrees C no viable cells were detected after 8 days in cranberry juice or after 22 days in grapefruit juice while at 35 degrees C no viable cells were detected after 5 and 15 days, respectively. However, in apple juice, although a 5 log reduction occurred, viable cells could be detected after 29 days. P. cyclohexanicum was able to multiply in both tomato and pineapple juices. In tomato juice, there was a 2 log increase in viable counts after 8 days at 30 degrees C but no increase at 35 degrees C, while in pineapple juice there was a 1 log increase in numbers over 29 days with no significant difference between numbers of viable cells present at 30 and 35 degrees C. The organism survived at 50 degrees C for 10 min in culture medium without a significant loss of viability while similar treatment at 60, 70 and 80 degrees C resulted in approximately a 3-4 log reduction, with no viable cells detected after treatment at 85 or 90 or 95 degrees C but, when pre-treated at intermediate temperatures before exposure to higher temperatures, some cells survived. However, in orange juice a proportion of cells survived at 95 degrees C for 10 min without pre-treatment and there was no significant difference between numbers surviving with and without pre-treatment. The results from this study demonstrate that P. cyclohexanicum is able to grow in a number of juices, other than orange juice, and able to survive a number of high temperature procedures. Therefore, if initially present in the raw materials P. cyclohexanicum might survive the pasteurization procedures used in the fruit juice industry, contaminate and consequently spoil the final product.     

Prevalence, concentration, spoilage, and mitigation of Alicyclobacillus spp. in tropical and subtropical fruit juice concentrates

The presence of Alicyclobacillus in fruit juices and concentrates poses a serious problem for the juice industry. This study was undertaken to determine the (i) prevalence, concentration, and species of Alicyclobacillus in tropical and subtropical concentrates; (ii) efficacy of aqueous chlorine dioxide in reducing Alicyclobacillus spp. spores on tropical and subtropical fruit surfaces; and (iii) fate of and off-flavor production by Alicyclobacillus acidoterrestris in mango and pineapple juices. One hundred and eighty tropical and subtropical juice concentrates were screened for the presence and concentration of Alicyclobacillus spp. If found, the species of Alicyclobacillus was determined by 16S rDNA sequencing and analysis with NCI BLAST. Of these samples, 6.1% were positive for Alicyclobacillus, and nine A. acidoterrestris strains and two Alicyclobacillus acidocaldarius strains were identified. A five-strain cocktail of Alicyclobacillus spp. was inoculated onto the surface of fruits (grapefruit, guava, limes, mangoes, oranges and pineapple), which were then washed with 0, 50, or 100 ppm aqueous chlorine dioxide. Significant reductions due to chlorine dioxide were only seen on citrus fruits. A five-strain cocktail of A. acidoterrestris was inoculated into mango and pineapple juices. Microbial populations were enumerated over a 16-day period. Aroma compounds in the juice were analyzed by GC-olfactometry (GC-O) and confirmed using GC-MS. GC-O of mango juice identified previously reported medicinal/antiseptic compounds. GC-O of pineapple juice revealed an unexpected “cheese” off-aroma associated with 2-methylbutyric acid and 3-methylbutyric acid.     

Factors associated with the likelihood of Giardia spp. and Cryptosporidium spp. in soil from dairy farms

A study was conducted to identify factors associated with the likelihood of detecting Giardia spp. and Cryptosporidium spp. in the soil of dairy farms in a watershed area. A total of 37 farms were visited, and 782 soil samples were collected from targeted areas on these farms. The samples were analyzed for the presence of Cryptosporidium spp. oocysts, Giardia spp. cysts, percent moisture content, and pH. Logistic regression analysis was used to identify risk factors associated with the likelihood of the presence of these organisms. The use of the land at the sampling site was associated with the likelihood of environmental contamination with Cryptosporidium spp. Barn cleaner equipment area and agricultural fields were associated with increased likelihood of environmental contamination with Cryptosporidium spp. The risk of environmental contamination decreased with the pH of the soil and with the score of the potential likelihood of Cryptosporidium spp. The size of the sampling site, as determined by the sampling design, in square feet, was associated nonlinearly with the risk of detecting Cryptosporidium spp. The likelihood of the Giardia cyst in the soil increased with the prevalence of Giardia spp. in animals (i.e., 18 to 39%). As the size of the farm increased, there was decreased risk of Giardia spp. in the soil, and sampling sites which were covered with brush or bare soil showed a decrease in likelihood of detecting Giardia spp. when compared to land which had managed grass. The number of cattle on the farm less than 6 mo of age was negatively associated with the risk of detecting Giardia spp. in the soil, and the percent moisture content was positively associated with the risk of detecting Giardia spp. Our study showed that these two protozoan exist in dairy farm soil at different rates, and this risk could be modified by manipulating the pH of the soil.     

Development of multiplex loop-mediated isothermal amplification-RFLP (mLAMP-RFLP) to detect Salmonella spp. and Shigella spp. in milk

A multiplex loop-mediated isothermal amplification-RFLP (mLAMP-RFLP) was developed and validated for simultaneous detection of Salmonella strains and Shigella strains in milk. In this system, two sets of LAMP primers were designed to specifically target invA of Salmonella spp. and ipaH of Shigella spp. Under isothermal conditions at 63 °C, ladder pattern of DNA bands could be amplified within 60 min in the presence of genomic DNAs of Salmonella strains and Shigella strains, which could be distinguished between Salmonella spp. and Shigella spp. simultaneously based on the different ladder pattern of DNA bands and subsequent restriction enzyme analysis. The overall analysis time was approximately 20 h including the enrichment of the bacterial cells, which greatly saved detection time. The sensitivity of mLAMP was found to be 100 fg DNA/tube with genomic DNAs of Salmonella strains and Shigella strains, comparatively, multiplex PCR was 1 pg DNA/tube. The mLAMP allowed the detection of milk sample artificially contaminated by Salmonella strains and Shigella strains at initial inoculation levels of approximate 5 CFU/10 mL. In conclusion, the mLAMP described here can potentially facilitate simultaneous monitoring of Salmonella and Shigella in a large number of food samples, which could be used as a primary screening method and as a supplement to classical detection method.     

Development and validation of a mathematical model to describe the growth of Pseudomonas spp. in raw poultry stored under aerobic conditions

Poultry meat spoils quickly unless it is processed, stored, and distributed under refrigerated conditions. Research has shown that the microbial spoilage rate is predominantly controlled by temperature and the spoilage flora of refrigerated, aerobically-stored poultry meat is generally dominated by Pseudomonas spp.

The objective of our study was to develop and validate a mathematical model that predicts the growth of Pseudomonas in raw poultry stored under aerobic conditions over a variety of temperatures.

Thirty-seven Pseudomonas growth rates were extracted from 6 previously published studies. Objectives, methods and data presentation formats varied widely among the studies, but all the studies used either naturally contaminated meat or poultry or Pseudomonas isolated from meat or poultry grown in laboratory media. These extracted growth rates were used to develop a model relating growth rate of Pseudomonas to storage or incubation temperature. A square-root equation [Ratkowsky, D.A., Olley, J., McMeekin, T.A., and Ball, A., 1982. Relationship between temperature and growth rate of bacterial cultures. J. Appl. Bacteriol. 149, 1–5.] was used to model the data. Model predictions were then compared to 20 Pseudomonas and 20 total aerobes growth rate measurements collected in our laboratory. The growth rates were derived from more than 600 bacterial concentration measurements on raw poultry at 10 temperatures ranging from 0 to 25 °C. Visual inspection of the data and the indices of bias and accuracy factors proposed by Baranyi et al. [Baranyi, J., Pin, C., and Ross, T., 1999. Validating and comparing predictive models. Int. J. Food Micro. 48, 159–166.] were used to analyze the performance of the model.

The experimental data for Pseudomonas showed a 4.8% discrepancy with the predictions and a bias of + 3.6%. Percent discrepancies show close agreement between model predictions and observations, and the positive bias factor demonstrates that the proposed model over-predicts growth rate, thus, can be considered fail-safe. Both Pseudomonas spp. as well as total aerobes may be considered good indicators of poultry spoilage.

A properly constructed and validated model for Pseudomonas growth under aerobic conditions can provide a fast and cost-effective alternative to traditional microbiological techniques to estimate the effects of storage temperature on product shelf-life. The model developed here may be used to determine the effect of both initial Pseudomonas concentration and storage temperature on shelf-life of poultry meat under aerobic storage conditions over temperatures from 0 to 25 °C.     

Prevalence of Bacillus spp. in different food products collected in Argentina

The prevalence of Bacillus spp. in 279 samples of different food products collected in Argentina was studied. Bacillus spp. was confirmed in 28 out of 70 honey samples, 29 out of 29 flour samples, 15 out of 50 cheese samples, and 30 out of 30 spice samples, while Bacillus spp. was not found in fresh anchovy. Among the 70 honeys studied, Bacillus cereus, Bacillus pumilus, Bacillus laterosporus and Paenibacillus larvae subspp. larvae showed an incidence of 23%, 4%, 8% and 38%, respectively. More diversity of Bacillus species was found in rye flours than in white flours, Bacillus subtilis being the predominant species isolated from rye flour. B. cereus had an incidence of 50% in Port Salut Argentino cheeses. Meanwhile, B. pumilus was identified in both Port Salut and Quartirolo cheeses with an incidence of 50% and 25%, respectively. All the spices analysed showed Bacillus mycoides as the sole aerobic spore-forming bacilli isolated. The association of the presence of B. cereus, B. subtilis and Bacillus licheniformis with both the potential spoilage of foods and foodborne outbreaks is well known. In this study, Bacillus spp. had an incidence of 38% among all the samples analysed, therefore the monitoring of those species should be routinely done in microbiological food analyses.     

A note on the incidences of Salmonella spp., Listeria spp. and Escherichia coli O157:H7 serotypes in Turkish sausage (Soudjouck)

The incidence of Salmonella spp., Listeria monocytogenes and Escherichia coli O157:H7 was determined in 100 Turkish sausage (soudjouck) samples collected from shops and markets in the Afyon province, Turkey. Salmonella spp. were detected in 7% of the samples. All of the isolates were S. enterica Paratyphi B. In addition, Listeria spp. were detected in 9% of the samples. Its distribution was 7% L. monocytogenes and 1% each of L. ivanovii and L. innocua. Serological study of the seven L. monocytogenes isolates showed that three of these were 1/2 ab, three were 5/6 ab and one was 1 ab. E. coli O157:H7 was not detected in any of the samples. The pH values of the samples ranged from 4.8 to 6.5. In conclusion, increasing number of listeriosis and salmonellosis cases in Turkey and the contamination levels found indicate that risk assessment and improved preventive measures are required for these sausages.     

Two novel analytical methods based on polyclonal and monoclonal antibodies for the rapid detection of Cronobacter spp.: Development and application in powdered infant formula

Cronobacter spp. are opportunistic pathogens, and infections are associated with a high mortality rate. In the current study, monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) were generated using heat-inactivated C. sakazakii strain ATCC29544 as the immunogen. Following assay optimization, an indirect enzyme-linked immunosorbent assay (ELISA) based on pAbs and a sandwich ELISA based on mAbs and pAbs were established for the detection of Cronobacter spp. The indirect ELISA detected all species of Cronobacter assayed, and the limit of detection (LOD) was established as 10⁵ cfu/mL. In contrast, the sandwich ELISA was specific for C. sakazakii and had greater sensitivity than the indirect ELISA (LOD of 2 × 10⁴ cfu/mL). Following 10 h of enrichment, Cronobacter spp. were detected using either of the two analytical methods in samples inoculated with 1 cfu/100 g powdered infant formula (PIF). The results from this study demonstrated that both of these novel ELISAs were specific, sensitive, and rapid assays for the screening of pathogenic Cronobacter spp. in PIF.     

A brief study on the degradation kinetics of seven organophosphorus pesticides in skimmed milk cultured with Lactobacillus spp. at 42 °C

Seven organophosphorus pesticides (OPPs) including dimethoate, fenthion, malathion, methyl parathion, monocrotophos, phorate and trichlorphon were added to skimmed milk. The milk was inoculated with one strain of Lactobacillus spp. including L. bulgaricus, L. paracasei and L. plantarum, and cultured at 42 °C for 24 h to investigate their impacts on degradation kinetics of the OPPs. The residual OPPs in the milk were extracted by an extraction solvent and quantified in a gas chromatography after purification. Degradation rate constant and half live periods of the OPPs were calculated from a first-order reaction kinetics model. The result shows that the selected Lactobacillus spp. exhibited some acceleration on OPPs degradation totally. Dimethoate and methyl parathion were more stable but malathion was the most labile. Both L. bulgaricus and L. plantarum had stronger acceleration on the degradation of the OPPs studied.     

Direct application of supercritical carbon dioxide for the reduction of Cronobacter spp. (Enterobacter sakazakii) in end products of dehydrated powdered infant formula

The objective of this study was to develop a viable new method for inactivation of Cronobacter spp. that could be applied directly to dehydrated powdered infant formula (PIF) using supercritical carbon dioxide (SC-CO₂). Samples inoculated with Cronobacter spp. were subjected to SC-CO₂ treatment under various conditions (temperature: 63, 68, and 73°C; pressure: 15, 20, and 25 MPa; time: 10, 20, and 30 min). The survival of Cronobacter spp. was assayed, as were any changes in the quality of the treated PIF. Inactivation of Cronobacter spp. by SC-CO₂ was enhanced as temperature and pressure conditions increased (>6.32 log₁₀ cfu/g). In a validation assay using low-level inoculation (3.21 log₁₀ cfu/g), treatment at 73°C and 15 MPa for 30 min, 20 MPa for 20 and 30 min, or 25 MPa for 20 and 30 min reduced Cronobacter spp. to undetectable levels, with no recovery of cell viability. There was no significant change in water activity, pH, and color of the treated PIF. Overall, the optimum conditions for elimination of Cronobacter spp. were determined to be 73°C and 20 MPa for 20 min. These parameters for effective SC-CO₂ treatment are feasibly applicable to end product of dehydrated PIF. The results of our study may contribute to the development of an efficient method for improving the microbiological safety of PIF.     

Survey of Listeria monocytogenes, Salmonella spp. and Escherichia coli O157:H7 in raw ingredients and ready-to-eat products by commercial real-time PCR kits

Real-time PCR (RTiPCR) assays including enrichment stage were evaluated for the rapid detection of Listeria monocytogenes, Salmonella spp. and Escherichia coli O157:H7 in raw ingredients and ready-to-eat products using molecular beacon probes available as commercial kits (WARNEX Genevision, Canada & AES Chemunex detection system, France). The accuracy of the assays was evaluated analyzing 1032 naturally contaminated food samples in combination to the conventional cultural methods. Presence/absence testing of the above pathogens was performed in 25 g samples of each product. In case of L. monocytogenes of 39 positive RTiPCR samples, 37 were confirmed by the cultural method (based on McNemar's test the difference between the two methods is insignificant). The highest incidence of L. monocytogenes in food products was found in desserts and the second highest in frozen pastries. None of the samples were cultural positive but negative in the RTiPCR test. One among the 343 investigated samples was positive for Salmonella spp. by RTiPCR and the cultural method. Out of 333 samples analyzed for E. coli O157:H7 no positive sample was detected. RTiPCR-based methods proved to be powerful tools for fast, sensitive and accurate pathogen detection in raw food ingredients and ready-to-eat products.     

Use of global sensitivity analysis in quantitative microbial risk assessment: application to the evaluation of a biological time temperature integrator as a quality and safety indicator for cold smoked salmon

The aim of this study was to apply a global sensitivity analysis (SA) method in model simplification and to evaluate (eO)^®, a biological Time Temperature Integrator (TTI) as a quality and safety indicator for cold smoked salmon (CSS). Models were thus developed to predict the evolutions of Listeria monocytogenes and the indigenous food flora in CSS and to predict TTIs endpoint. A global SA was then applied on the three models to identify the less important factors and simplify the models accordingly. Results showed that the subset of the most important factors of the three models was mainly composed of the durations and temperatures of two chill chain links, out of the control of the manufacturers: the domestic refrigerator and the retail/cabinet links. Then, the simplified versions of the three models were run with 10⁴ time temperature profiles representing the variability associated to the microbial behavior, to the TTIs evolution and to the French chill chain characteristics. The results were used to assess the distributions of the microbial contaminations obtained at the TTI endpoint and at the end of the simulated profiles and proved that, in the case of poor storage conditions, the TTI use could reduce the number of unacceptable foods by 50%.     

Changes in the dynamics of Coxiella burnetii infection in dairy cattle: An approach to match field data with the epidemiological cycle of C. burnetii in endemic herds

This study aimed to evaluate changes in the epidemiological status of Coxiella burnetii in dairy cattle herds to better understand the epidemiology of the infection and to predict its evolution. Bulk-tank milk (BTM) and serum samples were collected from 94 dairy cattle herds and analyzed by ELISA (BTM and sera) and PCR (BTM) in study 1 (S₁). Two years later (study 2; S₂), the same farms were visited with a similar sampling approach. To estimate seroconversion during this period, blood samples were collected from the maximum possible number of animals surveyed in S₁. Environmental samples were collected in S₂ to identify active shedding. Farms were allocated into 3 different categories in each study according to PCR and ELISA results: category A, with BTM ELISA and PCR positive herds and at least 1 seropositive animal; category B, with BTM ELISA or PCR positive herds or individual sera positive; and category C, with all negative results among herds. Changes in herd category between S₁ and S₂ were grouped in 9 classes. Two statistical models, one to search for drives of within-herd changes in C. burnetii infection status and another to look for variables modulating individual changes in C. burnetii antibody level, were built. Several herds in category A in S₁ remained in that category 2 yr later, indicating that C. burnetii can remain within a herd for a long time. Most of the herds with seroconversion and detection of the bacterium in the environment belonged to category A, suggesting active and recent infections. Changes in the epidemiological status of herds were driven by local densities of domestic ruminants, showing the implication of neighbor reservoirs; whereas individual changes in antibody levels were modulated by variation in the epidemiological status of herds. Observed changes in epidemiological status allowed depiction of the hypothesized life cycle of C. burnetii within dairy cattle herds, which should be tested by future long-term series studies on C. burnetii infection to help fitting control measures (e.g., vaccination) to within-herd C. burnetii status.