Aerobic Spore Counts Research Articles

Effect of bactofugation of raw milk on total bacterial and aerobic spore counts and on microbial diversity of sporeformers

This study describes the effect of bactofugation (10,000 × g in a continuous flow of 10,000 L/h) of three batches of raw milk on total bacterial count (TBC) and aerobic spore count, and it also shows the effect on the microbial diversity of spore-forming bacteria by analysing their genetic variability through molecular approaches. Given that milk must be preheated to approximately 55 °C before being bactofuged, for comparison, the three batches were evaluated at different stages as refrigerated raw, preheated, and bactofuged milk. For preheated milk, it was found that bactofugation caused a significant reduction (p < 0.05) of 99.52% (from 4.5 × 106 to 2.1 × 104 CFU/mL) in the mean TBC and of 95% (from 333 to 17 CFU/mL) in the aerobic mesophilic spore count. Due to the effect of bactofugation on preheated milk, a reduction of 82% was observed in both TBC and aerobic spore count. With respect to diversity, 107 isolates from raw milk, prior to bactofugation, and 16 isolates from bactofuged milk were recovered and grouped into 40 and 8 clusters, respectively. The predominant species detected in raw and preheated milk were Bacillus toyonensis (63% - 20 clusters) and Lysinibacillus fusiformis (15% - 8 clusters). Proportionally, B. toyonensis (69% - 6 clusters) and L. fusiformis (25% - 1 cluster) were predominant in bactofuged milk. B. pumilus, L. varians, B. flexus, B. invictae, and B. megaterium, bacteria with a known milk spoilage potential, were isolated from milk prior to bactofugation, and they reduced to undetectable levels in bactofuged milk. Bactofugation of milk, therefore, reduces the TBC and aerobic spore count, with a significant effect in reducing the microbial diversity of spore-forming bacteria, proportional to their incidence in raw milk. Therefore, bactofugation can be an alternative to increase the shelf life and technological potential of milk.

Occurrence of Clostridium perfringens vegetative cells and spores throughout an industrial production process of black soldier fly larvae (Hermetia illucens)

The main use of black soldier fly larvae (Hermetia illucens) is currently as an animal feed ingredient. While the bacterial community of the larvae has been characterised repeatedly via sequencing, microbiological safety assessment based on culture-dependent techniques is still scarce. This study focused on the occurrence of the spore-forming foodborne pathogen Clostridium perfringens during rearing and consecutive processing of the larvae, based on observations in a single rearing facility. C. perfringens vegetative cells and spores were determined, in addition to total viable counts, total aerobic spore counts and intrinsic parameters including pH, water activity and moisture content. All samples were obtained from an industrial production plant. In a preliminary experiment, substrate ingredients and dried larvae were analysed, but the larvae were produced with a previous batch of the substrate mixture. A second, more detailed, experiment was performed where all samples were collected sequentially from the same production run (substrate ingredients, substrate mixture, starting larvae, harvested larvae, residue, dried larvae and stored dried larvae). In the two experiments, (presumptive) C. perfringens, as determined on tryptose sulphite cycloserine agar, was found at low numbers in the ingredients and in the second experiment it was also found in the substrate mixture. Over the two experiments, total C. perfringens counts (i.e. vegetative cells plus spores) ranged between 3.0±0.1 and <1.2±0.5 log cfu/g and C. perfringens spores ranged between 2.5±0.1 and <1.0±0.0 log cfu/g. Interestingly, vegetative cells and spores of C. perfringens were below the detection limit in all larvae samples. Therefore, it appears that at this production site and based on the samples investigated, the pathogen did not colonise the larvae. However, these results indicate that insect producers should monitor this pathogen among others, and install good hygiene practices to avoid contamination.

Combination of microwave and gamma irradiation for reduction of aflatoxin B1 and microbiological contamination in peanuts (Arachis hypogaea L.)

Aflatoxin B1 (AFB1) is a natural carcinogen commonly present in food and feed which has deleterious effects on human and animal health. Combination treatment of microwave heating and gamma irradiation has been investigated. Artificially spiked peanuts (Arachis hypogaea L.) were used with a concentration of 300 μg/kg of AFB1 to evaluate the treatment efficacy. Qualitative and quantitative analysis of AFB1 was carried out by one dimensional thin layer chromatography and enzyme-linked immune-sorbent assay (ELISA), respectively. Gamma irradiation (5, 7 and 9 kGy) alone reduced 20 to 43% of AFB1, while, only low power microwave heating (360, 480 and 600 W) reduced it by 59 to 67%. The synergistic effect of a combination treatment at 7 and 9 kGy of irradiation at any of the chosen microwave power levels and sequence of treatment was able to reduce >95% in artificially spiked peanuts. Knowing the limitation of ELISA, an Ames test was employed to determine the mutagenicity of AFB1 in combination-treated (7 kGy and 600 W) artificially spiked peanut samples, resulting in an 80-85% reduction of its mutagenicity. Quality parameters of peanuts in terms of moisture content, water activity, hardness, colour, peroxide value and free fatty acid were evaluated and observed to be retained after combination treatments. Post-treatment sensory analysis in respect of appearance, colour, texture, taste, aftertaste and overall acceptability gave satisfactory scores. The microbiological safety of treated peanuts (exclusive and combination of 7 kGy and 360-600 W) was assessed in terms of total bacterial count, aerobic spore count, and yeast and mould count. The microbiological load was completely eliminated after exclusive gamma irradiation or the combination treatments. Combination treatment (7 kGy and 600 W) demonstrated 71 to 87% reduction in AFB1 from naturally contaminated peanuts and is recommended for further commercial application.

Spoilage potential of spore-forming bacteria from refrigerated raw milk

Aerobic bacterial spores are an important group of microorganisms in raw milk. These microbes are thermoduric, whereas the vegetative forms are thermophilic, thermoduric and psychrotrophic and reduce the shelf life of pasteurized milk. In Brazil, there are a lack of studies on the load of aerobic spores in raw milk; thus, little is known about the spoilage activity of these organisms. The aim the present study was to quantify the aerobic spores in Brazilian refrigerated raw milk of dairy region of Castro, Paraná state, assess the potential proteolytic and/or lipolytic isolates and identify the microorganisms derived from the germination. Twenty milk samples were evaluated, and the aerobic spore count was performed after plating the samples following heat treatment at 80°C for 12 min. The activity proteolytic and lipolytic isolates were evaluated through subculture on milk agar and tributyrin agar, respectively, and these microorganisms were identified using partial 16S rRNA gene sequences that were compared through GenBank. The aerobic spore counts ranged from 1 to 3.7 log CFU.mL-1, with a mean of 1.75 (± 0.59) log CFU.mL-1. After spore germination, 137 aerobic bacterial isolates were obtained, 40 of which (29.2%) showed milk spoilage activity. Among these, 31 isolates (77.5%) were proteolytic and lipolytic, seven isolates (17.5%) were exclusively lipolytic and two isolates (5%) were only proteolytic. Based on the 16S rRNA gene analysis, Bacillus licheniformis (55%), Bacillus spp. (27.5%), Paenibacillus spp. (7.5%), Bacillus pumilus (5%), Bacillus circulans (2.5%) and Brevibacillus spp. (2.5%) were identified. Studies of Brazilian raw milk microbiota have not yet described B. circulans which are frequently detected in milk from other countries. Among the 22 B. licheniformis isolates, 21 microbes (95.5%) showed proteolytic and lipolytic activity, and one isolate (4.5%) exhibited only proteolytic activity. The two B. pumilus isolates were proteolytic and lipolytic, whereas the B. circulans isolate was only lipolytic. Among the 11 Bacillus spp. isolates, eight isolates (72.7%) were proteolytic and lipolytic, one isolate (9.1%) was proteolytic and the other two isolates (18.2%) were lipolytic. The three Paenibacillus spp. and Brevibacillus spp. isolates were primarily lipolytic. Therefore, to extend the shelf life of pasteurized milk, preventive measures must be adopted to reduce contamination with spores because one-third of these microorganisms exhibited proteolytic and/or lipolytic activity.

Identification and characterization of aerobic spore forming bacteria isolated from commercial camel’s milk in south of Algeria

The aim of this study was to evaluate aerobic spore-forming species contamination levels in raw and pasteurized camel’s milk. The isolated strains were also identified by 16S rDNA sequencing and characterized for their heat resistance. Camel milk (8 samples of which 4 were pasteurized) were collected from farms and markets from different regions in south east Algeria’s (Laghouat, Ghardaïa and Ain Aminas). Aerobic spore counts had 2 logCFU/mL in the raw samples. The 16S rDNA sequencing enables the identification of a selection of 20 isolates: Bacillus licheniformis (Five strains), Bacillus cereus (Nine strains), Bacillus subtilis (Three strains), Bacillus pumilus (One strain), Bacillus sonorensis (One strain), Geobacillus stearothermophilus (One strain). All B. cereus strains belonged to the phylogenetic group III of B. cereus, according to the classification proposed by Guinebretière et al. (2008), and supposedly represent potential strains of pathogenic B. cereus.The heat resistance (D-value i.e. the decimal reduction time) and heat sensitivity (zT values i.e. the temperature increase that lead to a ten-fold reduction of the D value) of spores of B. cereus, B. licheniformis, B. pumilus, B. sonorensis, B. subtilis were determined. The D100°C-values were ranged 1.09–9.55min whereas the z-values 6.45–8.50°C.

English

Egg white curd cheese is a curdled protein concentrate made from egg whites. Its advantages are: high protein content, a taste similar to cow's milk curd cheese, minimum carbohydrates, and no fat or cholesterol. Thus, it offers an attractive substitute for cow's milk curd cheese to many followers of a “modern lifestyle” as well as those living with health problems (sensitivity to milk products). Both the internal and sensory characteristics of egg white curd cheese were studied. In addition, the level of potentially dangerous microbes in the product was calculated. The fresh product was in excellent microbiological condition, and over a storage period of three weeks, no Salmonella or Staphylococcus aureus could be detected in it. The levels of Enterobacteriaceae and all live aerobic spore counts were completely satisfactory. In summary, it can be stated that we have succeeded in developing a microbiologically stable product which is applicable for numerous diets, has a flavor resembling mild cow's mild curd cheese, lacks enhancers and preservatives, and can even be consumed as it is.   Key words: Eggwhite, curdcheese, Salmonella, paleolithic diet.

Production of an 18% protein liquid micellar casein concentrate with a long refrigerated shelf life

Our objective was to develop a process to produce a high-concentration liquid micellar casein concentrate (18% protein, MCC18) with a long refrigerated shelf life. The MCC18 is a novel milk protein ingredient produced by fractionating skim milk using microfiltration (MF). To achieve a long refrigerated shelf life, the processing of MCC18 was designed to maximize the removal of low-molecular weight compounds [e.g., lactose and nonprotein nitrogen (NPN)] that can be easily metabolized by microbes, while minimizing the microbial count in the final product. The production of MCC18 was done over a period of 5 d. The experiment was replicated 3 times in different weeks with a different batch of raw milk. Raw whole milk was pasteurized and separated to produce skim milk. Skim milk was ultrafiltered (UF) to remove more than half of the lactose and NPN. The UF milk retentate was diluted with water and then MF in 3 stages to remove approximately 95% of the serum protein and further remove lactose and NPN. The retentate from the last stage of MF was UF to concentrate the protein to 18% and batch pasteurized. The MCC18 was collected immediately after processing in sterile plastic vials and stored at 4°C. The average MCC18 contained 21.78% total solids, 18.27% true protein, 0.31% NPN, and 0.13% lactose. The MCC18 at the day of processing contained a mean aerobic bacterial count of 2.1 log cfu/mL and mean aerobic spore count of 2.3logcfu/mL. The MCC18 formed a solid gel at temperatures <22°C, but the MCC18 reverted back to a liquid when warmed from 4°C temperature to >22°C. This provides a unique opportunity in ingredient handling and packaging and eliminates the challenges encountered in reconstitution of dried milk protein ingredients. The MCC18 produced in this study maintained a bacteria count <20,000 cfu/mL for 16wk when stored at a refrigeration temperature of 4°C. Further study is needed to determine if changes occur in the organoleptic and functional properties of MCC18. We envision that the conversion of skim milk to MCC and its coproducts (serum protein concentrate and lactose concentrate) could be used as an alternative to the production of nonfat dry milk to balance milk production seasonality, specifically the components of skim milk portion.

Assessment of the microbial safety and quality of cooked chilled foods and their production process

Refrigerated processed foods of extended durability (REPFEDs) are a heterogeneous group of food products. This study assesses the microbial safety and quality along the production process in five REPFED companies. Samples were taken of raw materials (n=123), intermediate products (n=123), end products at production day (n=45) and at end of shelf life (n=90), food contact surfaces (n=226) and worker's hands/gloves (n=92). Samples are analysed for total psychrotrophic aerobic count, aerobic spore count, sulphite reducing Clostridia, Bacillus cereus and Listeria monocytogenes.Both L. monocytogenes and B. cereus were detected on the raw materials. Nine of 72 raw materials tested were positive (in 25g) for L. monocytogenes and all but one of these raw materials were raw or minimally processed animal products. Three of 123 raw materials contained high counts (>4log CFU/g) of B. cereus, all of these samples were dried herbs. During production both food contact surfaces (90/226) and gloves (43/92) contained increased levels of total psychrotrophic aerobic counts (≥3log CFU/25cm2). This points out a potential source of bacterial recontamination. However, only a four and six of 223 food contact surfaces were positive (per 25cm2) for L. monocytogenes and B. cereus respectively. None of the gloves sampled contained L. monocytogenes and only 2 sets of gloves were positive for B. cereus. Of the 123 intermediate products tested twelve tested positive for L. monocytogenes (in 25g) and 5 showed elevated counts of B. cereus (ca. 2.5log CFU/g). Despite the presence of L. monocytogenes in the raw materials, the production area and in some of the intermediate products, none of the end products were positive for L. monocytogenes and only 9 of 135 samples (6.7%) showed to have low numbers of B. cereus (<2.7log CFU/g). This results show that the current pasteurization processes and the food safety management system are adequate to guarantee the production of microbiologically safe foods but that some improvements can still be made with regard to supplier selection, cleaning and disinfection, hygiene training and setting the shelf life duration.

Changes of microbiological and physicochemical properties in Chinese infant formula caused by high heat treatment applied on concentrated milk

In 2010, China published highly strict food safety standard for infant formula (GB 10765 2010). However, many manufacturers in China found it difficult to meet the new microbiological standard. The aim of this study was to find the appropriate processing conditions for producing Chinese infant milk with reduced microbial load. Concentrated milk was heated at 95 °C for 15 s (LHT) and 110 °C for 4 s (HHT), and their effects on the microbiological quality and physicochemical properties of the infant formula milk powder were investigated. Compared to LHT, HHT made significant effects on the bacterial, thermophilic, and aerobic spore counts. In addition, the total bacterial count of the infant formula milk powder after HHT met the requirements of the safety standard (GB 10765 2010). In terms of nutritional content, both powders met the requirement (GB 10765 2010) with no significant differences observed. There were also no significant differences observed between the two powders in terms of particle morphology, wettability, free fat content, thermal characteristics of fat, or milk fat globule membrane permeability. Although some slight differences (P ≤ 0.05) were found in solubility, dispersibility, viscosity, particle size of the reconstituted powder, and protein characteristics, the acceptability of the product under both heat treatments was found to be similar. The knowledge derived from this work can potentially provide an effective method for processing infant formula milk powder in China.

Characterization of mesophilic bacilli in faeces of feedlot cattle

To determine the identity and composition of mesophilic Bacillus spp. in faeces sampled from feedlot cattle. Faecal samples from 10 feedlot cattle were analysed. The total aerobic spore count increased from 4.6 x 10(4) CFU g(-1) (before feedlotting, day 0) to 1.6 x 10(6) CFU g(-1) (feedlot for day 76). A total of 150 randomly selected spore isolates (60 each from days 0 and 76 cattle, 30 from feed) were speciated using a Bacillus group-specific PCR-amplified ribosomal DNA restriction analysis technique (Wu et al. 2006). At day 0, Bacillus subtilis and Bacillus cereus predominated with a prevalence of 58.3% and 26.7%, respectively, whereas three species, B. subtilis (50.0%), Bacillus licheniformis (27.6%) and Bacillus clausii (20.0%) predominated in day 76 faecal samples. Of these, only the first two species were present in feed samples at a frequency of 70% and 30% respectively. All B. cereus isolates on day 0, possessed at least one of three enterotoxin genes (nheA, nheB and nheC) but these were completely eliminated after a period of feedlotting. All isolates of B. licheniformis were genotypically heterogeneous according to pulsed-field gel electrophoresis analysis. Cattle faeces contain large numbers of Bacillus spores representing different mesophilic species. Stable faecal populations of particular Bacillus spp. mimicking those found in feed, were subsequently established by feedlotting. The results obtained and methods used in this study will help to investigate the indigenous Bacillus composition in the gastrointestinal tract of cattle and will further guide the administration of Bacillus probiotics.

The use of nisin as a preservative in fish sausage stored at ambient (28 ± 2 °C) and refrigerated (6 ± 2 °C) temperatures

Summary The efficacy of nisin at three different concentrations, 12.5, 25 and 50 ppm, on the keeping quality of fish sausage in synthetic casing at ambient (28 ± 2 °C) and refrigerated (6 ± 2 °C) temperatures was assessed. Gel strength, expressible water content, total volatile base nitrogen, total plate count and aerobic spore counts were affected by the storage temperatures and nisin concentrations used. Fish sausage treated with 50 ppm of nisin was acceptable after storage at ambient temperature for 20–22 days compared with the control, which were acceptable only for 2 days. The keeping quality of the sausages, at refrigerated temperature, varied from 30 days in the control to 150 days in 50‐ppm nisin‐treated samples. The residual nisin decreased slowly in samples stored at refrigerated temperature, whereas, in fish sausages stored at ambient temperature, the decrease was rapid. Nisin at 50‐ppm level showed a significant effect (P ≤ 0.05) on the gel strength and overall acceptability scores both at ambient and refrigerated temperature.

Developments in microbiological risk assessment for drinking water.

1. SUMMARYThis paper considers the development of microbiologicalrisk assessment models for pathogenic agents in drinkingwater with particular reference to Cryptosporidium parvum,rotavirus and bovine spongiform encephalopathy (BSE).The available evidence suggests that there is potential forconsiderable variation in exposures to C. parvum oocyststhrough drinking water, during both outbreak and non-outbreak conditions. This spatial/temporal heterogeneityarises both from variation in oocyst densities in the rawwater and fluctuations in the removal efficiencies of drinkingwater treatment. In terms of risk prediction, modelling thevariation in doses ingested by individual drinking waterconsumers is not important if the dose–response curve islinear and the oocysts act independently during infection.Indeed, the total pathogen loading on the population asrepresented by the arithmetic mean exposure is sufficient forrisk prediction for C. parvum, BSE and other agents of lowinfectivity, providing the infecting particles (i.e. oocysts orBSE prions) are known to act independently. However, formore highly infectious agents, such as rotavirus, ignoring1. Summary, 1912. Introduction, 1922.1 Overview of the risk assessment approach, 1923. Pathogen exposures through drinking water, 1923.1 Pathogen exposures under non-outbreak condi-tions, 1933.1.1 Variation in micro-organism counts withinlarge volume samples, 1933.1.2 Effect of treatment on the spatial distribu-tion of micro-organisms, 1933.1.3 Implication for Cryptosporidium exposures todrinking water consumers under non-out-break conditions, 1953.2 A model for Cryptosporidium oocyst concentrationsin drinking water during an outbreak, 1964. Dose–response curves. Estimating the risk to humansfrom ingesting low pathogen doses, 1974.1 Cryptosporidium parvum, 1974.1.1 Experiments with salmonellas in mice suggestthat pathogens act independently and do notco-operate during infection, 1984.1.2 Acquired protective immunity for Cryptospo-ridium parvum, 1994.1.3 Virulence of different strains of Cryptospori-dium parvum, 1994.2 Human rotavirus, 1994.3 Bovine spongiform encephalopathy, 2004.3.1 The human oral ID

Microbiological and Chemical Quality of Raw Milk in New York State

Between December 1993 and January 1996, samples of raw milk from bulk tanks were collected by licensed milk haulers at the time of pick-up from 855 randomly selected farms in New York State (representing ∼10% of all dairy farms in the state). The milk was examined for microbial and chemical qualities. Bacterial numbers were determined by standard plate count, laboratory-pasteurized count, coliform count, heat-resistant spore-forming psychrotroph count, aerobic spore count (mesophilic), rapid psychrotrophic count, and preliminary incubation count. The frequency distributions for these counts are presented. Paired correlation analyses between the microbiological parameters showed low correlations between test results; no correlation coefficients were >0.8. The four highest positive correlation coefficients were found between standard plate count and rapid psychrotrophic count (0.7685), rapid psychrotrophic count and preliminary incubation count (0.6648), standard plate count and preliminary incubation count (0.5800), and aerobic spore count and laboratory-pasteurized count (0.5393). All other correlation coefficients were <0.5. Milk freezing points and acid degree values were determined for all samples. Frequency distributions for these results are also presented.

Observations on the effect of raw milk quality on the keeping quality of pasteurized milk

Raw milk was stored for up to 14 d at 4 degrees C and pasteurized on days 1, 3, 4, 7, 9 and 14. Precautions were taken to eliminate post-pasteurization contamination. The pasteurized milks were stored at 4 degrees C and analysed at weekly intervals for standard plate counts (SPC), psychotrophic counts (PC) and aerobic spore counts (ASC). The initial raw milk quality was very good and the keeping quality of all the pasteurized milks tested was greater than 22 d. In some cases the milk still had acceptable SPC after 42 d storage, which shows the keeping quality that can be achieved when the process is well controlled. However, the best keeping quality resulted from milk pasteurized on the third and fourth days. Even milk pasteurized on the seventh and ninth had superior keeping quality to that pasteurized on the first day. The lactoperoxidase anti-microbial system in raw milk may be most active around days 3 and 4.

Enumeration of Aerobic Spore-Formers and Bacillus cereus in Meat Product Additives

Microbial populations in 225 samples of meat product additives including spices, seasoning, proteins, starch, salt, sugar and colorants, were enumerated by means of aerobic plate counts (APC), aerobic spore counts (ASC), Bacillus cereus total counts (BcTC), and B. cereus spore counts (BcSC). Our data indicate that meat product additives with the exception of sugar and salt were contaminated mainly by aerobic spore-formers including B. cereus under spore-forming conditions. To improve the quality of meat products, it will be necessary to develop methods to reduce the bacterial contamination of meat product additives.