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Laboratory of Epidemiology and Veterinary Microbiology, Group of Bacteriology and Biotechnology, Pasteur Institute of Tunisia (IPT), University of Tunis El Manar (UTM), 2092 Tunis, Tunisia
Laboratory of Epidemiology and Veterinary Microbiology, Group of Bacteriology and Biotechnology, Pasteur Institute of Tunisia (IPT), University of Tunis El Manar (UTM), 2092 Tunis, Tunisia
Laboratory of Epidemiology and Veterinary Microbiology, Group of Bacteriology and Biotechnology, Pasteur Institute of Tunisia (IPT), University of Tunis El Manar (UTM), 2092 Tunis, Tunisia
Laboratory of Epidemiology and Veterinary Microbiology, Group of Bacteriology and Biotechnology, Pasteur Institute of Tunisia (IPT), University of Tunis El Manar (UTM), 2092 Tunis, Tunisia
Laboratory of Epidemiology and Veterinary Microbiology, Group of Bacteriology and Biotechnology, Pasteur Institute of Tunisia (IPT), University of Tunis El Manar (UTM), 2092 Tunis, Tunisia
Laboratory of Animal Resources and Food, National Institute of Agronomy, University of Carthage, Tunis (INAT) Tunisia, 43, Rue Charles Nicole, Cité Mahrajène, Le Belvédère, 1082 Tunis, Tunisia
Laboratory of Epidemiology and Veterinary Microbiology, Group of Bacteriology and Biotechnology, Pasteur Institute of Tunisia (IPT), University of Tunis El Manar (UTM), 2092 Tunis, Tunisia
Spore-forming bacteria, principally Bacillus species, are important contaminants of milk. Because of their high heat resistance, Bacillus species spores are capable of surviving the heat treatment process of milk and lead to spoilage of the final product. To determine the factors influencing the contamination of milk, spore-forming bacteria occurrence throughout the UHT milk production line during winter, spring, and summer was studied. The obtained results confirm that the total viable rate decreases rapidly throughout the production line of UHT milk showing the efficiency of thermal treatments used. However, the persistent high rate of spore-forming bacteria indicates their high heat resistance, especially in spring and summer. In addition, a significant variation of the quality of raw milk according to the location of the collecting centers was revealed. The molecular identification showed a high degree of diversity of heat-resistant Bacillus species, which are isolated from different milk samples. The distribution of Bacillus species in raw milk, stored milk, bactofuged milk, pasteurized milk, and UHT milk were 28, 10, 16, 13, and 33%, respectively. Six Bacillus spp. including Bacillus licheniformis (52.38%), Bacillus pumilus (9.52%), Bacillus sp. (4.76%), Bacillus sporothermodurans (4.76%), Terribacillus aidingensis (4.76%), and Paenibacillus sp. (4.76%) were identified in different milk samples.
Occurrence and resistance of potentially highly heat resistant spore forming bacteria in milk products and at dairy farms. PhD Thesis. Centre for Agricultural Research, Ghent, Department of Animal Product Quality, University of Gent,
Belgium2004
). These bacteria are of particular concern because they are capable of forming endospores and can thus survive pasteurization and other heat treatments commonly used to process raw food materials (
). The heat resistance of aerobic spore-formers isolated from dairy products was examined to give an overview of occurring highly heat-resistant spores (HRS). These spores have a special position among total microflora of milk with regard to their ability to survive thermal treatment of milk and subsequently to propagate in final products (
). The highly heterogeneous genus Bacillus comprises the largest species group of endospore-forming bacteria. Because of their ubiquitous nature, Bacillus spores can penetrate food production at several stages, resulting in significant economic losses and posing a potential risk to consumers due to the capacity of some Bacillus strains for toxin production (
). The major spore-forming bacilli have contaminated and spoiled treated-UHT or sterilized milk, especially Bacillus licheniformis, Bacillus cereus, Geobacillus stearothermophilus, Bacillus coagulans, Bacillus sporothermodurans, Brevisbacillus brevis, Paenibacillus lactis, and Bacillus sphaericus (
Occurrence and resistance of potentially highly heat resistant spore forming bacteria in milk products and at dairy farms. PhD Thesis. Centre for Agricultural Research, Ghent, Department of Animal Product Quality, University of Gent,
Belgium2004
). In addition, several entry points of theses microorganisms have been identified at the farm level including concentrate feeds, silage, bedding, manure, soil, wash water, clusters, teat cups, and filter cloths (
). The processing plant has also been identified as a source of spore-forming bacteria, and there might be potential for milk contamination due to the presence and persistence of Bacillus and Paenibacillus spp. in processing environments (
An improved understanding of the sources of potentially HRS throughout the production line of UHT milk is necessary to prevent or reduce their presence in final product and to increase product shelf life (
) were evaluated. Although the incidence of B. sporothermodurans and other heat-resistant bacteria in Tunisian milk (raw milk, pasteurized milk, and UHT milk) and the characterization of their phenotype and genotype properties have been evaluated by
, the factors influencing their incidence and their origin have not previously been studied. The present study aims to evaluate the quality of different types of milk. In addition, the factors influencing the presence of Bacillus and related genera in a milk chain in Tunisia and the source of these bacteria in packaged fluid milk were determined.
MATERIALS AND METHODS
Milk Sampling
Forty-one samples were taken at different stages during the UHT milk manufacturing. Twenty-one samples of raw milk have been provided from different collected centers situated in northern and northwest of Tunisia during winter, spring, and summer periods.
Milk collection was achieved in a dairy plant in Tunisia and the different sampling points are tankers of raw milk originated from different collecting centers, raw milk storage tank, bactofuged milk, standardized milk, pasteurized milk, and UHT milk (Table 1).
Table 1Sampling plan of milk during 3 seasons and from different manufacturing steps of UHT Tunisian milk
Determination of Milk Quality and Isolation of Bacteria
The quality of different types of milk samples was evaluated using 2 parameters: total and spore-forming bacteria counts. In fact, the total flora presented in different samples of milk was determined by plating on plate count agar after serial dilutions prepared in peptone water (0.01%). The determination of spore-forming bacteria from UHT milk was obtained by spreading 0.1 mL of product on brain-heart infusion agar supplemented with 1 mg/L of vitamin B12. After incubation for 48 or 72 h at 37°C, the plates were examined and the counts of individual microbial groups were expressed as colony-forming units per milliliter of milk. Typically, colonies representing each visually distinct morphology were selected and transplanted several times to obtain pure culture. Only strains representing gram-positive characters were used in this study. Furthermore, isolation of these bacteria from raw, bactofuged, standardized, and pasteurized milk was realized after 4 heat treatments at 100°C with varying the duration of treatment (Table 2) to destroy vegetative bacteria and make isolation of heat-resistant bacteria that survive the heat pre-treatment more easily. The isolated bacteria from the third and fourth heat treatment (100°C for 40 min and 100°C for 50 min) were heated another time at 100°C for 40 min to select the highly heat-resistant bacteria, so 21 isolated bacteria survived treatment at 100°C for 40 min.
Table 2Heat treatments for the isolation of high resistant bacteria from different types of Tunisian milk
Microbial cultures were stored at (−20°C) on brain-heart infusion agar supplemented with 20% glycerol.
Phenotypic Characterization of Isolates
One hundred twelve isolates were examined for colony and cell morphology, and for motility. Colony morphology was described using standard microbiological criteria, followed by Gram staining and microscopic observation (
Forty 5 isolates showing different phenotypic characteristics were selected. Bacterial DNA was extracted from pure bacteria culture according to the methods provided by GF-1 Bacterial DNA extraction Kit (Vivantis Technologies, Subang Jaya, Selangor, Malaysia) user's guide protocol. Therefore, the molecular identification of selected isolates was conducted by different PCR primers. The first and second were species-specific PCR BSPO and gapC (CarthaGenomics Advanced Technologies, Iman Abou Hanifa city, Tunisia), which allow the characterization, respectively, of B. sporothermodurans and Paenibacillus sp. (
The third PCR primer is for the amplification of housekeeping gene rpoB (encoding the β subunit of RNA polymerase) based on available gene sequences for different Bacillus spp. (
). This gene present in all gram-positive bacteria had previously been shown to be an appropriate target for DNA sequencing-based characterization of broad groups of gram-positive bacteria (
). Because rpoB is generally less well conserved among closely related organisms than those for 16S rDNA, many authors hypothesize that sequences from these genes would provide increased discrimination among the isolated Bacillus species over 16S rDNA sequence data. The rpoB fragment was amplified using primers described by
The PCR reaction mixture of each PCR contained 5 µL of 10× PCR buffer (with MgCl2), 0.2 µL of 25 mM deoxynucleotide triphosphates, 0.5 µL of each primer stock solution (25 mM), 0.2 µL of Taq polymerase (5 U/µL), 5 µL of chromosomal DNA, and 13.6 µL of sterilized distilled water in a total volume of 25 µL. The PCR conditions released in this study for each gene are given in Table 3.
Table 3Primers and polymerase chain reaction (PCR) cycling conditions
The identification of selected isolates of Bacillus species was confirmed by sequencing the 16S rRNA genes after PCR amplification, using the universal primers forward Bact16F27N (5′-AGAGTTTGATCCTGGCTCAG-3′) and reverse 16R1525 (5′-CTACGGCTACCTTGTTACGA-3′; Invitrogen), as described by
. The PCR reaction mixture contained 5 µL of 10× PCR Buffer (with MgCl2), 0.2 µL of 25 mM deoxynucleotide triphosphates, 0.5 µL of each primer stock solution (25 mM), 0.2 µL of Taq polymerase (5 U/µL), 5 µL of chromosomal DNA, and 13.6 µL of sterilized distilled water in a total volume of 25 µL.
The PCR was performed in a thermal cycler (Bio-Rad T100, Bio-Rad, Hercules, CA). Thirty thermal cycles were carried out as following: denaturation at 94°C for 45 s, hybridation at 55°C for 1 min, and extension at 72°C for 2 min. The first cycle was preceded by initial denaturation at 94°C for 3 min, and the last one was followed by a final extension step at 72°C for 7 min.
Five microliters of each PCR product were electrophoresed on 1.5% (wt/vol) agarose gel using Tris borate-EDTA. Gel was stained with Run-Safe DNA Gel Strain (7 µL/100 mL; Thermo Fisher Scientific, Waltham, MA) and visualized under UV light.
The amplicons were sequenced in both orientations on the MegaBACE 1000 capillary sequencer (Amersham BioSciences, Little Chalfont, UK), in the Genomic and Biomedical Ontogenetic Laboratory at the Pasteur Institute of Tunisia. The sequence data were edited and analyzed using into the BioEdit version 5.0.9 and the RDP Sequence Aligner programs (
). Obtained sequences were compared with available databases using the GenBank BLASTN combined with Ez-Taxon (http://eztaxon-e.ezbiocloud.net/) search tools.
Statistical Analysis
Data were analyzed using the SAS v. 9.1.3 program (SAS, 1990, SAS Institute Inc., Cary, NC). Analysis of variance and Duncan's multiple range method were used to compare any significant differences between solvents and samples. Values were expressed as means ± standard deviations. Differences were considered significant at P < 0.05. All the analyses were carried out in triplicate, and the values were the average of 3 replicates.
RESULTS AND DISCUSSION
Study of the Microbiological Quality of Milk
The microbiological quality of different types of collected milk from spring, winter, and summer was evaluated by the determination of total flora and aerobic-spore-forming bacteria counts. Figure 1 shows the mean log (cfu/mL) of the total flora and heat-resistant bacteria in all raw milk samples collected from different origins. The microbial quality of raw milk, particularly the incidence of HRS, depends on its origin. The rate of total flora is almost the same in all samples of raw milk (106 cfu/mL), but the level of aerobic-spore-forming bacteria differs between samples. The HRS is present only in 3 samples. Our findings are in agreement with preview reports described by
who worked on raw milk samples collected from different local points of the Van Province in Turkey, found that climatic conditions, seasonal variation, and regional differences are the most important sources of variation in microbial composition of milk (
). Moreover, many researchers found that regional incidence of spores in raw milk can be linked to the pasturing cattle, the milking equipment and the farm bulk tanks (
showed that each farmer's milking practices may contribute to the diversity of bacteria found in the milk, which they produce after analysis of different samples of bulk milk collected from 67 dairy farms in the Savoie regions of France.
Figure 1Total and heat-resistant spore (HRS) counts in different samples of raw milk. Values given are means (error bars represent SD) of 3 independent experiments.
The farm environment (silage, feed, animal faces, bedding, soil, and so on) is associated with poor hygienic practices and affects the quality of raw milk (
The second factor influencing the microbiological quality of milk is seasonal variation. The mean log (cfu/mL) of total flora and heat-resistant bacteria during the manufacturing process of UHT milk at different seasons is summarized in Figure 2. The results demonstrated a decreasing number of total flora during the UHT milk process (106 cfu/mL in raw milk to 102 cfu/mL in bactofuged milk). This result proved the efficiency of heat treatment in reducing the total flora. The incidence of the HRS throughout the lines of production of UHT milk was lower in winter and higher in spring and summer mostly in pasteurized and bactofuged milk (10–25 cfu/mL of HRS, respectively). Our findings are in agreement with
suggested that one possible reason responsible for the seasonal distribution of psychrotrophic bacilli in raw milk is that they are derived from summer pasture and enter milk due to increased contamination of the cow's udders. In general, the occurrence of Bacillus sp. in raw milk is usually attributed to seasonal effects (
). Hay and dust are considered to be sources of these bacteria during winter months, whereas dirty teats by soil are the sources during the humid summer months.
Figure 2Effect of seasons and dairy processing in the total and heat-resistant spore (HRS) counts of milk. Values given are means (error bars represent SD) of 3 independent experiments.
On the other hand, microorganisms can enter into the milk chain through dairy processing. Results shown in Figure 3 indicate that the total viable count rate decreases rapidly throughout the production line of UHT milk (from 106 to 1 cfu/mL in UHT milk), showing the efficiency of thermal treatments used. However, the existence of aerobic spore-forming bacteria in different segments of UHT milk production lines is considerable. Also, the increasing number of spore-forming bacteria between raw and UHT milk (1–8 cfu/mL of HRS, respectively) proves the possible contamination of milk by these spores in dairy industries. So, additional sources in the factory, such as equipment, packaging materials (
) are a significant sources of contamination of milk and dairy products with spore-forming bacteria.
Figure 3Total and heat-resistant spore (HRS) counts throughout the UHT milk production line. Values given are means (error bars represent SD) of 3 independent experiments.
The majority of isolates, obtained after a heat treatment of 40 min at 100°C, present biochemical and microbiological characteristics that match completely with Bacillus species. They were positive to the Gram reaction and catalase tests. Variable results were obtained for oxidase reaction and all isolates hydrolyzed casein. This phenotypic identification was followed by a molecular study to identify the isolated species. Twenty-one isolates were selected for molecular identification. The amplification of BSPO gene demonstrate that one isolate from raw milk harbored this gene. Only one isolate from UHT milk reacted positively in gap C-PCR. However, all strains reacted positively in rpoB-PCR, which proves that all isolates belonged to the genus Bacillus in concordance with phenotypic identification results. Table 4 shows the results of the sequence alignment and the comparison of partial 16S rDNA genes of the strains analyzed in this study with sequences of the National Center for Biotechnology Information databases. Phylogenetic analysis indicated that all isolated strains are related to 6 species of the Bacillus genus. Despite the small number of samples and identified species, our data present a prospective picture on spore-forming bacteria biodiversity in Tunisian milk.
Table 4Molecular identification of aerobic spore-forming bacteria isolated from Tunisian milk
According to Table 5, B. licheniformis is the most commonly isolated species of Bacillus, present in milk at various processing stages, and this genus represents 52.38% of all isolates. Our findings are in agreement with
found that B. licheniformis was the most commonly isolated species of Bacillus found in milk at all stages of processing and was ubiquitous in the farm environment. However, previous studies have shown that some Bacillus species, such as B. licheniformis, have been linked to potential food poisoning (
). Of the 4 Bacillus species isolated from UHT milk, we detected the presence of Paenibacillus sp. (4.76%), which was isolated for the first time in UHT milk produced in Tunisia. These findings are in agreement with the results of many authors such as
Occurrence and resistance of potentially highly heat resistant spore forming bacteria in milk products and at dairy farms. PhD Thesis. Centre for Agricultural Research, Ghent, Department of Animal Product Quality, University of Gent,
Belgium2004
who signaled the presence of Paenibacillus sp. in both raw and heat-treated milk. Moreover, Paenibacillus spores have been isolated from silage and feed concentrates, which may be the origin of this contamination.
Table 5Distribution of Bacillus species isolated from different milk samples
Interestingly, we demonstrate the presence of Terribacillus aidingensis (4.76%), a spore-forming, moderately halophilic bacterium. This species was isolated from field soil in Japan (
Complete genome sequence of Terribacillus aidingensis strain MP602, a moderately halophilic Bacterium isolated from Cryptomeria fortune in Tianmu Mountain in China.
), and this is the first time it was isolated from the dairy product (UHT milk). The main phenotypic characteristic of the genus Terribacillus is the formation of ellipsoidal endospores (
). Bacillus sporothermodurans, the highly heat-resistant bacteria, was isolated from raw milk (4.76%), and we found that 9.52% of the isolated strains were identified as B. pumilus and 4.76% as Bacillus sp. These results were partially in agreement with the findings of
who studied the incidence of heat-resistant spore formers from 3 types of Tunisian milk and reported the presence of 7 Bacillus species in both raw and pasteurized milk (B. sporothermodurans, B. cereus, B. subtilis, B. licheniformis, Brevibacillus brevis, B. sphaericus, and B. pumilus) and the persistence of 4 Bacillus species in UHT milk (B. sporothermodurans, B. cereus, B. sphaericus, and B. licheniformis).
Distribution of Different Species Isolated from Different Types of Milk
The distribution of Bacillus species in the different types of analyzed milk is summarized in Table 5. In the raw milk, 4 Bacillus species were present, and for the UHT milk the predominant isolated strains belonged to B. licheniformis. Bacillus species can be present in milk deriving from a variety of sources, but psychrotrophic strains of Bacillus sp. are introduced into milk as spores from pasture or as a result of improper cleaning of bulk tanks (
). In addition, we noticed the presence of spore-forming bacteria in bactofuged and pasteurized milk, and these results are in correlation with the findings of
who suggested that pasteurized milk may be spoiled by spore formers (e.g., Bacillus sp.) that have survived heat treatment or entered the milk process after heat treatment by recontamination of the milk. So, the presence of these spore-forming bacteria in pasteurized milk, which should have been inactivated by pasteurization, could be due to postpasteurization contamination or to their resistance to thermal treatments. Therefore, we note that significant numbers of the field strains are resistant to thermal treatments during the pasteurization operations and even after the sterilization process UHT milk (12 strains). Of these 12 heat-resistant strains, the species found are Paenibacillus and Terribacillus aidingensis in UHT milk. However, heat-resistant strains are also present belonging to pathogenic Bacillus sp. implicated in some cases of food poisoning, namely B. licheniformis (6 strains). Our findings indicate that a considerable diversity of Bacillus sp. exists in raw milk and some of these spore formers appear to persist over time in UHT processing. These findings are consistent with a variety of other reports that have shown that various bacteria can persist in some cases for extended periods of time in food processing plants. In addition, the contamination of milk after the high temperature sterilization may result from several sources, but 2 important ones are the seals in the homogenizer and the air supply to the aseptic packaging unit.
Heat resistance of microorganisms under seals. In Proceedings of the 24th International Dairy Congress (Melbourne). International Dairy Federation,
Brussels1994
showed that spores trapped under seals had enhanced heat stability, largely attributable to a very low water activity in their microenvironment, and could act as a reservoir of spore contamination.
CONCLUSIONS
This study revealed a large diversity of spore-forming species, which are able to survive heating for 40 min at 100°C. These results show clearly the persistence of a potential risk of foodborne illness due to UHT milk consumption despite the application of heat treatment at UHT. Therefore, it seems to be evident to talk about a direct link of contamination with highly HRS from the raw milk on the dairy farm to the final product in the dairy. It is important to conclude that raw milk used for the UHT milk should be chosen with extreme care and high microbiological qualities. Also, a chain management approach taking into account the entire chain from raw materials via processing to final products will be the most effective way to control and to reduce spores in various food production processes and to prevent spoilage of foods. Moreover, it is necessary to investigate new strategies to eliminate bacterial spores from UHT milk and other dairy products. Interesting research still needs to be conducted in this area.
ACKNOWLEDGMENTS
The authors acknowledge the financial support provided by the Tunisian Ministry of Higher Education and Scientific Research.
REFERENCES
Abo-elnaga H.I.
Hegazi F.Z.
Abo-elnaga I.G.
Spore-forming rods surviving boiling the raw milk and implicated in later spoilage of the product.
Heat resistance of microorganisms under seals. In Proceedings of the 24th International Dairy Congress (Melbourne). International Dairy Federation,
Brussels1994
Complete genome sequence of Terribacillus aidingensis strain MP602, a moderately halophilic Bacterium isolated from Cryptomeria fortune in Tianmu Mountain in China.
Occurrence and resistance of potentially highly heat resistant spore forming bacteria in milk products and at dairy farms. PhD Thesis. Centre for Agricultural Research, Ghent, Department of Animal Product Quality, University of Gent,
Belgium2004
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