Isolation, Characterization, and Identification of Bacterial Contaminants in Semifinal Gelatin Extracts

Abstract

Bacterial contamination of gelatin is of great concern. Indeed, this animal colloid has many industrial applications, mainly in food and pharmaceutical products. In a previous study (E. De Clerck and P. De Vos, Syst. Appl. Microbiol. 25:611-618), contamination of a gelatin production process with a variety of grampositive and gram-negative bacteria was demonstrated. In this study, bacterial contamination of semifinal gelatin extracts from several production plants was examined. Since these extracts are subjected to harsh conditions during production and a final ultrahigh-temperature treatment, the bacterial load at this stage is expected to be greatly reduced. In total, 1,129 isolates were obtained from a total of 73 gelatin batches originating from six different production plants. Each of these batches was suspected of having bacterial contamination based on quality control testing at the production plant from which it originated. For characterization and identification of the 1,129 bacterial isolates, repetitive-element PCR was used to obtain manageable groups. Representative strains were identified by means of 16S rRNA gene sequencing, species-specific gyrB PCR, and gyrA and rpoB sequencing and were tested for gelatinase activity. The majority of isolates belonged to members of Bacillus or related endospore-forming genera. Representative strains were identified as Bacillus cereus, Bacillus coagulans, Bacillus fumarioli, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, Bacillus sonorensis, Bacillus subtilis, Bacillus gelatini, Bacillus thermoamylovorans, Anoxybacillus contaminans, Anoxybacillus flavithermus, Brevibacillus agri, Brevibacillus borstelensis, and Geobacillus stearothermophilus. The majority of these species include strains exhibiting gelatinase activity. Moreover, some of these species have known pathogenic properties. These findings are of great concern with regard to the safety and quality of gelatin and its applications.

Incidence and Diversity of Potentially Highly Heat-Resistant Spores Isolated at Dairy Farms

“…In addition to confirming FAME identifications, ARDRA enabled nine additional identifications at the species level. ARDRA clusters 4,6,7,8,24,28,29, and 30 and strain R-6443 were identified as Bacillus smithii, B. circulans, Bacillus oleronius, B. sporothermodurans, Brevibacillus brevis, Aneurinibacillus aneurinilyticus, Aneurinibacillus thermoaerophilus, Virgibacillus proomii and Paenibacillus thiaminolyticus, respectively ( Table 4). The strains in ARDRA clusters 6, 7, 8, 12, 13, and 24 grouped together with the type strain of the respective species analyzed simultaneously in this study.…”

“…A total of 16,6,8, and 36 isolates of raw milk, the milking equipment, green crop, and fodder, respectively, could not be analyzed since they failed to grow according to the prescribed growth conditions for FAME analysis. These isolates were not further considered.…”

Antibiosis and dark-pigments secretion by the phytopathogenic and environmental fungal species after interaction in vitro with a Bacillus subtilis isolate

“…The PCR-reactions were performed with the following universal primers: 16S rRNA (8bF, 5'-ACTGGGATCCACAGGAGTGCAAACGACG A-3'; 518f, 5'-CCAGCAGCCGCGGTAAT-3'; 1406r, 5'-ACGGGCGGTGTGTRC-3'; and 1512r, 5'-CTGAAGCTTACGGYTAGCTTGTTACGAC TT-3'), gyrA (gyrA-f, 5'-CAGTCAGGAAATGC GTACGTCCTT-3' and gyrA-r, 5'-CAAGGTAA TGCTCCAGGCATTGCT-3'), and rpoB (rpoB-f, 5'-AGGTCAACTAGTTCAGTATGGAC-3' and rpoB-r, 5'-AAGAACCGTAACCGGCAACTT-3') (De Clerck et al, 2004). Each 50 µL PCR reaction mix contained 5 µL Taq buffer 10x, 3 µL MgCl 2 25 mM, 0.4 µL dNTPs 25 mM, 0.5 µL Taqpolymerase 5 U/mL (all reagents from Fermentas), 1.25 µL primer 20 mM, 50 ng genomic DNA and 36.1 µL H 2 O milli-Q.…”

“…Many misclassifications have been found in the phylum Firmicutes (Wang et al, 2007). De Clerck et al (2004) reported that the analysis of only the 16S rRNA gene was unsuitable for the species or sub-species identification of the genus Bacillus, particularly of the cereus and subtilis groups. Sequencing of other genes, such as gyrA and rpoB, was suggested to aid in molecular identification of the subtilis group because of the great homology among 16S rRNA sequences (Chun andBae, 2000, Earl et al, 2007).…”

Isolation and Characterization of a Bluegill-Degrading Microorganism, and Analysis of the Root Hair-Promoting Effect of the Degraded Products

“…is known as a protease producing strain and suppresses the fungal pathogen in soil. 22,23) Moreover, the genus Brevibacillus is not listed as an infectious or toxin-producing microorganism. 24) Phytohormones, which contribute to plant growth and/or plant physiology, have been studied extensively.…”