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Microorganisms Mar 2022Acrylamide is a toxic compound that is formed in cooked carbohydrate-rich food. Baking, roasting, frying, and grilling are cooking methods that cause its formation in...
Acrylamide is a toxic compound that is formed in cooked carbohydrate-rich food. Baking, roasting, frying, and grilling are cooking methods that cause its formation in the presence of reducing sugar and asparagine. To prevent acrylamide formation or to remove it after its formation, scientists have been trying to understand acrylamide formation pathways, and methods of prevention and removal. Therefore, this study aimed to: (1) screen newly isolated LAB for acrylamide removal, (2) optimize conditions (pH, temperature, time, salt) of the acrylamide removal for selected LAB isolates using Box-Behnken design (BBD), (3) investigate the acrylamide removal abilities of selected LAB isolates under the in vitro digestion conditions using INFO-GEST2.0 model, and (4) explore the mechanism of the acrylamide removal using scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDS), zeta potential, transmission electron microscopy (TEM) measurement, and Fourier transform infrared spectroscopy (FTIR). Forty strains were tested in MRS broth, where and had the highest capability of acrylamide removal by 39% and 26%, respectively. To enhance the binding ability, both strains were tested under controlled conditions of pH (4.5, 5.5 and 6.5), temperature (32 °C, 37 °C and 42 °C), time (14, 18 and 22 h), and NaCl (0%, 1.5% and 3% /) using Box-Behnken design (BBD). Both strains removed more acrylamide in the range of 35-46% for and 45-55% for . After testing the bacterial binding ability, both strains were exposed to a simulated gastrointestinal tract environment, removing more than 30% of acrylamide at the gastric stage and around 40% at the intestinal stage. To understand the mechanism of removal, LAB cells were characterized via scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDS) and transmission electron microscopy (TEM) techniques. Cell charges were characterized by zeta potential and functional groups analyzed by Fourier transform infrared spectroscopy (FTIR). Results indicated that increasing cell wall thickness improved acrylamide adsorption capacity. Both FTIR and EDS indicated that functional groups C=O, C-O, and N-H were associated with acrylamide adsorption.
PubMed: 35336133
DOI: 10.3390/microorganisms10030557 -
Veterinary Sciences Aug 2022Changes in the gut microbiome can be associated with diseases and affect the overall health of an individual. In the current study, the gut microbiome profile of dogs...
Changes in the gut microbiome can be associated with diseases and affect the overall health of an individual. In the current study, the gut microbiome profile of dogs diagnosed with advanced stages of multicentric lymphoma was compared with that of healthy dogs and analyzed. For this purpose, dogs from veterinary hospitals diagnosed with lymphoma were selected and were further narrowed down to cases of stage IV multicentric lymphoma. Fecal samples from the selected sick and healthy dogs were collected and analyzed using MiSeq sequencing. The gut microbiota in the two groups of dogs was statistically analyzed and compared. The results revealed significant differences in the microbial populations present in sick and healthy dogs. Phylum Actinobacteria and two species ( and ) were found in high proportions in sick dogs and may be considered as potential biomarkers for canine stage IV multicentric lymphoma. Further investigations need to be conducted to understand the mechanisms they might be involved in.
PubMed: 36006324
DOI: 10.3390/vetsci9080409 -
Food Technology and Biotechnology Dec 2023Heat-stabilised defatted rice bran (HSDRB) is a primary by-product of rice bran oil extraction industry and a nutritious source of protein. However, despite the unique...
RESEARCH BACKGROUND
Heat-stabilised defatted rice bran (HSDRB) is a primary by-product of rice bran oil extraction industry and a nutritious source of protein. However, despite the unique nutritional profile of rice bran protein, the protein-rich by-product, HSDRB is underutilised as a low-value animal feed. Research on protein extraction from HSDRB by enzymatic hydrolysis has attracted the attention of numerous scientists. However, a cost-effective extraction method is required to mitigate the high costs associated with the use of enzymes. Therefore, we have presented an alternative economical and natural approach for protein extraction from HSDRB by solid-state fermentation (SSF) with heterofermentative microbes.
EXPERIMENTAL APPROACH
SSF of HSDRB with two types of traditional Asian fermentation starters, namely loog-pang and koji, were evaluated for enzyme production and their efficacy in extracting proteins from HSDRB. For this purpose, HSDRB fermentation was carried out for 0, 12, 24, 48, 72 and 96 h followed by 24-hour hydrolysis to evaluate the extracted rice bran protein. In addition, microbiome diversity in the fermentation starters was also determined by metagenomic sequencing of 16S rRNA and internal transcribed spacer to identify bacteria and fungi, respectively.
RESULTS AND CONCLUSIONS
The microbial community in the fermentation starters showed the dominance of lactic acid bacteria (LAB) such as in loog-pang and , and in koji, while yeast species and dominated the fungal diversity in loog-pang and koji starters, respectively. The results suggest that loog-pang and koji can produce cellulase, neutral and acid proteases during fermentation. Despite the discrepancy in their microbial diversity and the enzyme activity during SSF, both starters could effectively increase protein extraction from HSDRB. A positive relationship between the SSF duration and extracted protein was observed. During SSF with loog-pang and koji after 72 h followed by 24-hour hydrolysis, 65.66 and 66.67 % protein was extracted from HSDRB, respectively. The amino acid analysis of the protein hydrolysate produced by the non-fermented and fermented methods showed no difference and had an abundance of glutamic and aspartic acids, leucine, arginine, alanine and glycine amino acids, which accounted for approx. 58 % of the total amino acids.
NOVELTY AND SCIENTIFIC CONTRIBUTION
Loog-pang and koji (traditional Thai and Japanese fermentation starters, respectively) were found to be effective in extracting proteins from HSDRB by SSF although they are inexpensive microbial enzyme sources. Future research aimed at scaling up HSDRB protein extraction for usage in industrial applications can draw on our results.
PubMed: 38205047
DOI: 10.17113/ftb.61.04.23.8255 -
Journal of Bacteriology Dec 2013A 94-kb integrative conjugative element (ICESluvan) transferable to Enterococcus faecium and Enterococcus faecalis from an animal isolate of Streptococcus lutetiensis...
ICESluvan, a 94-kilobase mosaic integrative conjugative element conferring interspecies transfer of VanB-type glycopeptide resistance, a novel bacitracin resistance locus, and a toxin-antitoxin stabilization system.
A 94-kb integrative conjugative element (ICESluvan) transferable to Enterococcus faecium and Enterococcus faecalis from an animal isolate of Streptococcus lutetiensis consists of a mosaic of genetic fragments from different Gram-positive bacteria. A variant of ICESluvan was confirmed in S. lutetiensis from a patient. A complete Tn5382/Tn1549 with a vanB2 operon is integrated into a streptococcal ICESde3396-like region harboring a putative bacteriophage exclusion system, a putative agglutinin receptor precursor, and key components of a type IV secretion system. Moreover, ICESluvan encodes a putative MobC family mobilization protein and a relaxase and, thus, in total has all genetic components essential for conjugative transfer. A 9-kb element within Tn5382/Tn1549 encodes, among others, putative proteins similar to the TnpX site-specific recombinase in Faecalibacterium and VanZ in Paenibacillus, which may contribute to the detected low-level teicoplanin resistance. Furthermore, ICESluvan encodes a novel bacitracin resistance locus that is associated with reduced susceptibility to bacitracin when transferred to E. faecium. The expression of a streptococcal pezAT toxin-antitoxin-encoding operon of ICESluvan in S. lutetiensis, E. faecium, and E. faecalis was confirmed by reverse transcription (RT)-PCR, indicating an active toxin-antitoxin system which may contribute to stabilizing ICESluvan within new hosts. Junction PCR and DNA sequencing confirmed that ICESluvan excised to form a circular intermediate in S. lutetiensis, E. faecalis, and E. faecium. Transfer between E. faecalis cells was observed in the presence of helper plasmid pIP964. Sequence analysis of the original S. lutetiensis donor and enterococcal transconjugants showed that ICESluvan integrates in a site-specific manner into the C-terminal end of the chromosomal tRNA methyltransferase gene rumA.
Topics: Anti-Bacterial Agents; Antitoxins; Bacterial Proteins; Bacterial Toxins; Base Sequence; Gene Expression Regulation, Bacterial; Glycopeptides; Molecular Sequence Data; Operon; Plasmids; Streptococcus; Vancomycin; Vancomycin Resistance
PubMed: 24078615
DOI: 10.1128/JB.02165-12 -
Veterinary World Sep 2019Little information about the stability and changes of sheep ruminal microbiota due to pathogen intervention in the rumen simulation technique (RUSITEC) is available....
BACKGROUND AND AIM
Little information about the stability and changes of sheep ruminal microbiota due to pathogen intervention in the rumen simulation technique (RUSITEC) is available. This study aimed to investigate the effect of administration of a novel isolated strain on rumen microbiology and physiology. In addition, the isolation of pigment-producing is described.
MATERIALS AND METHODS
Microbial strains were isolated from sheep rumen digesta. An isolated strain of was evaluated in the RUSITEC system fed with mixed cattle feed and compared with an in-house developed probiotic formulation (PF), PF 1, containing , , and . The parameters of volatile fatty acid, lactic acid, pH profiling, and the coliform anti-pathogenicity were evaluated to determine the effect of on rumen function and physiology.
RESULTS
Administration of reduced the coliform count by 31.20% from 7.2×10 colony-forming units (CFU)/mLto 1.7×10 CFU/mL. Agar diffusion assays revealed the extracellular antimicrobial activity of against coliforms; and with 12 and 14 mm zones of inhibition, respectively. Simultaneously, an increase of 61.62% in the rumen yeast count was noted. The physiological changes resulted in a 5% reduction in acetic acid concentration from 431 to 405 mg/L.
CONCLUSION
The present research indicates that is highly capable of altering rumen physiology and function on colonization and is a key transition microbe to be studied during rumen intervention studies. A decrease in the coliform count could be attributed to extracellular production of a bacteriocin-like substance, as illustrated through agar diffusion assays.
PubMed: 31749568
DOI: 10.14202/vetworld.2019.1362-1371 -
Journal of Clinical Microbiology Sep 2011All Streptococcus bovis blood culture isolates recovered from January 2003 to January 2010 (n = 52) at the Hospital Universitario Ramón y Cajal were reidentified on the...
All Streptococcus bovis blood culture isolates recovered from January 2003 to January 2010 (n = 52) at the Hospital Universitario Ramón y Cajal were reidentified on the basis of their genetic traits using new taxonomic criteria. Initial identification was performed by the semiautomatic Wider system (Fco. Soria-Melguizo, Spain) and the API 20 Strep system (bioMérieux, France). All isolates were reidentified by PCR amplification and sequencing of both the 16S rRNA and sodA genes and by mass spectrometry using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS; Bruker, Germany). Results of 16S rRNA/sodA gene sequencing were as follows: Streptococcus gallolyticus subsp. gallolyticus, 14/14 (number of isolates identified by 16S rRNA/number of isolates identified by sodA gene sequencing); Streptococcus gallolyticus subsp. pasteurianus, 24/24; Streptococcus spp., 7/0; Streptococcus infantarius subsp. infantarius, 0/2; Streptococcus lutetiensis, 0/5; Leuconostoc mesenteroides, 4/0; and Lactococcus lactis, 3/3. MALDI-TOF MS identified 27 S. gallolyticus isolates but not at the subspecies level, 4 L. mesenteroides isolates, 3 L. lactis isolates, and 6 S. lutetiensis isolates, whereas 12 isolates rendered a nonreliable identification result. Pulsed-field gel electrophoresis grouped all S. gallolyticus subsp. gallolyticus isolates into 3 major clusters clearly different from those of the S. gallolyticus subsp. pasteurianus isolates, which, in turn, exhibited no clonal relationship. The percentages of resistance to the tested antimicrobials were 38% for erythromycin, 23% for fosfomycin, 10% for levofloxacin, 6% for tetracycline, and 4% for co-trimoxazole. The most frequent underlying diseases were hepatobiliary disorders (53%), endocarditis (17%), and malignancies (12%). We conclude that sequencing of the sodA gene was the most discriminatory method and that S. gallolyticus subsp. pasteurianus appears to have a higher genetic diversity than S. gallolyticus subsp. gallolyticus.
Topics: Adult; Aged; Aged, 80 and over; Anti-Bacterial Agents; Bacteremia; Bacterial Proteins; Bacterial Typing Techniques; DNA, Bacterial; DNA, Ribosomal; Drug Resistance, Bacterial; Electrophoresis, Gel, Pulsed-Field; Female; Humans; Male; Microbial Sensitivity Tests; Middle Aged; Molecular Typing; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Streptococcal Infections; Streptococcus bovis; Superoxide Dismutase
PubMed: 21752968
DOI: 10.1128/JCM.00524-11 -
Microorganisms Oct 2020This study aimed to set-up a biotechnological protocol for manufacturing a reduced-fat Burrata cheese using semi-skimmed milk and reduced-fat cream, in different...
This study aimed to set-up a biotechnological protocol for manufacturing a reduced-fat Burrata cheese using semi-skimmed milk and reduced-fat cream, in different combinations with exopolysaccharides-synthesizing bacterial starters (, E1, or subsp. and . subsp. , E2) and carrageenan or xanthan. Eight variants of reduced-fat cheese (fat concentration 34-51% lower than traditional full-fat Burrata cheese, used as the control) were obtained using: (i) semi-skimmed milk and reduced-fat cream alone (RC) or in combination with (ii) xanthan (RCX), (iii) carrageenan (RCC), (iv) starter E1 (RCE1), (v) starter E2 (RCE2), (vi) both starters (RCE1-2), (vii) E1 and xanthan (RCXE1), or E1 and carrageenan (RCCE1). Post-acidification occurred for the RCC, RCX, and RCE2 Burrata cheeses, due to the higher number of mesophilic cocci found in these cheeses after 16 days of storage. Overall, mesophilic and thermophilic cocci, although showing cheese variant-depending dynamics, were dominant microbial groups, flanked by sp. during storage. Lactobacilli, increasing during storage, represented another dominant microbial group. The panel test gave highest scores to RCE1-2 and RCXE1 cheeses, even after 16 days of storage. The 16S-targeted metagenomic analysis revealed that a core microbiota (, , , sp., , , and sp.), characterized the Burrata cheeses. A consumer test, based on 105 people, showed that more than 50% of consumers did not distinguish the traditional full-fat from the RCXE1 reduced-fat Burrata cheese.
PubMed: 33096692
DOI: 10.3390/microorganisms8101618 -
Current Research in Microbial Sciences 2024Raw milk from native small ruminant breeds in Epirus, Greece, is a valuable natural source of autochthonous lactic acid bacteria (LAB) strains with superior...
Antilisterial activity of raw sheep milk from two native Epirus breeds: Culture-dependent identification, bacteriocin gene detection and primary safety evaluation of the antagonistic LAB biota.
Raw milk from native small ruminant breeds in Epirus, Greece, is a valuable natural source of autochthonous lactic acid bacteria (LAB) strains with superior biotechnological properties. In this study, two bulk milks (RM1, RM2) from two local sheep yards, intended for traditional Kefalotyri cheese production, were preselected for bacteriocin-like antilisterial activity by in vitro tests. Their antagonistic LAB biota was quantified followed by polyphasic (16S rRNA gene sequencing; IGS for ; a multiplex-PCR for ) identification of 42 LAB (RM1/18; RM2/24) isolates further evaluated for bacteriocin encoding genes and primary safety traits. Representative isolates of the numerically dominant mesophilic LAB were (10) in both RMs, (7) in RM2, and (1) in RM1; the subdominant thermophilic LAB isolates were (8), (6), (3), (1), (1), (2), (1) and (1). Based on their and profiles, six strains (8 isolates) were atypical lying between the subspecies and whereas two strains profiled with subsp. that is first-time reported in Greek dairy food. Two RM1 strain biotypes (3 isolates) showed strong, enterocin-mediated antilisterial activity due to possession. One from RM1 possessed and , while additional nine RM2 isolates of the group processed or singly. All showed direct (cell-associated) antilisterial activity only, as also both strains from RM2 did strongly. Desirably, no LAB isolate was β-hemolyrtic, or cytolysin-positive, or possessed for vancomycin resistance, or and virulence genes. However, all three from RM2 possessed and/or virulence genes. In conclusion, all strains, the two safe, enterocin A-B-P-producing strains, and the two antilisterial strains should be validated further as potential costarter or adjunct cultures in Kefalotyri cheese. The prevalence of α-hemolytic pyogenic streptococci in raw milk, mainly in RM2, requires consideration in respect to subclinical mastitis in sheep and the farm hygiene overall.
PubMed: 38116185
DOI: 10.1016/j.crmicr.2023.100209 -
Veterinary World Jul 2018A critical prerequisite for studying rumen microbial community by high throughput molecular biology methods is good quality community DNA. Current methods of extraction...
BACKGROUND AND AIM
A critical prerequisite for studying rumen microbial community by high throughput molecular biology methods is good quality community DNA. Current methods of extraction use kits designed for samples from the different origin for rumen. This puts stress on the development of a relevant manual method for DNA extraction. The objective of this study was to modify the existing methods of community DNA extraction and thereby systematic comparison of their efficiency based on DNA yield, purity, 16S rRNA gene sequencing, and identification to determine the optimal DNA extraction methods whose DNA products reflect targeted bacterial communities special to rumen.
MATERIALS AND METHODS
Enzymatic method, Chemical method, Enzymatic + Chemical method, and Enzymatic + Chemical + Physical method were modified toward evaluation of community DNA extraction from solid, squeezed, and liquid fractions of goat rumen digesta. Each method was assessed critically for nucleic acid yield and its quality. The methods resulting in high nucleic acid yield, optimal purity ratios with intact band on agarose gel electrophoresis were optimized further. Optimized methods were studied using standard polymerase chain reaction (PCR) with universal bacterial primers and 16S rRNA primers of targeted rumen bacteria. Methods denoting the presence of targeted rumen bacteria were assessed further with 16S rRNA gene sequencing and identification studies. It led toward methods efficacy estimation for molecular biology applications. Effect of rumen sample preservation on community DNA extraction was also studied. Their mean standard deviation values were calculated to understand sampling criticality.
RESULTS
Modified Chemical method (Cetrimonium bromide) and Enzymatic+Chemical+Physical (ECP) method (Lysozyme-Cetrimonium bromide-Sodium Dodecyl Sulfate-freeze-thaw) could extract 835 ng/µl and 161 ng/µl community DNA from 1.5 g solid and 2 ml squeezed rumen digesta with purity ratios of 1.8 (A/A) and 2.3 (A/A) respectively. Comparative analysis showed the better efficiency of ECP method and chemical method toward freshly squeezed rumen digesta and solid rumen digesta. However, sample preservation at -80°C for 1.5 months drastically affected the yield and purity ratios of community DNA. New protocol revealed targeted microbial community having Gram-positive as well as Gram-negative bacteria such as , , , and .
CONCLUSION
To date, this is the first report of modified methods wherein least chemicals and steps lead toward PCR and 16S rRNA gene sequencing quality community DNA extraction from goat rumen digesta. Detection of targeted rumen bacteria in solid and squeezed rumen digesta proves their strongest association with rumen fiber mat. It also marks the presence of distinct microbial communities in solid and squeezed rumen fractions that in turn differs the performance of each different method employed and yield of nucleic acid obtained. It also leaves a possibility of the presence of complex microbial consortia in squeezed rumen digesta whose DNA extraction methods need more attention. Finally, manual protocols of community DNA extraction may vary in different ruminant which suggests undertaking rigorous research in their establishment.
PubMed: 30147271
DOI: 10.14202/vetworld.2018.990-1000 -
The ISME Journal Nov 2008Alimentary carbohydrate overload is a significant cause of laminitis in horses and is correlated with drastic shifts in the composition of hindgut microbiota. Equine...
Alimentary carbohydrate overload is a significant cause of laminitis in horses and is correlated with drastic shifts in the composition of hindgut microbiota. Equine hindgut streptococcal species (EHSS), predominantly Streptococcus lutetiensis, have been shown to be the most common microorganisms culturable from the equine caecum prior to the onset of laminitis. However, the inherent biases of culture-based methods are estimated to preclude up to 70% of the normal caecal microbiota. The objective of this study was to evaluate bacterial population shifts occurring in the equine caecum throughout the course of oligofructose-induced laminitis using several culture-independent techniques and to correlate these with caecal lactate, volatile fatty acid and degrees of polymerization 3-7 fructo-oligosaccharide concentrations. Our data conclusively show that of the total microbiota present in the equine hindgut, the EHSS S. lutetiensis is the predominant microorganism that proliferates prior to the onset of laminitis, utilizing oligofructose to produce large quantities of lactate. Population shifts in lactobacilli and Escherichia coli subpopulations occur secondarily to the EHSS population shifts, thus confirming that lactobacilli and coliforms have no role in laminitis. A large, curved, Gram-negative rod previously observed during the early phases of laminitis induction was most closely related to the Anaerovibrio genus and most likely represents a new, yet to be cultured, genus and species. Correlation of fluorescence in situ hybridization and quantitative real-time PCR results provide evidence supporting the hypothesis that laminitis is associated with the death en masse and rapid cell lysis of EHSS. If EHSS are lysed, liberated cellular components may initiate laminitis.
Topics: Animals; Bacteria; Biodiversity; Cecum; Escherichia coli; Fatty Acids, Volatile; Gastrointestinal Tract; Horse Diseases; Horses; Lactic Acid; Oligosaccharides; Streptococcus; Veillonellaceae
PubMed: 18580970
DOI: 10.1038/ismej.2008.67