Tesi sul tema "Dairy microbiology"

Giardia and Cryptosporidium are intestinal protozoan parasites that infect a wide range of host species, including humans. DNA sequencing of Giardia and Cryptosporidium isolates from human and animal sources has identified numerous species and genotypes, and has demonstrated that a number of Giardia and Cryptosporidium genotypes are shared between animals and humans. Therefore, livestock may act as a source of contamination of the food and water supply. The goal of this study was to optimize methods for the detection and molecular characterization of Giardia and Cryptosporidium. Achieving this objective involved the incorporation of immunomagnetic separation, as well as the evaluation of different methods, including microscopy, flow cytometry and polymerase chain reaction. A number of faecal samples from adult cattle and calves were collected from farms in Ontario and Prince Edward Island (PEI). Following DNA extractions from stool samples, a nested-PCR was used for Giardia to amplify a fragment of the 18S rRNA gene generating a 292-bp product. Nested-PCR protocol was also used for Cryptosporidium to amplify fragments of the heat-shock protein 70 (HSP-70) gene (ca. 325 bp). For Giardia, of 143 cattle samples analyzed by PCR, 32 (22.4 %) were positive. When IMS was incorporated into the methodology, 64 out of 143 (44.8 %) samples analyzed were positive for Giardia. For Cryptosporidum, out of 143 cattle faecal samples analyzed by the PCR method using the HSP-70 gene, 58 were positive (40.6 %), while using IMS, plus PCR, 60 samples were positive (42 %). Results from this study indicated that incorporation of IMS significantly improve the sensitivity of PCR for the detection of both Giardia (p<0.01) and Cryptosporidium (p=0.02). Among the other genes that were targeted, including the beta-giardin gene and glutamate dehydrogenase (GDH) for Giardia, and the Cryptosporidium oocyst wall protein (COWP) and 18S rRNA for Cryptosporidium, the 18S rRNA for Giardia , and HSP-70 for Cryptosporidium were found to be the "best genes". When different methods, including microscopy, flow cytometry and PCR-IMS were compared, the PCR method showed the highest sensitivity in detecting both parasites. Genotyping done by DNA sequencing showed that there was a high prevalence of zoonotic genotypes (Assemblage A for Giardia , and C. parvum bovine genotype for Cryptosporidium ) among the samples from both PEI and Ontario. In addition, a temporal study was done on calf samples from Ontario and showed that over time there was a decrease in Cryptosporidium infections, concomitant with an increase in Giardia infections.

Thesis (MSc)--University of Stellenbosch, 2003.
ENGLISH ABSTRACT: The effect of the bacteriocin-like peptide AS-48, produced by Enterococcus faecalis FAIRE 92, was tested against a mastitis isolate of Staphylococcus aureus in an in vivo and in vitro study. During initial tests peptide AS-48 showed no significant activity towards S. aureus, even with a ten-fold concentrated cell-free supernatant. Activity was obtained only after purification with Triton X-114 phase partitioning, followed by cation exchange chromatography. Titers for the purified peptide varied between 3200 and 12800 AU/ml. The purified peptide also exhibited activity towards Streptococcus agalactiae and Streptococcus dysgalactiae, but not against Escherichia coli. The size of peptide AS-48 was determined at 7150 Da, based on electronspray mass spectrometry and SDS-PAGE. Complete inhibition of cell growth was obtained by adding 1ml of the purified peptide (3200 AU/ml) to 100 ml of cells of S. aureus in the lag growth phase. When the same concentration of peptide AS-48 was added to a culture of S. aureus in mid-exponential growth, a slight decrease in viable cell numbers was recorded, which lasted for only 30 min. Cell growth commenced thereafter. In situ experiments in cows were done with purified peptide AS-48, encapsulated in liposomes. These in vivo studies were conducted by administering peptide AS-48 (6400 AU/ml) to different udder quarters. In a prevention trial, i.e. where quarters were pretreated with peptide AS-48, a reduction close to 90% in the viable cell numbers of S. aureus was recorded relative to the control quarters, which were not treated with the peptide. A 50% reduction in somatic cell count (SCC) was recorded. In the treatment trial, i.e. infected quarters treated with peptide AS-48, a reduction of up to 94% in viable cell numbers of S. aureus was recorded. In the same quarters, a reduction in SCC amounted to almost 80%. A recombinant strain was constructed by conjugating plasmid 92 (p92), encoding peptide AS-48, from Enterococcus faecalis FAIRE 92 to E. faecalis FA2/Ent, which produces enterocins 1071A and 1071B. Southern blot hybridization experiments revealed thepresence of plasmid p92 in the recipient strain without the loss of plasmid pEF1071, which encodes enterocins 1071A and 1071B. All three antimicrobial peptides, i.e. enterocin 1071A, enterocin 1071B and peptide AS-48, were produced in transconjugant FA2/Ent/AS-48. The spectrum of antimicrobial activity of the transconjugant was greater than that recorded for strains FA2/Ent and FAIRE 92, respectively and included E. faecalis, Bacillus cereus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus sakei, Leuconostoc cremoris, Leuconostoc pentosaceus, Staphylococcus carnosus and S. aureus. These organisms are not inhibited by strain FA2/Ent. However, low levels of peptide AS-48 was produced by strain FA2/Ent/AS-48. Further research in fermentation and gene expression will be needed before the transconjugant E. faecalis FA2/Ent/AS-48 may be used in the treatment of mastitis.
AFRIKAANSE OPSOMMING: Die effek van die bakteriosien-agtige, peptied AS-48, geproduseer deur Enterococcus faecalis FAIRE 92, is gedurende ‘n in vivo en in vitro studie teen ‘n mastitiese Staphylococcus aureus-isolaat getoets. Aanvanklike toetse met peptied AS-48, selfs tienvoudig gekonsentreerde selvrye supernatant, het geen beduidende aktiwiteit teen S. aureus getoon nie. Aktiwiteit is eers verkry na suiwering met Triton X-114 fase-skeiding gevolg deur katioon uitruilingschromatografie. Titers vir die gesuiwerde peptied het tussen 3200 en 12800 AE/ml gewissel. Die gesuiwerde peptied het ook aktiwiteit teen Streptococcus agalactiae en Streptococcus dysgalctiae getoon, maar nie teen Escherichia coli nie. Peptied AS-48 het ‘n molekulêre massa van 7150 Da, soos bepaal met elektronsproeimassa spektrometrie en SDS-PAGE. Totale inhibisie van selgroei is verkry deur 1 ml gesuiwerde peptied AS-48 (3200 AE/ml) by ‘n 100 ml kultuur van S. aureus in die sloerfase te voeg. Dieselfe konsentrasie peptied AS-48, toegevoeg tydens die mideksponensiële groeifase, het egter slegs ‘n klein vermindering in die aantal lewende selle teweeg gebring en het ook vir slegs ‘n 30 min geduur. Selgroei het hierna weer normaal voort gegaan. In situ eksperimente op koeie is uitgevoer met gesuiwerde peptied AS-48, geenkapsuleerd in liposome. Hierdie In vivo studies is onderneem deur peptied AS-48 (6400 AE/ml) in verskillende kwarte van die uier, kunsmatig of reeds geïnfekteerd met S. aureus, toe te dien. In ‘n voorkomings-eksperiment waar kwarte vooraf met peptied AS- 48 behandel is, is ‘n verlaging van byna 90% in die lewende seltelling van S. aureus relatief tot die kontrole kwarte, sonder behandeling met peptied AS-48, verkry. ‘n 50% verlaging in die somatiese seltelling (SST) is verkry. In die behandelings-eksperiment, waar geïnfekteerde kwarte met peptied AS-48 behandel is, is ‘n verlaging van byna 90% in lewende S. aureus selle gevind. In dieselfde kwarte is ‘n verlaging van byna 80% in die SST genoteer.‘n Rekombinante ras is gekonstrueer deur plasmied 92 (p92), wat kodeer vir peptied AS- 48, vanaf Enterococcus faecalis FAIRE 92 na E. faecalis FA2/Ent, wat enterosien 1071A en 1071B produseer, te konjugeer. Southern-klad hibridisasie het die teenwoordigheid van plasmied p92 in die ontvanger ras, sonder die verlies van plasmied pEF1071 wat enterosien 1071A en 1071B kodeer, getoon. Al drie antimikrobiese peptiede, nl. enterosien 1071A, enterosien 1071B en peptied AS-48, is deur die transkonjugant FA2/Ent/AS-48 geproduseer. Die spektum van antimikrobiese aktiwiteit van die transkonjugant vand die transkonjugant is breër as dié van rasse FA2/Ent en FAIRE 92, onderskeidelik en het ook E. faecalis, Bacillus cereus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus curvatus, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus sakei, Leuconostoc cremoris, Leuconostoc pentosaceus, Staphylococcus carnosus en S. aureus ingesluit. Hierdie organismes word nie deur ras FA2/Ent geïnhibeer nie. Lae vlakke van peptied AS-48 is egter deur ras FA2/Ent/AS-48 geproduseer. Verdere navorsing in fermentasie en geenuitdrukking is nodig voordat E. faecalis FA2/Ent/AS-48 in die behandeling van mastitis gebruik kan word.

An integrated wastewater treatment facility at a dairy in Glendale, Arizona, consisting of an upper subsystem (solids separators, anaerobic lagoons, and aerobic ponds) and lower subsystem (wetland subsystems) has been proven to be successful in reducing indicator organisms and potential pathogens (bacteria, enteric viruses, and parasites). The collection sump of the new integrated system collects all dairy wastewater and pumps it to solid separators, which then flows by gravity to anaerobic lagoons and aerobic ponds. The upper subsystem achieved significant microbial reductions of >98 percent for total coliform, >91 percent for coliphage, >95 percent for enterococci, >91 percent for Listeria monocytogenes, and >99.9 percent for Cryptosporidium . Additional reductions although limited were observed in the outflow from the wetland cells.

This project was designed to assess the potential for contamination of produce during irrigation with wastewater from animal operations. Dairy wastewater from the University of Arizona Campus Dairy Research Center was used to irrigate three different types of vegetable crops: lettuce, carrot, and bell pepper. This study was conducted over two consecutive years. The crops were planted in February and vegetables were harvested from May through July. Irrigation water and vegetable samples were examined for Escherichia coli, Clostridium perfringens, Listeria monocytogenes, and coliphage. In the dairy wastewater, E. coli concentrations averaged 5.7 x 10⁵ MPN/100 mL in the first year (2000), and 9.9 x 10⁷ MPN/100 mL in the second year (2001). C. perfringens concentrations were nearly the same in both years (1.7 x 10⁴ and 3.4 x 10⁴ CFU per 100 mL). Coliphage averaged 2.0 PFU/mL in 2000 and 1.3 x 10⁴ PFU/mL in 2001 in wastewater. E. coli was detected with greater frequency on carrots (100 and 96%) succeeded by lettuce (67 and 96%) and bell peppers (63 and 58%). The same was true for C. perfringens : carrots (100%), lettuce (86 and 88%), and bell peppers (100 and 50%). Coliphages were not detected on any of the vegetable crops except for average concentrations of 2 PFU/g on lettuce in the first year. L. monocytogenes was not detected on any of the vegetable samples. ANOVA test results indicates that E. coli and C. perfringens concentrations on three crops were statistically different (p < 0.0001) which suggest that the degree of contamination on the surface of the vegetables depends on where the edible portion of the crop is situated (above the soil or under the soil). The greatest contamination occurred on the carrots followed by lettuce and bell peppers. E. coli and C. perfringens were recovered from the carrots, bell peppers, and soil 55 days after wastewater irrigation of the plots had ceased. Positive correlations (p < 0.05) were found between E. coli and C. perfringens density and soil moisture content. The greatest risk of infection from pathogenic E. coli (O157:H7) occurs from consumption of lettuce and carrots. The annual risk of infection from consumption of all three vegetables was above the acceptable risk of 1:10,000 per year. The results of this study suggest that a more strict irrigation water quality standard for root and leafy vegetables might be appropriate to prevent the risk of infection in exposed population.

The genus Staphylococcus contains at least 47 species and 23 subspecies. Bacteria in this genus are ubiquitous; many are commensals on human and animal skin and can be opportunistic pathogens. In dairy cattle, staphylococci are the leading cause of intramammary infections (IMI) and mastitis. Mastitis is the inflammation of the mammary gland, and is one of the leading infectious diseases causing production losses in the dairy industry. Based on the ability to clot blood plasma in vitro, members of the genus can be divided into two groups: coagulase positive staphylococci (CPS) and coagulase negative staphylococci (CNS). In the dairy industry, Staphylococcus aureus is the most common CPS causing mastitis and is considered a major mastitis pathogen compared to the CNS, which as a group have been described as minor mastitis pathogens. The CNS species are increasingly recognized as an important cause of bovine mastitis, although the relative role of some species is still uncertain. Our understanding of the local and global epidemiology of CNS mastitis is improving with application of more accurate DNA sequence-based species identification methods and techniques to discriminate between strains within species. These factors have led to a shift in perspective, with the CNS being recognized as a heterogeneous group where some species are more important than others in bovine mastitis. The major goals of this thesis were to describe Staphylococcus mastitis epidemiology, and to identify phenotypes that may contribute to persistence in various niches on selected dairy farms in Vermont. We conducted 2 field studies on 2 groups of farms in Vermont. In the first study, we collected S. aureus isolates from bulk tank milk of 44 certified organic dairy farms. In the second field study, we completed quarter milk, cow skin, and environmental sampling of 5 herds that make farmstead cheeses. In both studies, we used non-selective and selective agar medium to isolate staphylococci from the farm sources. From these studies, we collected 1,853 Staphylococcus spp. isolates. We used PCR-amplicon sequence-based species identification to describe Staphylococcus species diversity on these selected Vermont dairy farms. S. aureus isolates were strain-typed using an established Multilocus Sequence Typing (MLST) scheme. A novel MLST scheme was developed to investigate the molecular epidemiology of S. chromogenes, one of the leading CNS species causing bovine mastitis in this and other studies. We also evaluated antibiotic resistance and biofilm formation phenotypes and genotypes of staphylococci to test the hypothesis that these phenotypes may be associated with strain types. In the study of organic dairy farms, 20 S. aureus strain types (STs) were identified, including ten novel STs. The majority of STs belonged to lineages or clonal complexes (CCs) previously identified as cattle adapted (e.g. CC97 and CC151). Associations between ST and carriage of beta-lactam resistance and biofilm forming capacity were identified among the S. aureus isolates from these farms. In the 5-herd study, a total of 27 different staphylococci species were identified from various niches including humans, but only five species; S. chromogenes, S. aureus, S. haemolyticus, S. simulans, and S. xylosus were commonly identified to cause IMI. S. aureus and S. chromogenes strain types were niche specific.

In this study, strains of probiotic bacteria have been selected for tolerance to low pH, bile, sucrose, oxygen in media and low storage temperatures. Lactobacillus acidophilus 2401 and Bifidobacterium infantis 1912 were selected as strains able to survive in these conditions. These two strains were then offered further protection from the adverse conditions of food processing and storage by microencapsulation in a calcium alginate and starch gel matrix. Encapsulation in calcium alginate increases survival in yoghurt. In cheddar cheese the free L. acidophilus 2401 and B. infantis 1912 cells survived better than the encapsulated cells, probably due to the dense nature of the cheddar cheese matrix combined with the encapsulation restricting the flow of the nutrients and metabolites between the outside environment and the cells. In ice cream survival was high, probably due to the high fat and solids nature of the ice cream combined with the low storage temperature. The trial results of the laboratory scale production was consistent with the survival results for yoghurt and cheddar cheese. Incorporation of encapsulated probiotic bacteria into ice cream and cheddar cheese was acceptable by sensory standards and largely unnoticeable in comparison with the same foods without capsules. The capsules were visible and able to be felt on the tongue when eaten in yoghurt causing the product to be disliked by the panellists.

In this study, strains of probiotic bacteria have been selected for tolerance to low pH, bile, sucrose, oxygen in media and low storage temperatures. Lactobacillus acidophilus 2401 and Bifidobacterium infantis 1912 were selected as strains able to survive in these conditions. These two strains were then offered further protection from the adverse conditions of food processing and storage by microencapsulation in a calcium alginate and starch gel matrix. Encapsulation in calcium alginate increases survival in yoghurt. In cheddar cheese the free L. acidophilus 2401 and B. infantis 1912 cells survived better than the encapsulated cells, probably due to the dense nature of the cheddar cheese matrix combined with the encapsulation restricting the flow of the nutrients and metabolites between the outside environment and the cells. In ice cream survival was high, probably due to the high fat and solids nature of the ice cream combined with the low storage temperature. The trial results of the laboratory scale production was consistent with the survival results for yoghurt and cheddar cheese. Incorporation of encapsulated probiotic bacteria into ice cream and cheddar cheese was acceptable by sensory standards and largely unnoticeable in comparison with the same foods without capsules. The capsules were visible and able to be felt on the tongue when eaten in yoghurt causing the product to be disliked by the panellists.
Master of Science (Hons)

Thesis (M.Sc.) (Hons.) -- University of Western Sydney, Hawkesbury, 2000.
A thesis presented for the fulfilment of Master of Science (Honours), Centre for Advanced Food Research, School of Science, Food and Horticulture, University of Western Sydney, Hawkesbury, December 2000. Spine title : Survival of probiotic bacteria in dairy foods. Bibliography : leaves 228-244.

Thesis (MSc (Microbiology))--Stellenbosch University, 2008.
Mastitis is considered to be the most costly disease affecting the dairy industry. Management strategies involve the extensive use of antibiotics to treat and prevent this disease. Prophylactic dosages of antibiotics used in mastitis control programmes could select for strains with resistance to antibiotics. In addition, a strong drive towards reducing antibiotic residues in animal food products has lead to research in finding alternative antimicrobial agents. Streptococcus macedonicus ST91KM, isolated from bulgarian goat yoghurt, produces the bacteriocin macedocin ST91KM with a narrow spectrum of activity against Grampositive bacteria. These include mastitis pathogens Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus and Staphylococcus epidermidis as well as Lactobacillus sakei and Micrococcus varians. Macedocin ST91KM is, according to tricine-SDS PAGE, between 2.0 and 2.5 kDa in size. The activity of macedocin ST91KM remained unchanged after 2 h of incubation at pH 2.0 to 10.0 and 100 min at 100 °C. The peptide was inactivated after 20 min at 121 °C and when treated with pronase, pepsin and trypsin. Treatment with α-amylase had no effect on activity, suggesting that the mode of action does not depend on glycosylation. Precipitation with 60 % saturated ammonium sulphate, followed by Sep-Pak C18 separation recovered 43 % of macedocin ST91KM. Amplification of the genome of strain ST91KM with primers designed from the sequence of the macedocin prescursor gene (mcdA) produced two fragments (approximately 375 and 220 bp) instead of one fragment of 150 bp recorded for macedocin produced by S. macedonicus ACA-DC 198. Strain ACA-DC 198 was not available. However, the DNA fragment amplified from strain LMG 18488 (ACA-DC 206), genetically closely related to strain ACADC 198, revealed 99 % homology to the mcdA of S. macedonicus ACA-DC 198 (accession number DQ835394). Macedocin ST91KM may thus be a related bacteriocin described for S. macedonicus. The peptide adsorbed equally well (66 %) to L. sakei LMG13558 and insensitive cells, e.g. Enterococcus faecalis BFE 1071 and FAIR E92, and Streptococcus caprinus ATCC 700066. Optimal adsorption of macedocin ST91KM was recorded at 37 °C and 45 °C and at pH of 8 - 10. Addition of solvents decreased adsorption by 50%, suggesting that the receptors to which the bacteriocin binds have lipid moieties. The addition of MgCl2, KI and Na2CO3 completely prevented adsorption of macedocin ST91KM to the target cells, possibly due to competitive ion adsorption on the bacterial cell surface. The peptide has a bacteriocidal mode of action, resulting in lysis and the release of DNA and β-galactosidase. Atomic force microscopy of sensitive cells treated with macedocin ST91KM have shown deformation of the cell structure and developing of irregular surface areas. Antimicrobial susceptibility patterns were evaluated against eighteen mastitis pathogens. All isolates tested were resistant to methicillin and oxacillin, but had minimum inhibitory concentrations (MICs) falling in the intermediate and susceptible range against erythromycin. S. agalactiae and S. epidermidis had the highest sensitivity to macedocin ST91KM. A teat seal preparation containing macedocin ST91KM effectively released bacteriocin inhibiting the growth of the bacterial pathogen. Macedocin ST91KM could form the basis for an alternative dry cow therapy to prevent mastitis infections in dairy cows, as it is effective against pathogens that display resistance to conventional antibiotic therapy.

Thesis (MSc)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: This study describes: the evaluation of the current, and potential assay methods for the quantification of cholesterol, cholesteryl esters and free fatty acids in milk and the application thereof ; an account of the difficulties associated with the usage of FoodPro® Cleanline, an enzyme preparation used as processing aid, during ultra-high temperature processing of milk ; the development of activity assays which can be used for the kinetic characterization of glycerophospholipid cholesterol acyltransferase, the active enzyme in FoodPro® Cleanline ; the development of an accurate and facile activity assay, and the validation thereof, which can be used for the validation of enzyme activity prior to dosage of milk with FoodPro® Cleanline.
AFRIKAANSE OPSOMMING: Hierdie studie beskryf: die evaluering van die huidige, en potensiële, metodes vir die kwantifisering van cholesterol, cholesteriel esters en vryvetsure in melk, sowel as die toepassing van hieridie metodes ; 'n verduideliking van die moeilikhede wat ondervind word gedurende die gebruik van FoodPro® Cleanline, 'n ensiempreparaat vir gebruik as 'n verwerkingshulpmiddel, tydens ultrahoë-temperatuurprosessering van melk ; die ontwikkeling van aktiwiteitsbepalings metodes vir gebruik in kinetiese karakterisering van gliserofosfolipied cholesterol asieltransferase, die aktiewe ensiem in FoodPro® Cleanline ; die ontwikkeling van 'n akkurate, eenvoudige aktiwiteitsbepalings metode, en bevestiging van hierdie metode, wat gebruik kan word vir kwalitieitskontrole alvorens die dosering van melk met FoodPro® Cleanline.

Listeria monocytogenes is a foodbome pathogen that can cause severe illness and even death. It is found in dairy and meat products. The focus is on rapid detection since conventional methods are time consuming (4-5 days). Pre-enrichment steps, as part of those methods, are time consuming. Our objective was to develop a detection system without a pre-enrichment step, giving a final result within 2 to 4 h. In the concept of "the need for speed," a detection system with an antibody-based capture technique, followed by polymerase chain reaction (PCR), was developed. Glass beads coated with a Listeria polyclonal antibody were added to the food sample. After a static incubation/capturing step, beads-cell complexes were separated from the food, and boiled to lyse the cells and release the DNA. In a final PCR/electrophoresis step the DNA samples were analyzed. The use of a flow-based capturing system (ImmunoFlow) was also investigated. Using a bead-antibody complex in this ImmunoFlow setup has several advantages, including the possibility of concentrating the microorganisms out of large food samples (with flow through setup), the exclusion of a pre-enrichment step, and the potential for automation. Besides buffer solution (Tris), different kinds of milk, e.g., pasteurized, Ultra High Temperature (UHT), and raw milk, were also investigated. The detection limit in buffer solution was 1 x 106 CFU/ml no matter if the ImmunoFlow system or the static incubation was used. For the different pasteurized milk samples, the detection limit varied between 1 x 107 and 1 x 108 cells/ml in the static procedure. For UHT and raw milk, however, capturing of Listeria monocytogenes cells was not possible in the static or the ImmunoFlow setup. In conclusion, we developed a rapid and specific detection system for Listeria monocytogenes at high concentration in pasteurized milk using a static capturing procedure. The total test time for this detection system is less than 4 h, which is much faster than the present detection systems (which are using an enrichment step prior to testing). Implementing a real-time PCR system after capture would further reduce this detection time.

Enterococcus species are integral members of the gastrointestinal microfloral of humans, animals, birds, as well as insects. Their presence in water and food has been greatly associated with faecal contamination. This study was aimed at evaluating the incidence of Enterococcus species in cow dung and environmental water sources in three commercial dairy farms. In addition, their antibiotic profiles were determined as well as resistance and virulence genes. Furthermore, the genetic relatedness of the isolates was determined by molecular typing method (RAPD PCR). Three hundred and thirty four water and faecal samples consisting of 117, 116 and 101 were collected from Seven Star Middle Drift and Fort Hare Dairy trusts respectively. Of the 334 samples collected, 289 were of faecal origin and 45 from water sources within the farms. All samples were screened for enterococci using culture base growth media and molecular methods targeting the tuf gene. Speciation was done using species-specific primers and the incidences of various species within the farms determined. Furthermore resistance to antibiotics and multidrug-resistant phenotypes were established using the disk diffusion method. Genes coding for virulence and resistance were also determined. From the samples collected, 313 (289 faecal and 24 water) presumptive enteroccocci were isolated, 305 of 313 (97.45 percent) were confirmed as Enterococcus of which 239 of 305 (78.38 percent) were identified as E. hirae, 15 of 305 (4.92 percent) as E. faecium, 12/305 (3.93 percent) as E. durans, 6 of 305 (1.97 percent) as E. faecalis and 33 of 305 (10.82 percent) were unidentified. Out of the five virulence genes that were targeted in the study only gelE (71.80 percent of 219/305) and ace (27.2 percent 83/305) were present in the isolates. Phenotypic resistance to antibiotics was observed is in all twelve antibiotics tested with multidrug resistance phenotypes detected in some enterococcal isolates most predominant in Seven Star and Middledrift dairy trust. Finally RAPD profiles of the isolates showed high relatedness between the strains from water and cow dung sources in all three commercial dairy farms suggesting possible contamination from cow dung to the water sources or vice versa.

Mastitis and milk quality affect every dairy farmer across the globe. Sand bedded freestalls are the industry standard for cow comfort, welfare, and the control of environmental mastitis. Compost bedded packs may be a viable alternative to the sand bedded freestall. Compost bedded packs are maintained at a consistent level of moisture, nutrients, and aeration to favor compost microorganisms. Greater bacteria counts in bedding have traditionally been associated with increased mastitis rates and mastitis pathogens can be found in the pack and on the teats of cattle housed in even well managed compost bedded pack barns. In spite of this, herd SCC often remains low in well managed herds. The relationship between stress and comfort in the housing environment was a primary focus of this research. Cows housed in environments with low stress and high comfort may be better able to defend themselves against pathogens. Establishing changes in immune function in response to housing environment would improve milk quality by contributing to the knowledge of how mastitis-causing pathogens are contracted. An additional goal of this research was to determine the effect of compost bedded pack barns on thermoduric bacteria populations. Due to the increased temperatures associated with composting, thermoduric bacteria capable of surviving pasteurization are of potential concern in compost bedded packs. This research will investigate potential differences in thermoduric bacteria counts between compost bedded packs and sand bedded freestalls.

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1990.
Vita. Abstract. No film copy made for this title. Includes bibliographical references (leaves 149-165). Also available via the Internet.

High pressure (HP) kinetics of the microbial destruction and changes in the physicochemical characteristics of milk and pork were studied. Raw milk samples containing indigenous microflora of approximately 106 CFU/mL were heat sealed in dual peel sterilization pouches and subjected to HP treatment from 150--400 MPa with holding times ranging 5--120 min. The kinetic parameters (rate constant, k and decimal reduction time, D) for the microorganisms, alkaline phosphatase, color and viscosity were evaluated, based on first order kinetics and the pressure dependence of kinetic parameters was evaluated using pressure destruction time (PDT) and Arrhenius models. Kinetic data was well described by the first order model (R 2 > 0.90).
The application of pressure pulse was explored for pressure destruction of microorganisms as well as changes in physical-chemical characteristics of pork chops. Pork chops (2 days post-rigor) were subjected to HP treatment from 200--350 MPa for 0--120 min. Results showed that pressure changes of pork variables followed a dual effect consisting of an instantaneous pressure kill (IPK) with the application of pressure pulse (no holding) and a subsequent first order rate of destruction during the pressure hold time. The IPK values were pressure dependent and increased with pressure level. Parameters k and D indicated a higher rate of pressure destruction of microorganisms compared to quality attributes.
Kinetics of pressure destruction of Listeria monocytogenes Scott A were studied in relation to those of indigenous microorganism of milk and pork. The IPK was more pronounced with L. monocytogenes than with indigenous microflora. However, the kinetic parameters (k and D values) indicated a larger pressure resistance for L. monoctyogenes. HP processes were developed based on the standard plate count (SPC) kinetic data for indigenous microflora of milk as well as L. monocytogenes in milk and pork. The results showed that SPC kinetics permitted good estimation of microbial destruction in low pressure-lethality processes of milk and pork but its application at higher pressure-lethality levels were inaccurate. On the other hand, processes established based an destruction of L. monocytogenes were more predictable. Pressure pulse application to microbial lethality was also well predicted.
The shelf-life of milk and pork increased with the level of applied pressure lethality, but Q10 values suggested that low storage temperature was nevertheless required to control microbial growth and maintain quality. Storage of HP treated park offered some improvement in the texture but resulted in large color changes and drip losses. L. monocytogenes were not detected in any of the stored milk samples HP treated to achieve a lethality ≥10D.

Rumen microbiota enable dairy cattle to breakdown fiber into useable energy for milk production. Rumen bacteria, protozoa, and fungi ferment feedstuff into volatile fatty acids (VFA), the main energy source, while methanogens utilize fermentation by-products to produce methane. Milk fat contains several bioactive rumen-derived fatty acids (FA), including odd-chain FA (OCFA) and branched-chain FA (BCFA), important for maintenance of human health. The overarching dissertation goal was to determine which factors affect rumen methanogen and protozoal community structures and their metabolism products, while defining relationships between rumen microbiota and animal performance. Results presented contribute to the goals of providing new knowledge to dairy farmers, maintaining ruminant health, and enhancing bioactive FA in milk. The first objective was to use next-generation sequencing techniques to determine if lactation stage and dairy breed affect rumen methanogen and protozoal community structures and protozoa cell FA compositions in Jersey, Holstein, and Holstein-Jersey crossbred cows at 3, 93, 183, and 273 days in milk (DIM). A core methanogen community persisted by lactation stage and breed. At 3 DIM, methanogen 16S rRNA gene sequences formed distinct clusters apart from 93, 183, and 273 DIM, reflective of the dietary transition period post-partum. The starch-utilizing protozoal genus Entodinium, was more abundant in Holsteins than in Jerseys and Holstein-Jersey crossbred cows and positively correlated with milk yield. Jerseys had greater iso-BCFA contents in protozoa and milk and protozoa of the genus Metadinium. The second objective was to determine if supplementation of mixed cool-season grasses with annual forages (AF) alters the forage, microbial, and milk FA contents during typical periods of decreased pasture growth in Northeastern US. In short-term grazing (21d) of AF, ruminal VFA and major rumen-derived FA were not altered in bacterial and protozoal cells, suggesting little alteration of biohydrogenation and maintenance of ruminant health. In spring, milk contents of iso-15:0 and 17:0 per serving of whole milk were greater in control (CON)-fed cows, while contents of 12:0 and 14:0 per serving were greater in AF-fed cows. Contents of de novo FA and OCFA per serving of whole milk were greater in summer AF-fed cows than CON-fed cows, while total contents and BCFA did not differ, suggesting post-ruminal FA modifications in adipose tissue and the mammary gland. The third objective was to characterize and relate the rumen microbiota from CON- and AF-fed cows to animal performance. Rumen protozoal taxa were not altered, while less abundant bacterial taxa (< 5%) were different in both periods. The protozoal genus Diplodinium was positively correlated with feed efficiency and milk fat yield. In spring, AF-fed cows had greater abundances of the methanogen species Methanobrevibacter millerae, whereas CON-fed cows had greater abundances of the methanogen species Methanobrevibacter ruminantium, potentially as a result of differences in substrate availability. In conclusion, the work presented identifies several factors that influence rumen microbiota, rumen microbial FA, and milk FA, while providing new information to dairy farmers, researchers, and consumers.

There is an increasing consumer demand for dairy products which are safe and free from additives. Microbial starter strains, in combination with other factors, were studied for their contribution to the control of unwanted microbes, and maintaining the quality of soft cheese. The technological and functional characteristics of the starter culture strains Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris, and probiotic bacterial strains Bifidobacterium animalis subsp. lactis BB12, Lactobacillus acidophilus LA-5 and Lactobacillus casei Shirota were investigated. The tests included the milk fermentation, resistance to salt and heat, bile and acid resistance, and growth at a range of temperatures. The probiotic strains differed in their resistance to salt, bile salts and acid. Inhibitory interactions between probiotic bacterial strains with each other and with starter culture strains were not detected. The probiotic bacteria and starter culture strains used have an ability to grow together on homofermentative and heterofermentative differential agar and fermentation of fructose in different levels. Non-starter cheese (NSC), cheese with starter strains (SCS), and cheese with starter and probiotic strains (PSC) were manufactured. The levels of mesophilic aerobic and lactic acid bacteria, moulds and yeasts, and Enterobacteriaceae were evaluated in all cheeses. Their contents of fat, total solids, salt and pH value were tested during 21 days of storage at 2-5°C. Starter culture strains contributed to maintaining the quality of all cheeses, through decreasing the viable count of some undesirable microbes. Cheeses differed in the intensity of the crumbliness attribute, and in preference and intensity of colour attribute. The colour of starter soft cheese, which was tested using a colorimeter, was closer to the colour of probiotic soft cheese than those cheeses which were manufactured without starter culture. The microbial status, storage conditions, rancidity, and the sensory characteristics of unripened soft cheese, which was manufactured with starter culture strains only, were determined during the storage for 50 days at 2-5°C, as well as during their shelf life for the product. Modified Atmosphere Packaging (MAP) contributed to slowing the growth of unwanted microbes, and decreased the values of TBA, TVB-N and TMA in soft cheese. Consequently, delaying the undesirable changes and maintaining the quality of the product and extending its shelf life, when compared with vacuum, brine, and air packaging methods, under the same storage conditions. Potential effects of inulin on the cheese quality and sensory characteristics of probiotic soft cheese were investigated. The cheeses differed in their loads of lactic acid bacteria, in addition to the total solids and water activity. The levels of probiotic bacterial strains were higher in probiotic soft cheese that manufactured with inulin than in cheese without inulin, with a potential in the formation synbiotic between the probiotic strains LA-5 and BB12 and inulin. Both cheeses were recorded to have high acceptance in the cheese attributes, in terms of appearance, aroma, colour texture and the overall acceptance. The presence of inulin increased the hardness of cheese under vacuum packaging, after storage for 14 days at 2-5°C.

Thesis (M. Tech. (Environmental health: Food safety )) - Central university of Technology, Free State, 2013
Food processing plants and agricultural environments have a long-standing history of being known to provide a conducive environment for the prevalence and distribution of microorganisms which emanate as a consequence of activities undertaken in such premises. Microorganisms in the aforementioned environments may be found in the atmosphere (airborne), and/or on food contact surfaces. Airborne microorganisms from food handlers and in food products and raw materials (as part of bioaerosols) have in the past been implicated as having a potential to cause adverse health effects (especially in indoor environments) and therefore also to have economic implications. Recently their effect on food safety has received increased interest. The recent international interest in bioaerosols in the food industry has played a role in rapidly providing increased understanding of bioaerosols and their effects in different food processing environments. However, there is still a lack of research on the actual impact of bioaerosols over time in most of the food premises especially in Southern Africa and other developing countries. The overall purpose of this dissertation was to assess possible microbial contaminants and the role of selected environmental parameters on these microbes at a dairy farm plant in central South Africa. In relation to the purpose of the study, the objectives of this dissertation were to investigate and establish the food handler’s food safety knowledge, attitude, behaviour and practices. The sub-objective was to investigate the prevalence and distribution of microbial contaminants (both airborne and food contact surface populations), and concomitant environmental parameters. The microbe isolates from both investigations (i.e. air samples and food contact surfaces) were identified to strain level using matrix-assisted laser desorption ionization – time of flight mass spectrometry (MALDI-TOF MS). The findings of this study in relation to food handlers’ food safety knowledge, attitude, behaviour and practices indicated a dire need for training of employees as well as improved health and hygiene measures as emphasised by some of the identified strains. The environmental parameters (both indoor and outdoor) were similar, with no relationship established between airborne microbes’ prevalence and environmental parameters. The samples of the airborne microbial populations in both indoor and outdoor environments were similar. Airborne microbial counts at the dairy farm plant over the entire duration of the study ranged between 1.50 x 101cfu.m-3and 1.62 x 102cfu.m-3. Microbial counts on food contact surfaces ranged between 2.50 x 102 cfu.cm-2 and 1.10 x 105 cfu.cm-2 over the entire duration of the study. A wide variety of microorganisms (from air and food contact surfaces) such as the Gram-positive bacteria, Gram-negative bacteria, as well as fungi were present at the dairy farm plant. A number of the isolated genera have previously been associated with agricultural environments whilst others are associated with hospital environments. The positively identified strains were from genera such as Aeromonas, Arthrobacter, Candida, Pseudomonas, Pantoea, Citrobacter, Staphylococcus, Bacillus, Escherichia, Rhodococcus and Rhodotorula, amongst others. The isolation of microorganisms associated with food spoilage and foodborne disease outbreaks, which are known as indicator organisms such as Escherichia coli, Staphylococcus and Bacillus from both air and surface samples, signified possible faecal contamination and could be attributed to poor health and hygiene practices at the dairy farm plant. Despite the isolation of microorganisms associated with food spoilage and foodborne disease outbreaks, the isolation of microorganisms not usually associated with the food processing industry (usually associated with hospital environments) was an enormous and serious concern which suggested a need for further investigations at dairy farm plants as the implications of these pathogenic microorganisms in food is not known. The isolation of similar microorganisms from both the air samples and surface swabs suggests that airborne microbes have a potential of settling on food contact surfaces, therefore having a potential to contaminate dairy products which are known to be more prone to contamination and which, because of their nutritional status, serve as a good substrate for the growth of microorganisms.

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In dairy farming, one of the factors that raise the cost of production is feed, which can reach 70% of the total cost. With this, it is necessary to look for alternatives to reduce these costs without sacrificing productivity. An alternative is to enrich food resources of the animals, through the use of byproducts of industrial manioc starch. These alternative foods may constitute an interesting strategy, because in addition to being acquired by a low commercial value, may have high energy content and can also give an appropriate final destination byproduct of the cassava industry, contributing to the environment. The residue obtained during the processing of cassava has moisture content between 75% and 85%. The animal productivity can be affected negatively, depending on consumption and digestibility of nutrients. This study aimed to evaluate the chemical, microbiological, and detect the presence of mycotoxins in moist residue cassava starch (RMCS), and to evaluate the influence of five levels RMCS on intake and digestibility of nutrients, production and composition milk, and some blood parameters in dairy cows, Holstein, with average initial production of 30.65 kg milk / day. We observed a significant reduction in consumption (EE), and a linear effect for all nutrients studied, except for crude protein. Milk production was negatively affected, with a reduction to include RMCS. The chemical composition of RMCS did not change during storage, only significant variation in dry matter (DM). For populations of microorganisms, there was a significant increase only fungi and yeasts. Zearalenone levels were detected in 100% of the samples analyzed RUFM. The milk was reduced, but the efficiency of feed conversion was not affected, indicating that the residue can be used, however, it is necessary to adjust the levels of inclusion of the diet. The storage of RMCS proved effective under the conditions studied, since there were no changes in the nutritional value of the residue, but high counts of yeasts and fungi, and mycotoxins, may suggest that the material has a high risk of degradation
Na atividade leiteira, um dos fatores que elevam os custos da produção é a alimentação animal, que podem chegar a 70% do custo total. Com isso, é necessário buscar alternativas para redução desses custos, sem prejudicar a produtividade. Uma alternativa é o enriquecimento dos recursos alimentícios dos animais, através da utilização de subprodutos da indústria de fécula de mandioca. Esses alimentos alternativos podem constituir uma estratégia interessante, pois, além de serem adquiridos por um baixo valor comercial, podem apresentar alto teor energético, podem também, dar um adequado destino final ao subproduto da fecularia, contribuindo para preservar o meio ambiente. O resíduo obtido durante o processamento da mandioca apresenta teor de umidade entre 75% e 85%. A produtividade animal pode ser afetada de forma negativa, dependendo do consumo e da digestibilidade dos nutrientes da dieta. Esta pesquisa tem por objetivo avaliar a composição química, microbiológica, e também detectar presença de micotoxinas no resíduo úmido de fécula de mandioca (RUFM), além disso, avaliar a influência de cinco níveis de RUFM sobre o consumo e digestibilidade dos nutrientes, produção e composição do leite, e alguns parâmetros sanguíneos de vacas em lactação, da raça Holandesa, com produção média inicial de 30,65 kg de leite/dia. Nesta pesquisa observou-se a redução significativa no consumo de extrato etéreo (EE), e o efeito linear negativo para todos os nutrientes estudados, exceto para a proteína bruta. A produção de leite foi influenciada de forma negativa, ocorrendo redução com a inclusão de RUFM. A composição química do RUFM não sofreu alterações durante a estocagem, houve variação significativa apenas nos teores de matéria seca (MS). Para as populações de microrganismos, houve elevação significativa apenas de fungos e leveduras. Níveis de Zearalenona foram detectados em 100% das amostras de RUFM analisadas. A produção de leite sofreu redução, mas a eficiência na conversão alimentar não foi alterada, mostrando que o resíduo pode ser utilizado, no entanto, é necessário adequar os níveis de inclusão deste na dieta. A estocagem do RUFM mostrou ser eficiente nas condições estudadas, pois não houve alterações no valor nutricional do resíduo, porém, altas contagens de fungos e leveduras, e a presença de micotoxinas, podem sugerir que o material tenha alto risco de degradação

Packed-bed digesters are an alternative to covered lagoon digesters for methane production and anaerobic treatment of dilute wastewaters such as dairy barn flush water. The physical media of packed-beds retain biofilms, often allowing increased treatment rates. Previous studies have evaluated several types of media for digestion of dilute wastewaters, but cost and media fouling have setback commercial development. A major operational cost has been effluent recirculation pumping. In the present effort, a novel approach to anaerobic digestion of flush dairy water was developed at pilot-scale: broken walnut shells were used as a low-cost packed-bed medium and effluent recirculation was replaced by reciprocation mixing to decrease pumping costs and the risk of media clogging. Three packed-bed digesters containing walnut shells as media were constructed at the on-campus dairy and studied for about six months. Over that time, several organic loading rates (OLRs), measured as both chemical oxygen demand (COD) and volatile solids (VS) were applied to the new packed-bed digesters to allow modeling of methane production. The influence of temperature on methane production was also investigated. Additionally, the study measured solids accumulation in the walnut shell packed-bed as well as the effectiveness and durability of walnut shells as packing media. Finally, a simple economic analysis was developed from the methane model to predict the financial feasibility of packed-bed digesters at flush water dairies under similar OLR conditions. Three methane production models were developed from organic loading: saturation-type (following the form of the Monod equation), power and linear. The models were evaluated in terms of regression analysis and the linearity of experimental to predicted methane production. The best model was then chosen to develop the economic predictions. Economic predictions for packed-bed digesters were calculated as internal rate of return (IRR) using the methane models along with additional input variables. Comparisons of IRRs were made using electric retail rates of $0.10 to $0.20 per kilowatt-hour and capital cost subsidies from zero to 50%. Sludge accumulation in the packed-bed was measured via change in porosity, and walnut shell durability was measured as the change in mass of representative walnut shells over the course of the study. The linear-type model of methane production from volatile solids OLR best represented this data set. Digester temperature was not found to influence methane production in this study, likely due to the small daily average ambient temperature range experienced (14°C to 24°C) and the greater influence of organic loading. Porosity of the walnut shell packed-bed decreased from 0.70 at startup to 0.34±0.06 at the end of the six-month study, indicating considerable media fouling. Sludge accumulated in each digester from zero at startup to 281±46 liters at termination. Walnut shells in the packed-bed lost on average 31.4±6.3% mass during the study period which may be attributed to degradation of more readily bio-degradable cellulose and hemi-cellulose within the walnut shells. Given the predicted methane production and media life, at present, the economic outlook for packed-bed digesters at commercial dairies is quite dependent on utility electrical rates, available subsidies and future improvements to packed-bed digester technology. The predicted IRRs ranged from below 0% (at 0% capital subsidy and $0.10/kWh) up to 25% (at 50% capital subsidy and $0.20/kWh) at large dairies (3000 milking cows). Increases in organic loading were not shown to necessarily increase IRR, particularly at OLRs above 10 g/Lliquid-d (as COD or VS). Ultimately, to better assess the value of packed-bed digesters for flush dairies, additional study is needed on topics such as sludge accumulation prevention, long-term walnut shell degradation, dairy barn flush water mixing, and more detailed economic analysis.

Nesta pesquisa, estudaram-se as variações ocorridas na relação entre autólise de culturas lácticas e o desenvolvimento da proteólise de queijo Prato produzido em quatro regiões brasileiras: Santa Catarina (Queijo A), Goiás (Queijo B), São Paulo (Queijo C) e Minas Gerais (Queijo D). A análise quantitativa da população de bactérias lácticas durante a maturação mostrou perfis microbiológicos similares para todas as amostras de queijos examinadas. Após 5 dias de maturação, lactococos e estreptococos estavam presentes em números mais elevados do que lactobacilos mesofílicos e termofílicos, leuconostoc e fermentadores de lactato. Contudo, essas populações aumentaram consideravelmente no final do processo de maturação. Enterococos e fermentadores de citrato permaneceram em números relativamente reduzidos ao longo da maturação. A análise qualitativa mostrou a predominância de \"non starter lactic acid bactéria\" (NSLAB) nos queijos das quatro origens, principalmente de Lactobacillus sp. Outros gêneros foram identificados em menor proporção: Enterococcus sp., Pediococcus sp., Aerococcus sp., Tetragenococcus sp. e Streptococcus sp. O queijo C diferiu dos demais por não apresentar Pediococcus sp. e Streptococcus sp. As culturas lácticas adicionadas Lactococcus lacfis sp. e Leuconostoc sp. estavam presentes em populações menores. A autólise foi estudada pela determinação da atividade de aminopeptidases e detecção de autolisinas por zimogramas e de enzimas intracelulares por \"imunoblotting\". Uma maior liberação de aminopeptidases ocorreu no queijo D, seguido dos queijos C, B e A. Não foram detectadas bandas de atividade lítica nos zimogramas dos queijos A e B em todas as condições avaliadas. Nos zimogramas, detectou-se uma banda de 30 KDa nos queijos C e D a pH 7,4 e 44°C, e uma outra de 40 KDa, exclusiva no queijo D, ambas de fraca intensidade. A pH 6,8 e 42°C, detectou-se bandas de 40KDa de fraca intensidade no queijo C e forte no D, além de mais duas de fraca intensidade de 90KDa e 110KDa no queijo D. Na análise em \"imunnoblotting\" com o antisoro anti-Lc, foi observado apenas sinais fracos de reação positiva e em números inferiores àquelas reveladas com o citoplasma bruto de Lac. Lacfis subsp. lacfis (controle positivo), indicando que a autólise foi praticamente inexistente. Com o antisoro anti-D-LDH, também não se detectou sinais de reação positiva nos queijos A e B, enquanto nos queijos C e D, verificou-se sinais positivos de 37KDa, de forte intensidade e correspondentes à proteína D-LDH, indicando a lise de Lab. helveticus. A evolução da proteólise foi determinada quantitativamente durante a maturação e avaliada com base nos índices: NS-pH4,6/NT% e NNP/NT%, teor de tirosina, eletroforese (Uréia-PAGE) e quantificação de aminoácidos individuais livres. Não foram detectadas diferenças significativas entre os queijos A. B, C e D no início da maturação. Contudo, com a fragmentação das proteínas, ocorreu um aumento gradual desses índices, tendo-se observado valores mais elevados no queijo D, seguido dos queijos C, B e A. Os perfis eletroforéticos de proteínas foram similares para os queijos das quatro origens e mostraram claramente que o coagulante e a plasmina foram os responsáveis pela degradação inicial das caseínas. A taxa de degradação da αs1- e β-caseína apresentou a seguinte ordem: D > C ≥ B > A. O acúmulo de aminoácidos livres também foi mais rápido no queijo D, seguido dos queijos C, B e A. Portanto, a autólise de Lab. helveticus no queijo D acelerou a proteólise, diminuindo o período de maturação em 45% e não afetando negativamente o desenvolvimento de \"flavour\" e nem a textura. No final da maturação (45 dias), os compostos voláteis foram determinados por meio de cromatografia gasosa com espectrometria de massa (CG-MS). Com raras exceções, os queijos das quatro origens continham os mesmos compostos voláteis, embora em quantidades distintas. Os álcoois e ésters foram os compostos majoritários nos queijos A e B e benzaldeído, 3-metil-butanal-2 e hexanal nos C e D. O perfil de textura instrumental (TPA) e a análise sensorial descritiva e quantitativa foram realizados. Os queijos B, C e D apresentaram características mais típicas de queijo Prato, independentemente do fato de que o aroma de manteiga e o sabor doce serem mais acentuados no queijo D. O queijo A foi classificado como tendo as menores características de queijo Prato e apresentou maior nível de defeitos de \"flavour\", principalmente residual e amargor. Os queijos avaliados não apresentaram diferenças significativas quanto à elasticidade e coesividade. Pequenas alterações na composição físico-química dos queijos, principalmente os teores de umidade e de caseína, influenciaram nos parâmetros como a firmeza e a adesividade. O presente estudo demonstrou pela primeira vez a ausência de autólise de Lc. Lacfis sp. em queijo Prato de quatro origens, bem como a ocorrência de autólise de Lab. helveticus nos queijos de duas origens, C e D. A pronunciada autólise dessa espécie teve um impacto positivo na proteólise e foi a responsável pelo aumento da concentração de aminoácidos livres nesses queijos. As diferenças na evolução da proteólise observada entre os queijos C e D, com taxas mais baixas no queijo C, independentemente da autólise pronunciada de Lab. helveticus, foram atribuídas à falta de uniformidade na composição físico-química dos queijos, principalmente pH e os teores de sal na umidade (S/U).
This paper reports a study aimed at evaluating the variations that occur in the interrelationship between autolysis of lactic starter bacteria and the development of proteolysis in Prato cheese produced in four different regions of Brazil: Santa Catarina (Cheese A), Goiás (Cheese B), São Paulo (Cheese C) and Minas Gerais (Cheese D). Quantitative analysis of microbial population yielded similar microbiological profiles for all the cheese samples investigated. After 5 days ripening, lactococci and streptococci were present in higher numbers than mesophilic and thermophilic lactobacilli, leuconostoc and lactate fermenting bacteria. However, the populations of the latter species had considerably increased by the time the ripening process completed 45 days. Enterococci and citrate fermenting bacteria remained present in relatively low numbers throughout ripening. The findings from qualitative analysis confirmed the predominance of non-starter lactic acid bacteria (NSLAB) in the cheeses from four different origins, especially Lactobacillus sp. Other genera of non-starter lactic acid bacteria (NSLAB) were identified in smaller proportions: Enterococcus sp., Pediococcus sp., Aerococcus sp., Tetragenococcus sp. and Streptococcus sp. Cheese C differed from the cheeses in that it no evidence was found of the presence of Pediococcus sp. and Streptococcus sp. The bacteria of the lactic starter culture Lactococcus lactis sp. and Leuconostoc sp. were also found to be present, although in lower numbers. Autolysis was studied by: (1) determination of aminopeptidase activity; (2) detection of autolysins by zymograms and (3) detection of intracellular enzymes by immunoblotting. The release of aminopeptidase was highest in cheese D, followed by C, B and A. No bands of lytic activity were appeared in the zymograms of Cheeses A and B in all conditions evaluated. At pH 7,4 and 44°C, a low-intensity band of 30 KDa was found in cheeses C e D, whereas another low-intensity band was observed only in cheese D. At pH 6,8 and 42°C, bands of 40KDa were observed in cheese C (low intensity) and cheese D (high intensity), in addition to two more low-intensity bands of 90KDa and 110KDa in cheese D. Immunoblotting with antiserum anti-Lc produced only minor signs of positive reaction, evidenced by the formation of low-intensity bands of 100 Kda in cheeses A and B and two high-intensity bands of 75 Kda and 100 Kda in cheeses C and D. Since these were present in smaller numbers to those revealed with crude cytoplasm of Lac. lactis subsp. lactis, it was concluded that autolysis did practically non occur. Immunoblotting with antiserum anti-D-LDH also detected sings of positive reaction in cheeses A and B, whereas in cheeses C e D positive high-intensity signs of 37Kda were found relative to D-LDH protein, indicating lysis of Lab. helveticus. The evolution of proteolysis was determined quantitatively during the ripening process and evaluated on the basis of the following parameters: NS-pH4,6/NT% and NNP/NT% indexes, tyrosine content, electrophoresis (Urea-PAGE) and quantification of free amino acids. No significant differences were found between cheeses A, B, C and D in the ear1y stages of ripening. However, with the on-going fragmentation of proteins during ripening, a gradual increase of the ripening indexes occurred, with the highest values being observed in cheese D, followed by C, B e A. The electrophoretic profiles were similar for the four cheeses investigated and clear1y showed that the clotting agent or milk coagulant and plasmin were responsible for the initial breakdown of the caseins. The degradation rate of Q.sl- and p-casein followed the following order: D > C ≥ B > A. The buildup of free amino acids was also faster in cheese D, followed by cheeses C, B e A. At the end of the ripening process studied (45 days), the volatile compounds were identified using gas chromatography and mass spectrometry (GC-MS), whereas the instrumental texture profile was measured and evaluated by Texture Profile Analysis (TPA). Cheese samples were evaluated by descriptive and quantitative sensory analysis. With rare exceptions, the cheeses of four different origins contained the same volatile compounds, although in different quantities. Alcohols and esters were the predominant volatile compounds in cheeses A and B and benzaldehyde, 3-methyl-butanal-2 and hexanal in cheeses C and D. Autolysis of Lb. helveticus accelerated proteolysis in cheese D, thereby reducing ripening time by 45% without any negative effect on either flavor or texture development. Cheeses B, C and D exhibited the most typical Prato cheese characteristics, in spite of the fact that the buttery aroma and sweet taste were more pronounced in cheese D. Cheese A was rated as the cheese with the less typical overall Prato cheese profile and was also the one that exhibited the highest degree and number of flavor defects, notably aftertaste and bitterness. The cheeses investigated did not present any significant differences as to elasticity and cohesiveness. Minor changes in the physical-chemical composition of the cheeses - mainly related to the moisture and casein levels - influenced parameters such as firmness and adhesiveness. The present study demonstrates for the very first time the absence of autolysis of Lc. Lactis sp. in Prato cheese from four different origins, as well as the occurrence of autolysis of Lb. helveticus in two of the cheeses analyzed (cheeses C and D). The pronounced autolysis of this species had a positive impact on proteolysis and was responsible for the release of increased quantities of free amino acids in these cheeses. The differences in the evolution of proteolysis observed between cheeses C and D - lower rate of proteolysis in cheese C, in spite of pronounced autolysis of Lb. helveticus - were attributed to poor uniformity of the physical-chemical composition of this cheese, particularly as related to pH and the salt and moisture levels (S/M).

O objetivo deste trabalho foi verificar as possíveis correlações entre os índices de mastite e a qualidade microbiológica do leite cru, em 36 propriedades com atividade exploratória leiteira, no Estado de São Paulo, Brasil. Examinou-se 4662 quartos mamários de 1180 animais em lactação para se verificar a presença de mastite pelos Testes de Caneca de Fundo Escuro e CMT, e coletou-se uma amostra de cada quarto mamário positivo em pelo menos um dos testes para exame microbiológico. Para se avaliar a qualidade microbiológica do leite cru, coletou-se uma amostra de cada propriedade e fez-se a Contagem de microrganismos mesófilos aeróbios facultativos viáveis, Contagem de microrganismos psicrotróficos aeróbios facultativos viáveis, Contagem de microrganismos termófilos aeróbios facultativos viáveis, Contagem de Enterococcus spp., Contagem de Stapylococcus spp., Contagem de Streptococcus spp., Contagem de Corynebacterium spp., Contagem de bolores e leveduras, Número Mais Provável de coliformes totais e Número Mais Provável de coliformes fecais. Aplicou-se teste de Correlação de Pearson e Regressão Linear. Para comparação entre os índices de mastite, a melhor correlação foi entre o índice de resultados positivos ao teste CMT e os casos de mastite por causa infecciosa (r = 0,920 e R2 = 84,7%). Comparando-se o índice de mastite e a qualidade microbiológica do leite cru, o melhor resultado que apresentou correlação significativa foi entre o índice de casos de mastite por Staphylococcus spp. e a Contagem de microrganismos termófilos aeróbios facultativos viáveis (r = 0,522 e R2 = 27,3%). Pelo baixo número de análises que apresentaram resultado de Correlação significativa, notou-se que o índice mastite em rebanhos bovinos leiteiros não interfere na qualidade microbiológica do leite cru, nas condições estudadas neste experimento.
The purpose of this research was to investigate the possible correlations between the mastitis rate and raw milk microbiology quality, from 36 dairy farms, in São Paulo State, Brazil. It was investigated 4662 mammary quarters of 1180 lactant animals to examine mastitis cases using Tamis test and CMT. It was collected one sample of each positive mammary quarter in one test at least to microbiological analysis. To estimate the raw milk microbiological quality, it was colected one sample from each farm and realized one aerobic plate count of microorganisms mesophilic, aerobic plate count of microorganisms psychrotrophic, aerobic plate count of microorganisms termophilic, Enterococcus spp. plate count, Stahylococcus spp. plate count, Streptococcus spp. plate count, Corynebacterium spp. plate count, Yeast and Molds plate count, Most Probable Number of coliforms, Most Probable Number of fecal coliforms. It was used Pearson Correlation test and Linear Regression. In order to compare mastitis rates, the best correlation was between positives CMT test rate and the cases of mastitis caused by infections disease (r = 0,920 and R2 = 84,7%). Analysing the mastitis rate and the raw milk microbiological quality, the best result of correlation was between Staphylococcus mastitis rate and the aerobic plate count of microorganisms termophilic (r = 0,522 e R2 = 27,3%). Because of the low number of significant correlation, it was observed that the mastitis heard in dairy bovines herds do not interfere in raw milk microbiological quality, in the condition of this experiment.

Thesis (M. Tech. (Environmental health)) - Central University of technology, Free State, 2013
Introduction Dairy farms in central South Africa produce a substantial amount of milk, which is sold in Bloemfontein, Free State. Large volumes of unpasteurized (raw) milk is collected on the dairy farms, which undergoes further processing before it reaches the consumer at the end of the production line. There is a large proportion of the population that, in most cases unknowingly, consumes raw milk that has bacterial counts substantially higher than legal standards. Poor quality unpasteurized milk is either sold as fresh milk in the informal market, or as dairy products, such as cheese, manufactured from unpasteurized milk. Consumers are therefore, in most cases, unaware of the poor quality dairy products they consume. Milk quality is usually assessed in terms of bacterial content, which include Escherichia coli, coliforms and total bacterial count. The bacterial quality of milk is influenced by a number of factors, including farming practices, structural design of the milking shed, herd health and quality of water used in the dairy. If the highest level of hygiene practices is maintained, contamination of the milk by pathogenic microorganisms will be controlled, however, any drop in the vigilance of hygiene practices could result in unacceptable high levels of pathogenic microorganisms resulting in poor quality raw milk. Poor quality raw milk will inevitably result in poor quality pasteurized milk, containing unacceptably high levels of pathogenic organisms, which will eventually reach the consumer. Objectives The objectives of this study were to assess the quality of milk and influencing factors of milk produced on 83 dairy farms that supply milk intended for further processing to the greater Mangaung region, Central South Africa. Influencing factors investigated included, water quality and hygiene of milk contact surfaces, namely pulsator surfaces and milk pipeline surfaces. Methods Standard sampling procedures were followed when milk was sampled from bulk milk tanks, water at the point of use in the dairy, as well as collection of surface swabs. Escherichia coli, coliforms, total bacterial counts and somatic cell counts in milk were determined in terms of the regulations relating to milk and dairy products, and for water in terms of drinking water standards. These data were analysed and the factors that directly influence bacterial quality of milk were identified. Results 93% of the dairy farms displayed E. coli in their bulk milk containers, which did not comply with the legal standard. For coliforms, 86% of the milk samples did not comply with the legal standard. The total bacterial count of 85% of the milk samples did comply with the legal standard. The somatic cell count of 42% of the milk samples did not comply with the legal standard. The pulsator surfaces as well as the milk pipeline surfaces of 13% of the dairy farms displayed the presence of E. coli. 80% of the pulsator surfaces and 78% of the milk pipeline surfaces did comply with the legal standard pertaining to coliforms. The total bacterial count of pulsator surfaces revealed that 19% complied, whereas 29% of the milk pipeline surfaces complied with the legal standard. The water data further revealed that 31% of the dairy farms contained E. coli in the water used in the dairies. 63% of the dairy farms contained more than the allowable number of coliforms in their water. Chi-square tests revealed significant differences (p > 0.05) between the presence or absence of E. coli in milk and water; the presence or absence of E. coli in milk and milk pipeline surfaces; the presence or absence of E. coli in milk and pulsator surfaces and the presence or absence of E. coli in milk and the positioning of the cows in the milking shed. When milk quality indexes were calculated for all the farms, only four farms were classified with excellent milk, the remainder were all classified as producing poor quality milk. The hygiene quality indexes revealed that the hygiene practices on all the farms were not up to standard. Discussion and conclusion The study revealed that the milk produced for commercial processing and distribution in the greater Mangaung region of central South Africa was of poor quality. It is often mistakenly believed that the pasteurization process will remove all microorganisms from milk. As this is not the case, it is of major concern that milk delivered commercially is not of acceptable quality. Furthermore, it could be concluded that the quality of milk products from raw milk were also probably not of acceptable quality. The results further revealed that the possible contributing factors to the poor quality milk produced by the 83 commercial dairy farms were; poor quality water used in dairy sheds and contaminated milk contact surfaces. From this study it could be concluded that the overall status of milk production on the 83 commercial dairy farms studied, did not meet the standards required for milk quality, water quality and hygiene practices.

Three duodenally cannulated lactating Holstein cows fed cotton-seed meal (CSM), corn gluten meal (CGM) or blood meal (BM) as protein supplement were used in a 3 x 3 Latin Square experiment to determine microbial crude protein (MCP) in duodenal digesta. Diets, formulated to contain 15% crude protein (CP) on a dry matter basis, consited of 60% concentrate, 31% corn silage and 9% alfalfa hay. Chromium oxide was employed as flow marker. Microbial protein fraction of digesta CP (MCP/DCP) was estimated by three microbial markers: ¹⁵N, diaminopimelic acid (DAP) and ribonucleic acid (RNA). The isotopic method gave the most reliable results. Variability was higher with DAP and RNA. Results from RNA were lower (P < .01) and unreasonable. Based on ¹⁵N, MCP/DCP differed among treatments (P < .10) with means of 61.5, 59.4, and 50.0% for CSM, CGM, and BM, respectively, but differences were not significant for absolute amounts of total CP and MCP in duodenal digesta.

Estratégias de vedação tem sido adotadas com o intuito de reduzir a entrada de oxigênio para o interior dos silos. Aditivos químicos como o benzoato de sódio, que apresenta funções antimicrobianas também pode ser empregado para melhoria da estabilidade aeróbia de silagens. Porém, ainda não se sabe se pode causar efeitos adversos no consumo ou metabolismo dos animais alimentados e, consequentemente levar a alterações no desempenho animal. Desta forma no presente estudo, objetivou-se avaliar a influência de estratégias de vedação de silos trincheira sobre as perdas de MS e valor nutritivo de silagens de milho e, a adição de benzoato sódio na ração total no valor nutritivo para vacas leteiras. O experimento foi conduzido no Departamento de Zootecnia da Escola Superior de Agricultura \"Luiz de Queiroz\" (ESALQ/USP). A cultura do milho foi colhida com aproximadamente 35% de matéria seca (MS) e ensilada em silos trincheira (capacidade de 40 t). No momento do fechamento dos silos, dois tratamentos foram impostos: (1) lona dupla-face 200 μm protegida com bagaço de cana-de-açúcar (camada com espessura de 10 cm) (BG) e, (2) aplicação superficial de benzoato de sódio 150 g/m2 (diluído em água 1:4) imediatamente antes da vedação com lona dupla-face 200 μm (BZ). Vinte vacas Holandesas em lactação foram alocadas em cinco Quadrados Latinos 4 × 4 em períodos de 21 dias (14 d adaptação). As dietas experimentais continham (%MS): 8% de caroço de algodão, 9,5% de polpa cítrica, 18% de farelo de soja, 9,0% de milho moído seco, 2,5% premix mineral + vitaminas e 53% de silagem de milho: BG ou BZ ou silagem de milho BG + 0,15% de benzoato de sódio ou silagem de milho BZ mais 0,15% de benzoato de sódio. O benzoato de sódio foi diluído em água (0,3:1) e aspergido na ração total imediatamente antes de cada trato. Os dados foram submetidos à análise utilizando-se o procedimento MIXED do SAS, através de arranjo fatorial 2 × 2. A silagem com cobertura de bagaço de cana foi mais eficiente em reduzir a entrada de oxigênio durante o processo de fermentação e, consequentemente levou ao menor crescimento de microrganismos deterioradores e melhor conservação dos nutrientes da silagem, resultando em maior digestibilidade da MS. A adição de benzoato de sódio na dose de 0,15% na MN não altera o desempenho de vacas leiteiras.
Sealing strategies have been adopted to reduce oxygen entrance to silo. Chemical additives such as sodium benzoate have antimicrobial activity and it can also promote an aerobic stability on silage. Nevertheless it is still unknown if sodium benzoate supplementation on silage may affect animal consumption or cause deleterious effect on metabolism with influence on animal performance. The objective of this study was using different sealing strategies to assess dry matter loss and nutritional value on corn silage, and the influence of supplementing sodium benzoate on total mixed ration for dairy cattle. This trial was conducted at Animal Science Department of Luiz de Queiroz College of Agriculture (ESALQ/USP). Corn crop was harvested with 35% of dry matter (DM) and ensiled on horizontal silos (40 t capacity). A factorial design (2x2) for silo sealing and benzoate as an additive on dietary feed were evaluated. Silo sealing strategies were confected as follows: (1) Plastic film doubled-sized 200 μm covered with bagasse (layer thickness 10 cm) (BG) and 2) application of sodium benzoate on top surface of ensiled mass pulverizing 150 g m-2 (dilution of 1:4) sealing it immediately with plastic film double-sided 200 μm (BZ). After 343 days of storage, the silos were open and the lactation trial started. Two dietary treatments evaluated the addition sodium benzoate on feed mixture of total ration. Sodium benzoate was incorporated (+ 0.15 % on total feed) on corn silage from BG and BZ and no incorporation to BG and BZ were used as control treatment. Dietary sodium benzoate was diluted on water (0.3:1 ratio) and pulverized on total ration immediately before each meal. Feed formulation: 8% cottonseed meal, 9.5% citric pulp, 18% soybean meal, 9% dry corn meal, 2.5 % vitamin and mineral premix, and 53% of corn silage. Twenty Holstein cows lactating were allocated in five Latin squares (4 x 4) during 21 days (14 days to acclimate) and fed twice a day. Dry matter intake (DMI), milk yield and quality were recorded between day 15th and 21st from each experimental period. Data were subjected to MIXED procedure from SAS for factorial design (2x2). The silage with sugarcane bagasse coverage was more effective in reducing the oxygen input during the fermentation process and consequently led to lower growth of spoilage microorganisms and better conservation of silage nutrients, resulting in increased digestibility of dry matter. Adding 0.15 % of sodium benzoate on fresh matter diet doesn\'t affect the performance of dairy cows.

Duas formulações de queijo de leite cru e fermento endógeno foram produzidas e avaliadas após 30, 60 e 180 dias de armazenamento em câmaras de maturação de dois laticínios do Sudoeste do Paraná foram estudadas para avaliar as características, como uma das etapas de desenvolvimento de um queijo típico regional. A produção foi acompanhada desde a elaboração do fluxograma de processamento, coleta das amostras de queijo para realização de análises de proteína, lipídeos, umidade, cinzas, carboidratos, extrato seco total, gordura no extrato seco, calorias; atividade de água, textura instrumental (dureza, adesividade, elasticidade, coesividade, mastigabilidade e resiliência), cor (CIE Lab), análises microbiológicas (contagem de coliformes totais, contagem de coliformes termotolerantes e contagem de Staphylococcus coagulase positiva e pesquisa de Salmonella spp.); teste de aceitação relacionada às características sensoriais (cor, aparência, odor, textura, sabor) e intenção de compra. Essa pesquisa contribuiu com informações relevantes ao processo produtivo, tais como, a constatação da viabilidade do fermento lático liofilizado elaborado a partir das bactérias ácido láticas isoladas do leite da mesorregião do Sudoeste do Paraná e cujos resultados das análises dos queijos indicaram similaridade entre as formulações, no que se refere a caracterização física e físico-química, além de boa qualidade microbiológica, onde as discrepâncias entre as amostras dos laticínios não foram percebidas sensorialmente pelos provadores. Ajustes na padronização relacionados ao controle de qualidade tecnológica serão um fator de extrema importância para o sucesso das empresas e pequenos produtores envolvidos no projeto e que se dispõem a produzir o Santo Giorno, um queijo fino, com o grande diferencial de agregar características da região, com elevado padrão de qualidade higiênico-sanitária e com indicativos de grande aceitação pelo consumidor.
Two cheese formulations made from raw milk and endogenous yeasts with 30, 60 and 180 days of maturation in two dairy Paraná Southwest were studied to evaluate their quality through physical, physical-chemical, microbiological and sensorial characteristics, as one of the stages of development of a typical regional cheese. The production was accompanied from designing the flowchart processing, where the samples were collected to perform the analysis of proteins, lipids, moisture, ash, carbohydrates, total solids, fat in dry matter, calories; water activity, instrumental texture (hardness, adhesiveness, springiness, cohesiveness, resiliency and chewiness), instrumental color (CIE Lab); microbiological quality was assessed searching for total and thermotolerant coliforms, coagulase positive staphylococci and Salmonella spp.; the acceptance related to sensory characteristics of color, appearance, flavor, texture, taste and purchase intent was evaluated through the structured hedonic scale. This research contributed information relevant to the production process, such as the realization of the viability in freeze-drying lactic acid bacteria yeast isolated from milk in the Southwestern Mesoregion of Parana and the results of the analysis of the cheese showed similarity between formulations, regarding the physical, physical-chemical characterization, moreover good microbiological quality, where the differences between samples of dairy products were not perceived by sensory panelists. Adjustments in standardization related to technological quality control is an extremely important factor for the success of dairy companies and small producers involved in the project and that they have the option of producing the Santo Giorno, a fine cheese, with the great advantage of adding features of region, with high standard of sanitary conditions and with great consumer acceptance of indicative.

Background: Mastitis in dairy cattle is a common ailment worldwide. A cause of mastitis can be bacteria such as Escherichia coli. Mastitis is not a deadly ailment and sometimes the dairy cows show no symptoms but if certain virulence genes are present in the bacteria that cause the mastitis, the bacteria can be transmitted to humans and cause severe diseases. The potential presence of enterohemorrhagic Escherichia coli (EHEC) in particular would be a major concern for human health. Aim: The aim for this study was to analyze the presence of virulence genes known to be present in E.coli strains isolated from dairy cows with mastitis in Sweden. Method: A Qiagen BIO ROBOT EZ1 was used to purify DNA from 90 bacterial cultures. A panel of virulence genes were amplified and biotinylated from the purified DNA by PCR and an E.coli based DNA microarray was used to detect presumed virulence genes in E.coli. Result: There were no samples that had all the genes traditionally used to classify E.coli as EHEC or potential EHEC. 63 samples were analyzed without any problems but 27 samples were not fully analyzed. Conclusion: The DNA based microarray proved to be a reliable method to detect genes from pathogenic bacteria but it needed high concentration of purified DNA which was not always easy to obtain. There were some samples in this study that contained virulence genes.

Les méthodologies actuelles de caractérisation d'Escherichia coli producteur de toxine Shiga (STEC) nécessitent l'isolement de la souche, ce qui est compliqué par le fait qu'il n'existe pas de milieu d'isolement spécifique qui distingue clairement les STEC des E. coli commensaux non pathogènes. Par conséquent, obtenir des informations sur les souches en utilisant une approche métagénomique éviterait d'isoler les souches pour les caractériser complètement. Dans le cadre du projet, en collaboration avec le BfR en Allemagne, nous évaluerons si de nouvelles approches de métagénomique à lecture longue pourraient déterminer sans ambiguïté si des marqueurs spécifiques d'EHEC typiques (E. coli entérohémorragique) sont co-localisés dans une même souche. Les approches de séquençage hybrides de deuxième et troisième génération seront évaluées. Des pipelines bioinformatiques seront évalués pour analyser les résultats de l'analyse métagénomique. Ces méthodes seront appliquées dans une étude pilote pour étudier le microbiote du lait cru provenant d'exploitations laitières françaises et allemandes et pour identifier un microbiome commun associé aux STEC pathogènes. Nous essaierons de définir un système basé sur l'établissement d'un « score moléculaire » pour qualifier l'état des exploitations
Current methodologies for characterization of Shiga toxin-producing Escherichia coli (STEC) require strain isolation, which is complicated by the fact that there is no specific isolation medium that clearly distinguishes STECs from non-pathogenic commensal E. coli. Therefore, obtaining strain information using a metagenomics approach would avoid isolating a strain to fully characterize it. In the framework of the project, in collaboration with the BfR in Germany, we will evaluate whether new, long-read metagenomics approaches could unambiguously determine whether specific markers of typical EHECs (Enterohemorrhagic E. coli) are co-located in the same strain. Third generation hybrid sequencing approaches will be evaluated. Appropriate bioinformatic pipelines developed in collaboration with the BfR will be evaluated to analyze the metagenomic analysis results. These methods will be applied in a pilot study to study the microbiota of raw milk from French and German dairy farms and to tentatively identify a common STEC-associated microbiome. We aim to define a ‘molecular score' based system to identify the status of the farms, in line with the objective to better precise the notion of ‘STEC molecular risk assessment approach' at the farm level

The objective of this research was to investigate the effects of slow release urea on N metabolism in cattle. The ruminal behavior of Optigen®II and the effect of basal diet on the in situ degradability of urea and Optigen®II were evaluated. The effect of slow release urea and its interaction with degradable intake protein (DIP) level in the diet on N retention and excretion was evaluated utilizing 8 Holstein steers in a 4 x 4 Latin square experiment. In addition, the effect of slow release urea and DIP level on ruminal and systemic urea kinetics was evaluated using stable isotope techniques with 8 Holstein steers in a 4 x 4 Latin square experiment. Finally, slow release urea was evaluated under a practical beef production setting. The performance of slow release urea was compared to regular feed grade urea in a 42 day receiving study (288 Angus cross steers) as well as a 70 day growing study (240 Angus cross steers). High forage diets increased the ruminal degradation rate of both urea and slow release urea an increased the extent of degradation of slow release urea when compared to high concentrate diets. Lower DIP concentrations in the diet reduced systemic urea production, ruminal ammonia and plasma urea concentrations and urinary urea excretion under most circumstances but also led to a reduction in N retention, reduced diet digestibility, lower feed intake, lower growth rate and decreased feed efficiency. High DIP intakes increased N retention, growth rate, diet digestibility and improved feed efficiency but also lead to increased excretion on urea N in the urine. Slow release urea improved N retention and efficiency of N retention in high DIP diets when compared to urea and generally reduced plasma urea and ruminal ammonia concentrations. Compared to urea, slow release urea did not significantly improve the production of receiving cattle. However Optigen®II improved the feed efficiency when compared to urea on high concentrate diets but reduced feed efficiency on high forage diets.

Diversos estudos têm mostrado que alimentos adicionados de probióticos e prebióticos, em quantidades suficientes, trazem benefícios à saúde humana. Estes benefícios se devem, fundamentalmente, ao seu efeito na manutenção da microbiota intestinal benéfica. Paralelamente, ocorre aumento do consumo de derivados do açaí em grandes centros urbanos no Brasil e até mesmo no exterior, principalmente a sua polpa e derivados, que se deve, principalmente, ao alto valor calórico da fruta e à presença de pigmentos antioxidantes. O presente trabalho teve como principal objetivo desenvolver um mix de açaí que apresente aspectos nutricionais e sensoriais semelhantes ao produto tradicional, porém com propriedade funcional suplementar, por se tratar de um alimento probiótico, prebiótico ou simbiótico. Quatro tratamentos foram produzidos (três vezes cada um deles), todos contendo mix de açaí congelado: M1 (controle), M2 (com L. acidophilus La-5 + B. animalis subsp. lactis Bb-12), M3 (com inulina) e M4 (com L. acidophilus La-5 + B. animalis subsp. lactis Bb-12 + inulina) e armazenados a -18 °C por até 3 meses. Após 1, 7, 14, 21, 28, 56 e 84 dias de armazenamento, foram determinadas as populações de microrganismos probióticos, o pH e a cor instrumental dos produtos. O teor de umidade foi determinado após a fabricação dos produtos (dia 0). Adicionalmente, após 7, 42 e 84 dias de armazenamento, foi realizada análise sensorial (aceitabilidade). Foi determinada a composição centesimal dos produtos, a partir de amostras mantidas liofilizadas. Todos os resultados foram comparados, através de análise estatística. A diferenciação na composição centesimal e no teor de sólidos entre as formulações decorreu da adição da fibra prebiótica. Com relação à evolução da cor e do pH, não houve variação importante ao longo do tempo, assim como entre os diferentes mixes de açaí estudados. Os produtos obtiveram boa aceitabilidade sensorial por parte dos provadores (médias das notas acima de 7, na maioria das formulações). Houve maior aceitabilidade dos mixes suplementados com o ingrediente prebiótico e o mix simbiótico recebeu notas médias significativamente mais elevadas (p < 0,05) em dois períodos de armazenamento (7 e 84 dias), quando comparado aos demais. O tempo de armazenamento não influenciou a avaliação sensorial significativamente (p > 0,05). Durante o período de armazenamento proposto, os probióticos apresentaram sobrevivência satisfatória (populações superiores a 108 UFC por porção diária), sob o ponto de vista da legislação brasileira vigente, com exceção do B. animalis subsp. lactis no mix não suplementado de inulina. O mix de açaí apresentou-se como boa matriz para veicular os microrganismos probióticos testados e a inulina contribuiu para uma maior aceitabilidade sensorial do produto.
Some studies have been showing that food products supplemented with probiotics and prebiotics, in sufficient amounts, result in benefits to human health. These benefits are related, fundamentally, to their effect on the healthy intestinal microbiota maintenance. Concomitantly, consumption of açai food products, mainly its pulp and derivatives, is increasing in urban centers in Brazil and also abroad. The high caloric value and the presence of antioxidant pigments are responsible for its higher consumption. The main aim of the present study was to develop an açai mix with nutritional and sensory aspects similar to the conventional product, but with a supplementary functional property, by being a probiotic, prebiotic or synbiotic food. Four trials where produced (three times each), all containing frozen açaimix: M1 (control), M2 (supplemented with L. acidophilus La-5 + B. animalis subsp. lactis Bb-12), M3 (supplemented with inulin) and M4 (supplemented with L. acidophilus La-5 + B. animalis subsp. lactis Bb-12 + inulin) and stored at -18 °C for up to 3 months. After 1, 7, 14, 21, 28, 56 and 84 days of storage, the products were analyzed for probiotic microorganisms populations, pH and instrumental color. The moisture content was determined soon after production (on day 0). Additionally, after 7, 42 and 84 days of production, sensory evaluation (acceptability test) was carried out. Finally, compositional analyses proceeded from freeze-dried samples. Results were compared through statistical analysis. Differences in compositional analyses and moisture content between trials were attributed to the presence or absence of inulin. Instrumental color and pH evolution were not influenced by the storage period and also no differences between the different acaimixes studied were observed (p > 0,05). Products were well accepted by the panellists, and mean scores were often above 7. Prebiotic fiber inulin improved overall acceptability of açaimixes and the synbiotic mix obtained higher mean score (p < 0.05) in two storage periods (7 and 84 days), when compared to the others. The storage period did not influence sensory evaluation significantly (p > 0.05). During the storage period studied, probiotics showed a satisfactory survival (viability higher than 108 cfu for the products daily portion), according to the current Brazilian legislation, except for B. animalis subsp. lactis in the mix not supplemented with inulin. The açaimix showed to be a good matrix for the probiotic microorganisms tested and inulin contributed for a higher acceptability of the product.

A evolução da população de microrganismos mesófilos aeróbios, coliformes totais e fecais e Staphylococcus coagulase positiva foi estudada na linha de processamento do queijo Minas frescal em uma indústria de laticínios sujeita à fiscalização do Serviço de Inspeção Federal (S.I.F.). Três pontos do processamento foram avaliados individualmente: a pasteurização com amostras de leite cru e leite pasteurizado, a coagulação com amostras de leite pasteurizado, de coalhada cortada e de coalhada dessorada e a salga com amostras de queijos antes e depois da salga e da salmoura. A contagem de microrganismos psicrotróficos aeróbios foi incluída na etapa de salga porque é feita sob refrigeração. E depois foram coletadas amostras de um mesmo lote ao longo da linha. Totalizaram 11 coletas em um ano de estudo. Os resultados mostraram que a pasteurização, único ponto crítico de controle do processo, foi insuficiente para garantir a qualidade microbiológica do leite de acordo com os padrões microbiológicos vigentes, porque o leite cru apresentava péssima qualidade. Durante a coagulação houve crescimento médio de 1,70 ciclos log na população de mesófilos aeróbios e um crescimento médio >3,0 ciclos log na população de coliformes fecais. Durante a salga houve crescimento médio de 0,73 ciclos log na população de mesófilos aeróbios e um crescimento médio de 0,37 ciclos log na população psicrotróficos aeróbios. Ao longo da linha, verificou-se que houve crescimentos da ordem de 11,77 ciclos log de coliformes totais e de >9,68 ciclos log de coliformes fecais do leite pasteurizado até o queijo após a salga. Em relação ao Staphylococcus coagulase positiva não foi detectado aumento expressivo de sua população. Os resultados encontrados indicam que o queijo Minas frescal do laticínio estudado antes de ser embalado já se apresentava impróprio para o consumo, devido às altas contagens de coliformes totais e fecais podendo vir a causar riscos aos consumidores e que o próprio processo de fabricação foi responsável pela alta contaminação, constituindo um problema de saúde pública.
The evaluation of aerobic mesophilic, of total and fecal coliforms and Staphylococcus positive coagulase microorganisms population in a dairy processing line of "Minas frescal" cheese submitted to inspection of Federal Service was studied. Three processing steps were analysed individually: the pasteurisation through samples of raw and pasteurised milk; the coagulation through samples of pasteurised milk, cut and drained curd; and the salting through samples of cheeses before and after salting and brine itself. The aerobic psichrotrophic microorganim’s count was made because salting was carried out under refrigeration. After, all the samples of a same allotment were collected. In one year 11 collections were taken and analysed. The results showed that the pasteurisation, the unique control critical point in the process, was not able to ensure the microbial quality of milk in agreement to the microbial standards, due to the bad quality of the raw milk. On curding the aerobic mesophilic population had an average growth of 1.70 cycles log and the fecal coliforms had an average growth of > 3.0 cycles log. On salting the aerobic mesophilic microorganisms had an average growth of 0.73 cycles log as well the aerobic psichrotrophic population had an average growth of 0.37 cycles log. In the processing line, had an average growth of 11.77 cycles log on the population of total coliforms and >9.68 cycles log on the population of fecal coliforms from pasteurised milk until the cheese after salting. An expressive increase of the number of Staphylococcus positive coagulase was not detected. The results indicated that the manufactured "Minas frescal" cheese was improper for consumption even before packing because the high counting of total and fecal coliforms, being harmful to consumers and the processing was responsible for the high contamination, being a potential public health problem.

M. Tech. (Biotechnology, Department of Biosciences, Faculty of Applied and Computer Sciences), Vaal University of Technology.
The Organization for Economic Co-operation and Development (OECD) and the Food and Agriculture Organization (FAO) of the United Nations predict that milk production and the dairy sector will remain one of the fastest-growing agricultural subsectors over the coming decade. The global milk production is projected to expand over the 2011-2020 period at an annual rate of 2%. In South Africa alone, approximately 14 – 15 million litres of milk are wasted annually due to microbial spoilage. Therefore, the identification of the spoilage microorganisms in the milk products is necessary. This will contribute towards the design of appropriate measures to prevent wastage due to spoilage and in turn contribute towards sustainability of the sector. Accordingly, one hundred samples of spoiled full cream UHT milk were collected from two plants of each of the two largest milk processors. These samples were examined visually, and the pH was measured. A presumptive identification up to genus level was conducted by examining morphological features and conducting Gram-stain, catalase and oxidase tests. Species-specific identification was done by using the Analytical Profile Index and Biolog system. Molecular profiling was done by sequencing the rDNA genes. The main spoilage organisms identified in the samples were Pseudomonas, Micrococcus, Bacillus, Enterococcus and Lactobacillus. All organisms belonging to the five genera were psychrotrophs, which are commonly found in biofilms in UHT milk processing equipment. Therefore, according to the study, the spoilage bacteria apparently entered into the milk due to inadequate cleaning-in-place (CIP) processes. More importantly, further studies should be conducted in order to identify the spoilage microbes and how CIP processes can be improved.

The aim of this Ph.D. work was to investigate the physiological role and the technological relevance of the urease activity of the dairy bacterium Streptococcus thermophilus. It has been achieved a deeper comprehension of this peculiar enzymatic activity following different approaches. A milk-based medium that allows the discrimination between urease-positive S. thermophilus strains and urease-negative ones based on the colonies morphology had been developed; it was used as screening method looking for urease-defective mutants after UV mutagenesis of urease-positive strains of industrial interest. Moreover, a cytofluorimetric protocol for the evaluation of urease activity of various samples containing S. thermophilus was developed: the proposed applications are related to the evaluation of the urease activity of starter culture biomasses of S. thermophilus and to the enumeration of the S. thermophilus population in probiotic products containing this species, among others. We propose that the cytofluorimetric method should be seen as an innovative tool to put besides the standard ones to evaluate the quality of the products previously mentioned. The investigation of the physiological role of urease of S. thermophilus cells growing in milk highlighted that in presence of this enzymatic activity the overall metabolism of the species is boosted. Moreover, the cooperative role of urease, previously described for the yogurt consortium, has been extended also to the cooperation between a urease-positive S. thermophilus strain and a urease-negative one, supporting the proposal of the urease activity as an altruistic cooperative trait, which is costly for urease-positive species but provides a local benefit to the urease-negative species sharing the same environment, which can take advantage of the release of ammonia. At industrial level, urease activity is still considered more for its detrimental effects than for the positive effects exerted on S. thermophilus: we proposed different strategies to overcome this industrial problem. Firstly, we produced mutants of urease-positive strains of industrial interest, but they showed lower acidification rate compared to their wild types. So, we proposed to modify the production process of the biomasses with the aim of obtaining cells which carry a lower urease activity compared to those currently produced. In conclusion, the present work gives new insight in the comprehension of the urease activity of the dairy bacterium S. thermophilus, on how it can be exploited to improve the acidification performances of the strains and how it cannot, so far, be controlled during the milk acidification processes.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Ao nascimento, os bezerros exibem um trato gastrointestinal subdesenvolvido (TGI) cuja maturação é estritamente relacionada à colonização da microbiota. No entanto, pouco se sabe sobre os fatores que afetam o estabelecimento de comunidades de archaeas, bacterias e de fungos anaeróbicos no TGI dos bezerros, bem como as mudanças na estrutura e abundância desses grupos microbianos durante o período de transição da fase de pré-ruminante para verdadeiro ruminante. Para abordar essas lacunas no conhecimento, este trabalho empregou sequenciamento de próxima geração para caracterizar a microbiota do TGI de bezerros leiteiros mestiços (Holandês-Gir) durante o período pré-desmame. O primeiro estudo avaliou mudanças nas comunidades de archaeas metanogênicas, bacterias e fungos anaeróbicos no rúmen de bezerros leiteiros (n = 45) alimentados com duas dietas diferentes (M: somente leite cru (10% do peso vivo (PV)) e MC: leite cru (10% PV e concentrado ad libitum) e que foram abatidos aos 7, 28, 49, 63 dias de idade. No segundo estudo, caracterizamos as alterações nas comunidades bacterianas entre regiões GIT (rúmen, jejuno, ceco e cólon) de bezerros alimentados com MC (n = 17) que foram abatidos aos 7, 28, 49, 63 dias de idade. Os resultados do primeiro estudo revelaram que as comunidades de archaeas metanogênicas, bacterias e fungos coexistem no rúmen desde a primeira semana de vida, mas são afetadas diferentemente pela dieta e idade. A inclusão de concentrado na dieta de bezerros afetou significativamente a comunidade bacteriana do rúmen: observou-se um aumento na abundância de gêneros relacionados, direta e indiretamente, à degradação de amido (i.e. Megasphaera, Sharpea e Succinivribrio) e um decréscimo acentuado na abundancia de gêneros (i.e. Lactobacillus, Bacteroides e Parabacteroides) relacionados com a degradação de nutrientes do leite. Alterações na comunidade bacteriana, indiretamente afetaram a comunidade de metanogênicas: fermentação de carboidratos não fibrosos alterou padrões de fermentação (acetato:propionato) e disponibilidade de hidrogênio que por sua vez, favoreceu a colonização de Methanosphaera em vez de Methanobrevibacter. Na comunidade de fungos anaeróbicos, a abundância do genêro Caecomyces e família Neocallimastigaceae não variou significativamente com a dieta ou idade, provavelmente devido à alta variação inter-animal e baixo teor de fibra de concentrado usado em nosso estudo. In suma, este estudo mostrou que a manipulação da microbiota no rúmen em desenvolvimento é possível através da intervenção dietética. Nossos resultados podem ser úteis na elaboração de estratégias para promover a colonização de comunidades-alvo (isto é, produtores de butirato e utilizadoras de lactato) que estão ligadas ao desenvolvimento de papilas e equilíbrio do pH ruminal. Em relação ao segundo estudo, as comunidades bacterianas diferem qualitativa e quantitativamente entre os compartimentos (rúmen, jejuno, cécum e colón) do trato gastrointestinal e também respondem diferentemente ao avanço da idade que inclui a substituição progressiva da dieta líquida para a dieta sólida (i.e. aumento do consumo de concentrado). No rúmen, a comunidade bacteriana foi composta em sua maioria pelos gêneros Prevotella, Butyrivibrio e Ruminococcus cuja abundância aumentou proporcionalmente com a idade devido a maior disponibilidade de carboidratos não fibrosos no rúmen. No jejuno, o gênero Lactobacillus foi abundante desde a primeira semana de vida, mas sua dominância foi substituída por membros da família Clostridiaceae em bezerros mais velhos. As comunidades do ceco e do cólon foram compostas pelos gêneros Blautia, Paraprevotella, Prevotella Phascolarctobacterium and Succiniclasticum cuja abundância aumentou com a idade. Em resumo, nossos resultados mostraram que, embora comunidades bacterianas coexistam em regiões distintas do TGI, uma análise mais detalhada da estrutura, abundância e dinâmica dessas comunidades revela uma marcante segregação e sucessão ecológica no TGI de bezerros. Nosso estudo acrescenta novos insights sobre a colonização bacteriana no TGI de pré-ruminantes que podem servir como base para formulação de estratégias para promover a colonização de comunidades-alvo (i.e. bactérias probióticas) para melhorar a saúde e desempenho de bezerros leiteiros no período pré-desmame.
At birth, calves display an underdeveloped gastrointestinal tract (GIT) whose maturation is strictly related to microbiota colonization. However, little is known about the factors that affect the establishment of archaeal, bacterial and fungal communities in the GIT of calves, as well as the changes in their structure and abundance during calf development into a functional ruminant. To address these gaps in knowledge, this work employed next-generation sequencing to characterize the GIT microbiota of Holstein- Gyr crossbred dairy calves across pre-weaning development. The first study aimed to assess changes on the rumen archaeal, bacterial and fungal communities of crossbred dairy calves (n=45) across pre-weaning development (7, 28, 49, 63 days) on two different diets (M: only raw milk at 10% of body weight at birth (BW) and MC: raw milk (10% BW) plus starter concentrate ad libitum). In the second study, we characterized changes in the bacterial communities across GIT regions (rumen, jejunum, cecum and colon) of MC-fed calves (n=17) at 7, 28, 49, 63 days of age. The results of first study revealed that archaeal, bacterial and fungal communities co-occur in the rumen since early calf development but are impacted differently by pre-weaning diet and age. The inclusion of starter concentrate in the calf diet significantly affected rumen bacterial community by promoting increases of genera, direct and indirectly, related to degradation of readily fermentable carbohydrates (i.e. Megasphaera, Sharpea and Succinivribrio) and depressing those reliant on milk nutrients like lactose (i.e. Lactobacillus, Bacteroides and Parabacteroides). These bacterial changes resulted in apparent diet-driven archaeal differences due to altered fermentation patterns and availability of hydrogen in the rumen that favoured the colonization of members from genus Methanosphaera instead of Methanobrevibacter. No such differences were found for fungi community represented by members from genus Caecomyces and family Neocallimastigaceae, likely due to high inter-animal variation and low fibre content of concentrate used our study. Altogether, this study showed that manipulation of the microbiota in the developing rumen is possible through dietary intervention. Our results may be useful in designing strategies to promote colonization of target communities (i.e. butyrate- producers and lactate-utilizing) linked to functional development of the calf. In regards to second study, bacterial communities in the calf GIT differ qualitatively and quantitatively among compartments and respond differently to age advance that encompass the GIT development (i.e. rumen) and progressive replacement of milk- based to grain-diet (i.e. increase of starter concentrate intake). In the rumen, bacterial community was composed majority by members from genera Prevotella, Butyrivibrio and Ruminococcus whose abundance increased proportionally with age possibly due greater availability of readily fermentable carbohydrates in the rumen. Members from genus Lactobacillus were overrepresented in the jejunum but their predominance was replaced by members from Clostridiaceae family in older calves. The cecum and colon displayed similar abundance at taxa level and the abundance of genera Blautia, Paraprevotella, Prevotella, Phascolarctobacterium and Succiniclasticum increased significantly with age. In summary, our results showed that although there are bacterial communities “common” to distinct regions, a closer look at their structure, abundance and dynamic reveals marked segregation and ecological succession in the calf GIT. Our study adds new insights into bacterial colonization across GIT of pre-ruminant that may be considered in formulating strategies to promote the colonization of target communities aiming improve health (i.e. bacteria with probiotic capability) and performance of dairy calves in the pre-weaning period.

Staphylococcus aureus (S. aureus) is considered one of the most significant aetiological agent of intramammary infection in dairy ruminants, causing both clinical and subclinical forms accompanied with relevant economic losses due to reduced milk production and quality. S. aureus is also considered a major human foodborne pathogen. Milk and dairy products represent indeed potential sources of contamination by S. aureus preformed enterotoxins, which can be responsible of staphylococcal food-poisoning (SFP) outbreaks. S. aureus pathogenicity is also related to its capacity to quickly develop resistance against new antibiotics used in clinical practices. Among multidrug resistant strains, methicillin resistant Staphylococcus aureus (MRSA) represents a global health threat. In the last few years, the isolation of MRSA strains from both livestock and companion animals has gained growing attention, both in veterinary medicine and from a Public Health perspective. In particular, the presence of MRSA in livestock animals pose a potential risk of infection and/or colonization to humans both through direct contact, and through the consumption of contaminated food. However, there are still many unknowns regarding “MRSA epidemiology”, the risk factors involved in transinfection, and the direction of transmission between animals and humans. This is particularly true for small ruminants due to the fact that they are traditionally not included in national surveillance programs on antibiotic resistance, and related foodstuff are not routinely investigated for this purpose. MRSA population genetics and MRSA epidemiology, have been conventionally studied by using various molecular methods, such as multi-locus sequence typing (MLST) and typing of the S. aureus protein A (spa-typing), often in combination with the staphylococcal cassette chromosome mec (SCCmec) typing. Fingerprinting techniques such as pulsedfield gel electrophoresis (PFGE) have been also frequently implemented to study S. aureus infections in nosocomial settings, or in case of foodborne outbreaks. Currently, the MRSA clonal complex (CC) 398 is the most prevalent lineage among LivestockAssociated MRSA (LA-MRSA), but primarily “human” clones, such as the sequence type (ST) 1, have been also found in livestock. This work describes the genotypic and phenotypic characterization of S. aureus and MRSA isolates from dairy farms of Central Italy, with the main purpose to evaluate the potential zoonotic risks throughout the dairy production chain, in particular focusing on the small ruminants. The work was split in two parts: i) a preliminary study based on the characterization of S. aureus and MRSA strains isolated from a variety of milk and dairy products produced in Central Italy from different animal species; ii) a within-farm epidemiological investigation on the possible MRSA colonization of sheep and in-contact humans. In the first part, a total of 227 S. aureus colonies isolated from 54 samples of raw milk and dairy products of ovine, caprine, bovine and bubaline origin were tested for the presence of genes coding for staphylococcal enterotoxins (SEs/SEls) and for methicillin resistance. Ninety-three colonies from 31 of the 54 samples (57.4%), and from 18 (69.2%) of the 26 farms of origin tested positive for SEs/SEls genes. Most isolates harboured more than one toxin gene and 15 different toxinotypes were recorded. The most frequent were “sec” gene alone (28.6%), “sea, sed, ser, selj” (20%), “seg, sei” and “seh” (8.6%). The 77 colonies harbouring “classical enterotoxins” genes (sea-sed) were further tested for enterotoxin production, which was assessed for 59.2% of the colonies. Three MRSA isolates were detected in three different ovine milk/dairy product samples (1.3%) from two different sheep farms, both located in the province of Rome and apparently not epidemiologically related. All MRSA isolates belonged to spa type t127, ST 1, CC 1, SCCmec type IVa. In the second part, a within-farm epidemiological study was carried out in one of the two MRSA positive dairy sheep farms previously identified. A total of 556 individual milk samples were collected from all the lactating ewes of the herd, together with a bulk tank milk sample. Two weeks later, based on the results obtained from the individual milk samples, specimens (milk, nasal and wound swabs, udder skin samples) were further collected from two MRSApositive animals. Samples (nasal swabs, hand skin samples, and oropharynx swabs) were also collected from three persons in close contact with the animals (the farm-owner and two farm-workers). S. aureus was detected in 12 out of 556 (2.16%) individual samples, with 10 of them (10/556; 1.8%) positive for methicillin- susceptible S. aureus (MSSA) and two (2/556; 0.34%) for MRSA. MRSA was also isolated from the udder skin samples of both MRSA positive animals and from two out of the three persons examined. All the MRSA strains isolated from animal and human sources belonged again to spa type t127, ST 1, CC 1, SCCmec type IVa. However, pulsed field gel electrophoresis (PFGE) analysis performed on MRSA selected isolates of human and animal origin revealed that two closely pulsotypes, a human pulsotype (HP) and an ovine pulsotype (OP), circulated within the farm. The results of microarray analysis on selected strains representative of the two pulsotypes, revealed very similar profiles and only few differences in the gene composition (e.g. genes of the immune evasion cluster, IEC). The presence of IEC genes (sak, scn) was also confirmed by PCR analysis only in MRSA isolates showing the HP. MRSA selected isolates also displayed a very similar resistance phenotype being all resistant to cefoxitin, penicillin, erythromycin, clindamycin, streptomycin, kanamycin, and tetracycline. In conclusion, the occurrence of SEs/SEls positive isolates in a high proportion of milk and dairy product samples of Central Italy, including the detection also from “ready to eat” products of recently discovered SEs/SEls genes, is of concern and underline the need of standardised diagnostic methods to verify and quantify the presence of the “new” enterotoxins directly in food. The identification and genetic characterization of three MRSA isolates from two ovine farms represents the first Italian report on the occurrence of MRSA in ovine milk, and to the Author knowledge, the first genotyping of MRSA strains from ovine dairy products at international level. Although the prevalence was low, the isolation of MRSA from “ready to eat” food is of concern. The results of the within-herd study also demonstrated that the same MRSA strain (i.e., same sequence type, spa type, SCCmec type, PFGE and resistance profile) was able to persist over time at farm level, being detected two years after the first isolation, although the in-herd prevalence was very low. Yet, direction and routes of transmission of the MRSA farmspecific pulsotypes identified in this study remain unclear. Altogether, the results of these studies highlight the importance of applying effective biosecurity and hygiene practices at all times along the dairy production chain. Good hygiene and good manufacturing practices, as well as the use of microbiological testing at every level of the production chain should be implemented in order to control a possible spread of MRSA, and to minimize food poisoning risks.

ABSTRACT Within the complex bacterial community of traditional raw milk cheeses, lactic acid bacteria (LAB), naturally present in the milk as adventitious contaminants, play the key role in the cheese manufacture and ripening process through their acidifying, proteolytic and flavouring activities. Moreover, LAB contribute considerably to the microbial safety, since they produce organic acids and bacteriocins that extend shelf-lives of raw milk products. Numerous LAB genera and species such as Lactobacillus, Lactococcus, Streptococcus, Leuconostoc and Enterococcus are involved in these metabolic activities. This natural microbial biodiversity can be considered a heritage which has to be protected and preserved, since alterations in the composition of the beneficial autochthonous microbiota is of critical importance in maintaining the peculiar features of the traditional raw milk products. On the contrary, LAB may have also negative traits, such as the ability to produce toxic biogenic amines, possession of transmittable antibiotic resistance genes, and the potential for expression of putative virulence factors. Dairy safety aspects should include careful examination of these harmful features. Therefore, the study of micro-organisms at species and strain level and the monitor of their dynamics in the microbial community throughout cheese manufacture and ripening is helpful to improve the quality and safety of the final product. Classical microbiological and molecular techniques have been widely used to investigate the dairy biodiversity and to achieve the characterization of bacterial species present in a complex microbial dairy ecosystem, highlighting the advantages and the limitations of both methods. The aims of PhD thesis were: I) to apply a polyphasic approach based on different phenotypic and molecular techniques for the characterization of the LAB isolated from dairy products collected throughout the technological process of an artisanal raw milk cheese, such as “Formagèla Valseriana”; II) to evaluate the application of total microbial DNA extracted directly from dairy products as template in PCR 16S-23S rRNA region spacer analysis (RSA-PCR) in order to achieve a rapid knowledge about their microbial communities, bypassing the previous step of isolation in pure culture; III) to investigate the influence of equipment (thermocycler) and reagents (Taq polymerase) on results obtained from RSA analysis; IV) to explore the functional and safety aspects of E. faecalis strains isolated from dairy products collected in different North Italy regions through the study of their biodiversity, bacteriocin, volatic organic compounds and biogenic amines production, virulence factors and antibiotic resistance. Firstly, evolution of bacterial groups during the cheese-making process of “Formagèla Valseriana” was revealed by viable counting of LAB and non-LAB micro-organisms. A total of 143 presumptive LAB strains from milk culture, curd and cheese samples were randomly selected from agar plates and subjected to morphological and phenotypic characterization (microscopic examination, Gram staining, catalase test, growth at 15 and 45 °C, CO2 production from glucose) and molecular identification. To investigate LAB biodiversity, the taxonomic position of the isolates was established through RSA-PCR combined with a species-specific PCR assay. Strains characterized by atypical classes of spacer were identified by partial 16S rRNA sequencing. Random amplified polymorphic DNA analysis (RAPD-PCR) was carried out to explore the genetic diversity of LAB isolates. In addition, this study was intended to evaluate the application of microbial DNA extracted directly from dairy products as template in RSA-PCR in order to achieve a rapid knowledge about their microbial communities, bypassing the previous step of isolation in pure culture. As expected in raw milk cheeses, LAB (i.e. lactobacilli, lactococci and enterococci) were quantitatively the dominant group for all samples. The combination of various molecular procedures allowed many species to be detected. In particular, milk culture showed a prevalence of Streptococcus thermophilus, whereas the most detected LAB in curd belonged to the species Lactococcus lactis ss lactis, Lc. garvieae and Enterococcus durans. E. durans was the species most frequently isolated from cheese samples, even if several species (E. faecalis, E. gilvus, E. italicus, Lb. paracasei ss paracasei, St. bovis, Lb. plantarum, Lb. coryniformis and Ln. mesenteroides), which are indicative of biological richness, were also present. RAPD-PCR revealed a high biodiversity at species level, but it also showed the presence of significant genetic variability among the isolates belonging to the same species. RSA, using as template DNA extracted directly from milk culture, did not prove to be a suitable method for discrimination of microbial species in a complex matrix, since when microbial community was very heterogeneous and rich, it was difficult discerning LAB species or genera. Actually, the band of S. thermophilus represents the only one that could be frequently identified. The 143 LAB strains were then screened for their inhibitory activity against Listeria monocytogenes and Staphylococcus aureus and for their acidifying activity. Seven and six strains, mainly belonging to the Enterococcus genus, exhibited antagonistic activity against L. monocytogenes and S. aureus, respectively. With regard to acidifying activity, the majority of isolates (88.8%) were found to be low acidifying isolates, causing a pH decrease lower than 1.5 pH units, whereas only 16 strains were classified as medium acidifying isolates and could be considered the fastest acidifying strains. Yet, after 24 h 74.1% of the isolates showed a medium acidifying ability and 9.1% were classified as high acidifying strains. As revealed by screening the microbial population of “Formagèla Valseriana”, the Enterococcus genus is one of the most prevalent within dairy microbial community. However, enterococci are ascribed the most controversial nature, presenting both beneficial and negative features. They represent important nosocomial pathogens, but are also recognized to produce bacteriocin with antibacterial activity and to enhance flavour and aroma development in cheese. For this reason, 40 Enterococcus faecalis strains isolated from raw milk products (milk, curd and cheese) collected throughout the technological process of various North-west Italian traditional cheeses (Aosta Valley, Piedmont and Lombardy) over a 13 year period (1997-2009) were examined. The genetic biodiversity of E. faecalis isolates was assessed through RAPD-PCR and repetitive extragenic palindromic PCR (rep-PCR). Phenotypic and molecular protocols were applied in order to investigate the incidence of antibiotic resistance and virulence factors among strains. They were examined for their antimicrobial activity against 18 foodborne spoilage and pathogenic bacteria. Investigation was also made to identify their bacteriocinogenic potential by searching for bacteriocin-encoding genes (enterocin A, enterocin B, bacteriocin 31, enterocin P, enterocin Q, enterocin L50A-B, mundticin KS, enterocin CRL35, enterocin AS-48, and enterocin 1071A-B). In addition, they were inoculated in milk and submitted to head-space solid-phase-micro- extraction gas chromatography-mass spectrometry analysis in order to study their capability for production of volatile organic compounds (VOCs). Resistance to streptomycin (50%), quinupristin-dalfopristin (32.5%), tetracycline (25%), rifampicin (7.5%), chloramphenicol (5%) and erythromycin (2.5%) were evidenced. Ampicillin, ciprofloxacin, levofloxacin, mupirocin, nitrofurantoin, penicillin G and vancomycin were revealed effective antimicrobials against all the strains considered. Tetracycline resistance was also confirmed by detection of tet(M), tet(K), tet(L), tet(S) and transmissibility of resistance by integrase gene (int) of the Tn916/Tn1545 family of transposons. The high incidence of tet(M) gene, found in 90% of the isolates, doesn’t correlate with their phenotypic expression, thus highlighting the presence of silent genes. The phenotypic assay of haemolytic activity showed positive results (β-haemolysis) only in 2 strains. Among the 7 virulence determinants tested, only gel, asa1, esp and efaA genes were harboured. None other gene encoding for either different virulence factors (cylA, hyl, ace), or amino-acid decarbolylase activity (hdc, tdc, odc) was detected. Our results suggest that dairy enterococci don’t represent the major potential reservoir for the spread of the investigated detrimental traits in contrast to other strains of different origin. Considering their antimicrobial activity, all the E. faecalis tested were active against at least 1 of the 18 indicator strains. Remarkable inhibitory effects against harmful or detrimental microbial species, including Bacillus cereus (44.7% of strains), Escherichia coli (17.5%), L. monocytogenes (15%), St. aureus (2.6%), and Clostridium sporogenes (21.1%), was detected. Moderate antagonism towards LAB (65.8% of strains), especially Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus helveticus, was found, too. However, 31.6% of E. faecalis strains were reported as ideal preservatives. Of the 10 enterocin structural genes explored, only gene for enterocin AS-48 was identified, suggesting that the antimicrobial activity of the phenotypically positive isolates should necessarily be due to another enterogenic or non-enterogenic compound. A significant genetic variability was pointed out, but it was no possible to link the enterocin phenotypes and genotypes with the strain origin. No relation with the geographical origin or the period in which the strains were isolated was also found with regard to the VOCs production and very different enzymatic activities among strains was detected. The major volatile compounds produced were: ethanol, diacetyl, acetoin, acetic and benzoic acids. The results of the present study suggested that a polyphasic approach, combining different phenotypical and genotypical methods, may represent an essential tool to obtain a more effective, accurate and exhaustive investigation of the microbial typing. Finally, even if RSA-PCR was proven to be a rapid, reliable and cost-effective alternative to other PCR methods for the microbial identification and typing, the possibility to compare RSA-PCR results obtained with different thermal cyclers and Taq DNA polymerases has not been investigated effectively. Four models of thermal cyclers of 3 different commercial brands and 4 brands of Taq DNA polymerase were evaluated for their effects on the reproducibility of the RSA-PCR. Five reference LAB strains (St. thermophilus, Lb. helveticus, Lb. delbrueckii subsp. lactis, Lb. delbrueckii subsp. bulgaricus and Lb. fermentum) and a DNA mixture, obtained combining together the DNAs extracted from each reference strain in the same ratio, were used in the experiment. A wide variety of RSA-PCR profiles in terms of band number and size were observed in relation to thermal cycler and Taq DNA polymerase tested, highlighting the relevant influence of these two variables on RSA-PCR reproducibility. It is therefore recommended to choose, within each laboratory, appropriate operating procedures in accordance with one’s equipment and reagents.

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Bactérias ácido lácticas (BAL) foram isoladas do ambiente de produção de leite e avaliadas quanto ao potencial benéfico. Testes preliminares e análise por PCR foram aplicados para selecionar e identificar através de sequenciamento de rRNA 16S 15 cepas de BAL: Lactobacillus (n = 11; Lb. casei MSI1, Lb. casei MSI5, Lb. casei MRUV1, Lb. casei MRUV6, Lb. acidophilus MVA3, Lb. nagelli MSIV4, Lb. harbinensis MSI3, Lb. harbinensis MSIV2, Lb. fermentum SIVGL1, Lb. plantarum MLE5 e Lb. plantarum MSI2), Pediococcus (n = 2; P. pentosaceus MLEV8 e P. acidilactici MSI7) e Weissella (n = 2; W. paramesenteroides MRUV3 e W. paramesenteroides MSAV5). Todas as linhagens selecionadas apresentaram resistência ao baixo pH e à presença de sais biliares. O teste API ZYM foi realizado para caracterizar a atividade enzimática entre as cepas e foi observada elevada atividade β-galactosidase em 13 delas. Todas as cepas apresentaram alta taxa de sobrevivência ao suco gástrico e as condições intestinais simulados, capacidade de auto-agregação e co- agregação com micro-organismos indicadores e alta hidrofobicidade da superfície celular. A maioria das cepas foi positiva para os genes de adesão map e EFTu. Os resultados de deconjugação de sais biliares mostraram forte desconjugação para todas as cepas. Todas as cepas mostraram bons resultados para assimilar lactose. Após esta etapa de caracterização do potencial benéfico, as 15 BAL foram avaliadas quanto ao potencial de virulência e de resistência antimicrobiana. A produção de fatores de virulência (hemólise, gelatinase, lipase, desoxirribonuclease e aminas biogênicas: lisina, tirosina, histidina e a ornitina) foi avaliada por métodos fenotípicos, a 25 °C e 37 °C, bem como a resistência a 17 antibióticos. Os isolados foram também submetidos à análise de PCR para identificar a presença de 49 genes associados a fatores de virulência. Nenhuma das cepas apresentou atividade hemolítica, produção de gelatinase, lipase, desoxirribonuclease e aminas biogênicas. Das 15 cepas selecionadas, para 12 tipos de antibióticos no método de difusão em disco, todas as amostras foram resistentes à oxacilina e sulfa/trimetoprim, 14 foram resistentes a gentamicina, 11 foram resistentes a clindamicina, nove cepas foram resistentes à vancomicina, oito cepas para rifampicina, cinco foram resistentes a eritromicina, quatro foram resistentes à tetraciclina, duas cepas foram resistentes à ampicilina, uma cepa foi resistente ao cloranfenicol e nenhuma apresentou resistência ao imipenem. Para um teste quantitativo do antibiograma, 5 antibióticos em fitas Etest® (bioMérieux) foram selecionados. Todas as 15 cepas foram resistentes à vancomicina, duas para rifampicina, uma para gentamicina e uma para o cloranfenicol. Em relação aos genes relacionados com virulência, 19 dos 49 genes testados estavam presentes em algumas cepas. Após a caracterização do potencial virulento das 15 BAL, estas foram avaliadas quanto ao potencial tecnológico para aplicação na indústria de laticínios. Todas as cepas apresentaram capacidade de acidificação, atingindo valores de pH entre 0.73 e 2.11 em 24 horas: Lb. casei MRUV6 apresentou maior capacidade de acidificação (pH 2.11 após 24 h). Dez cepas foram capazes de produzir diacetil a 37 °C, com exceção de Lb. casei MSI1, Lb. harbinensis MSI3, Lb. fermentum SIVGL1, Lb. plantarum MLE5 e W. paramesenteroides MRUV3. Todas as cepas foram capazes de produzir exopolissacarídeos, e apenas duas cepas apresentaram atividade proteolítica (Lb. casei MSI5 e W. paramesenteroides MSAV5). Com base nessa caracterização, Lb. casei MRUV6 foi selecionado para produzir o leite fermentado, armazenado a 4 °C e 10 °C e monitorado até 35 dias de vida útil. As amostras foram submetidas a métodos fenotípicos e moleculares para avaliar a presença de Lb. casei MRUV6 (plaqueamento convencional e RT-PCR, verificando a expressão de gapdh, um gene housekeeping) e verificar a expressão do gene bsh, relacionado à resistência à sais biliares (RT-PCR). A população de Lb. casei MRUV6 se apresentou estável durante todo o período de armazenamento a 4 °C e 10 °C a níveis em torno de 9.9 log UFC/g e também pelo monitoramento da expressão do controle endógeno GAPDH. No entanto, o gene bsh não foi expresso durante o período de armazenamento. O estudo demonstrou o potencial uso da cepa de Lb. casei MRUV6 isolada de um ambiente lácteo para a produção de um produto lácteo fermentado e sua estabilidade durante o armazenamento a 4 °C e 10 °C. Todos os isolados do estudo apresentaram características benéficas, segurança para utilização em alimentos e potencial tecnológico para utilização na indústria de laticínios. Além disso, os mesmos podem ainda ser submetidos a estudos adicionais para avaliações in vivo e realizar a caracterização como probióticos.
Lactic acid bacteria isolated from dairy environment were evaluated for beneficial potential. Preliminary screening and PCR analysis were applied to select and identified through 16s rRNA sequencing 15 LAB strains: Lactobacillus (n = 11; Lb. casei MSI1, Lb. casei MSI5, Lb. casei MRUV1, Lb. casei MRUV6, Lb. acidophilus MVA3, Lb. nagelli MSIV4, Lb. harbinensis MSI3, Lb. harbinensis MSIV2, Lb. fermentum SIVGL1, Lb. plantarum MLE5 and Lb. plantarum MSI2), Pediococcus (n = 2; P. pentosaceus MLEV8 and P. acidilactici MSI7) and Weissella (n = 2; W. paramesenteroides MRUV3 and W. paramesenteroides MSAV5). All selected strains showed resistance to acidic pH and to presence of bile salt. API ZYM test characterized enzymatic activity of the strains and high β-galactosidase activity was observed in 13 strains. All strains presented high values for survival rate to simulated gastric and intestinal conditions, ability to auto and co-aggregate with indicators microorganisms and high cell surface hydrophobicity. Most of the strains were positive for map and EFTu beneficial genes. Strong bile salts deconjugation was applied for all strains and all strains showed good results for assimilating lactose. After this first part of the study, the 15 BAL were evaluated for potential virulence and antimicrobial resistance. The production of virulence factors (hemolysis, gelatinase, lipase, deoxyribonuclease and biogenic amines: lysine, tyrosine, histidine and ornithine) was assessed by phenotypic methods at 25 °C and 37 °C, as well as the resistance to 17 antimicrobials. The isolates were also subjected to PCR to identify the presence of 49 genes associated with virulence factors. None of the strains presented hemolytic activity or the production of gelatinase, lipase, deoxyribonuclease and tested biogenic amines. Of the 15 selected cultures, for 12 types of antibiotics in the disc diffusion method, all strains were resistant for oxacillin and sulfa/trimethoprim, 14 were resistant to gentamicin, 11 were resistant to clindamycin, nine strains were resistant to vancomycin, eight strains to rifampicin, five were resistant to erythromycin, four were resistant to tetracycline, two strains were resistant to ampicillin, one strain was resistant to chloramphenicol and none was resistant for imipenem. For a quantitative test of the antibiogram, five antibiotics were selected in Etest ® strips (bioMérieux). All 15 strains were resistant to vancomycin, two for rifampicin, one for gentamicin and one for chloramphenicol. Regarding the virulence related genes, 19 genes from 49 tested were present in some strains. Results showed that five cultures showed the presence of the int gene, four cultures showed the presence of the ant(4')-Ia gene, three cultures were positive for vanC2, cpd and tdc, two cultures for vanA, tet(K), tet(S), ermA, bcrR, mur-2ed, asa1 and ccf, and one culture was positive for vanC1, ermB, aph(3')-IIIa, aac(6’)-le-aph(2”)-Ia, bcrB and hyl. After characterizing the virulent potential of the 15 BAL, these strains were evaluated for the technological potential for application in the dairy industry. All strains presented acidification capacity, reaching pH values between 0.73 and 2.11 in 24 hours: Lb. casei MRUV6 presented the highest acidification ability (pH 2.11 after 24 h). Ten strains were able to produce diacetyl at 37 °C, except by Lb. casei MSI1, Lb. harbinensis MSI3, Lb. fermentum SIVGL1, Lb. plantarum MLE5 and W. paramesenteroides MRUV3. All strains were able to produce exopolysaccharides, and only two strains presented proteolytic activity (Lb. casei MSI5 and W. paramesenteroides MSAV5). Based on this characterization, Lb. casei MRUV6 was selected for producing fermented milk, stored at 4 °C and 10 °C and monitored until 35 days of shelf life. Samples were subjected to phenotypical and molecular methods to quantify the presence of Lb. casei MRUV6 (conventional plating and RT-PCR, by checking the expression of gapdh, a housekeeping gene) and to verify the expression of bsh gene, related to resistance to bile salts (RT-PCR). Lb. casei MRUV6 population was stable during storage period at 4 and 10 °C at levels around 9.9 log CFU/g, and by monitoring the expression of gapdh gene. However, bsh gene was not expressed during storage period. The study demonstrated the potential use of the beneficial strain Lb. casei MRUV6 isolated from a dairy environment for the production of a fermented milk product, and its stability during storage at 4 and 10 °C. All isolates from the study presented beneficial characteristics, safety for use in food and technological potential for use in the dairy industry. In addition, they may further be subjected to further studies for in vivo evaluations and characterization as probiotics.