published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

M.Tech. / Natural occurring components in human breast milk, cow milk and soy milk have shown anticarcinogenic abilities. The human breast milk protein, -lactalbumin, was found to induce apoptosis in cancer cells, embryonic cells and rapidly growing cells, when converted from its native form to a partial denatured apoptotic-inducing form. Moreover, radiation may cause irreversible changes of protein conformation at the molecular level. Native -lactalbumin is one protein that has shown a decrease in aromatic amino acid concentration and the formation of high and low molecular weight fractions when exposed to high doses of ionizing radiation. The effect of human breast milk, cow milk, soy milk and galactose (positive control) on SNO, A549 cancer cells and normal lymphocytes were investigated. Human breast milk was irradiated with low doses of Co60 ionizing radiation (0.1Gy, 1.0Gy and 5.0Gy) in order to establish the effect of these doses on the apoptotic-inducing ability of human breast milk. The techniques used included, Trypan blue dye exclusion (cell viability), haematoxylin and eosin stain (cell morphology), modified comet assay (halo) (DNA damage) and flow cytometry (apoptosis and necrosis). Findings showed that human breast milk, irradiated human breast milk and galactose induced apoptosis in the SNO, A549 cells and lymphocytes. The cell viability, cell morphology and DNA fragmentation patterns of irradiated human breast milk were similar to that of non-irradiated human breast milk, although the flow cytometry results did not correlate. Cow and soy milk did not induce apoptosis in the SNO, A549 cells and lymphocytes. The modified comet assay (halo) detected DNA damage as apoptotic or necrotic cells. A clear distinction could not be made between the two cell populations using this assay. Flow Cytometry discriminated and quantified apoptotic cells and necrotic/late apoptotic cells using Annexin V and Propidium Iodide (PI), respectively.

Selected public health criteria of pasteurised milk available to the consumer from milk -shops in a selected area of Pretoria compared with a national distributor's milk were evaluated. Pasteurised milk samples were obtained from five randomly selected milk-shops in the northwestern part of Pretoria over a six-week period from June to August 1998. Milk from a wellknown national distributor was also obtained from three supermarkets in the same area during the same time period to act as the control milk. Mean total aerobic bacterial counts, coliform counts and psychrotrophic bacterial counts were determined. The presence of Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Salmonella species, staphylococcal enterotoxins, inhibitory substances, alkaline phosphatase and somatic cells in milk were also determined. Of the 135 milk samples purchased from milk-shops, 87% were not fit for human consumption on the basis of the minimum standards prescribed in the Foodstuffs, Cosmetics and Disinfectants Act (Act 54 of 1972). In contrast, 100% of the 79 control milk samples passed all the safety criteria laid down in the Act. x Milk-shop milk quality varied between milk-shops and between sampling days. All milk-shop milk was sold as having been pasteurised, yet 38.5% of milk samples purchased failed the alkaline phosphatase test, indicating that they had not been pasteurised correctly or were contaminated with raw milk. The total aerobic plate counts were generally high for all milkshop milk samples ranging from 1.0 x 102 to 2.7 X 107 CFU/mQ with a median value of 41000 CFU/mQ, whereas for the control milk it ranged from 7.0 x 102 to 8.7 X 103 CFU/mQ, with a median value of 2 200 CFU/mQ. Coliform counts varied from 0 to 3.4 X 104 per mQ in milk-shop milk, with 68% of samples having counts lower than 20 coliforms/mQ, which is the maximum number allowed when the Petrifilm method of counting is used. Coliforms could not be detected in 1 mQ of control milk samples. E. coli was detected in 1 mQ of 17% of milkshop milk, 95% of which originated from milk which was alkaline phosphatase positive. Salmonella spp. could not be detected in 1 mQ in any of the E. coli-positive milk tested. Psychrotrophic bacterial counts done after pre-incubation of milk-shop milk were extremely high, and ranged from 3 x lOS to 2.2 X 108 CFU/mQ, with a median value of 2.4 x 107 CFU/mQ. In Europe the psychrotrophic count may not be greater than 100 000 CFU/mQ. None of the milk-shop milk passed this European standard, whereas 98.7% of the milk obtained from the national distributor fell within the prescribed parameters. S. aureus was isolated from 54 (40%) milk-shop milk samples, and four (7.8%) of 51 isolates tested produced staphylococcal enterotoxins A (SEA), B (SEB), D (SED) or a combination. Control milk did not contain any S. aureus and 15 milk samples tested for the enterotoxin gave a negative result. All control milk was negative for inhibitory substances, but these were detected in 54.1 % of milk-shop milk. Somatic cell counts varied between 1.2 x 104 and 1.6 x 106 cells/mQ in the milk-shop milk, with a median count of 4.2 x 105 cells/mt Only 18.7% of samples had counts above the legal limit of 500 000 cells/mQ. The national distributor's milk always had counts less than 150 000 cells/mQ. xi The results showed that milk-shop milk differed significantly (p < 0.05) from the national distributor's milk, and that the quality of milk purchased from milk-shop outlets was generally of a poor bacteriological quality. The presence of inhibitory substances, and the isolation ofE. coli and S. aureus (some of which were able to produce enterotoxins) indicated potentially unsafe milk and posed a serious public health risk to consumers. / Dissertation (MMed Vet (Hyg))--University of Pretoria, 2000. / Production Animal Studies / unrestricted

Call number: LD2668 .T4 1961 C65

Plasmin is the principal endogenous protease in bovine milk. Distribution of overall proteolytic potential among milk fractions was determined using Coumarin Substrate as a synthetic substrate. Casein containing fractions had a higher amidolytic potential. However, preparation of casein by acid treatment produced increased dissociation of plasminogen and plasmin from casein. The variable results obtained from milks of different cows could be due in part to the influence of inhibitors and activators of the fibrinolytic system present in milk. We have shown, for the first time, the occurrence of α₂-M in bovine skimmilk (using SRID) at a level (using ELISA) of 1.54 +/- 0.91 mg/ml. This inhibitor appeared to primarily associate with the acid whey fraction. A high level (< 1 mg/ml) of α₂-M was also detected in human skimmilk. The other major fibrinolytic inhibitor, α₂-AP, as well as the complex formed between this inhibitor and plasmin were also shown to occur in human milk. We used a coupled colorimetric assay to demonstrate the occurrence of a fibrin-independent plasminogen activator (similar to u-PA) in bovine skimmilk. The occurrence of a u-PA like activator in bovine milk was further confirmed in co-polymerized gel electrophoresis. Moreover, u-PA could also be detected in a sample of human skimmilk. However, the electrophoretic gel patterns also contained additional zones of clearing which may be due to the occurrence of other activators in bovine milk. These plasminogen activators may be fragments of u-PA, or t-PA (shown to occur in sow milk) which retain catalytic activity. The occurrence of such high levels of α₂-M (~ 4% of the total protein) and plasminogen activators may be of tremendous significance to the dairy industry, as they may not only influence plasmin-mediated proteolysis of milk proteins, but may also interfere with the action of milk clotting enzymes. / Master of Science

Thesis (MPA)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The aim of this thesis was to determine in terms of policy analysis why the bacteriological quality of milk in a particular area did not comply with statutory provisions during production. The policy issue is very complex due to the diversity of conditions that exist during the milking process, which may impact on the levels of bacteria in milk. Considering this diversity, various problematic conditions are likely to cause the bacteriological contamination of milk. Milk is regarded as safe for human consumption when laboratory quality tests indicate its bacteriological suitability. Intervention on the basis of milk control should assure that milk is produced and distributed within acceptable bacteriological levels. The ad hoc milk safety strategy of the West Coast District Municipality (WCDM) was found to be inappropriate for the effective control of hygienic conditions during milk production. The existing strategy does not provide for suitable programmes to deal with this complex issue of policy and the suggestion is that it should be reviewed. Judged on the basis of success factors, a decline in milk bacteriological quality for the WCDM area revealed factors that were not effectively controlled during milk production. During the research period the hygienic quality of milk from the WCDM area was regulated and judged primarily by means of laboratory quality tests. Food safety requires much more than merely relying on quality tests. It is imperative that milk laboratory analyses should in all instances be harmonised by means of an evaluation of hygiene and sanitary conditions during production. How the problem situation with regard to milk quality is approached defines the implementation of strategy. The Environmental Health Practitioners (EHPs) of the WCDM have been implementing the requirements of applicable legislation, but each according to an individual approach. Such modi operandi provide for inconsistency in the execution of any policy. Implementing policy should be relevant, specific and adequate and should be target based. Consequently, the modification of policy is required to the extent of changing the approach when it does not comply with the afore-mentioned criteria, strategy or policy governing milk safety. More could be done by the WCDM to manage problems at milking sheds that is associated with the bacteriological quality of milk. Management control strategy is perceived to be inadequate and coupled with a lack of willingness to tackle the crux of the problem. Implementing legislative policy necessitate an objective approach coupled with an appropriate strategy. The findings of the empirical analysis together with a discussion of the implementation of the WCDM milk control strategy explains the shortcomings that was experienced in that regard. Therefore, an improved policy framework is proposed for controlling milk safety at milking sheds. In addition, a strategic framework is also proposed to administer the milk safety policy by way of programmes. / AFRIKAANSE OPSOMMING: Die doel van hierdie tesis was om, in terme van openbare beleidsanalise, te bepaal waarom die bakteriologiese kwaliteit van melk in ’n spesifieke gebied tydens die produksie daarvan nie aan die wetlike standaarde voldoen het nie. Hierdie beleidsvraagstuk word as kompleks beskou weens die uiteenlopende toestande wat tydens die melkproses ontstaan en wat ‘n impak op die bakterievlakke van melk kan hê. Inaggenome van hierdie diversiteit kan die besmetting van melk waarskynlik deur verskeie probleemtoestande veroorsaak word. Melk word geag veilig te wees vir menslike gebruik wanneer laboratoriumkwaliteittoetse op die bakteriologiese geskiktheid daarvan aandui. Ingryping op grond van melkbeheer moet verseker dat melk binne die aanvaarde bakteriologiese vlakke geproduseer en verskaf word. Die ad hoc melkveiligheidstrategie van die Weskus Distrik se Munisipale (WDM) gebied is onvoldoende bevind vir die effektiewe beheer van higiëniese toestande tydens melkproduksie. Hierdie strategie maak nie voorsiening vir geskikte programme om hierdie komplekse beleidsvraagstuk te hanteer nie en die hersiening daarvan word voorgestel. Geoordeel aan die hand van suksesfaktore, dui ‘n afname in die kwaliteit van melk vir die WDM gebied op faktore wat tydens melkproduksie nie effektief beheer was nie. Die higiëniese kwaliteit van melk binne die WDM gebied was tydens die navorsingstydperk primêr deur middel van laboratoriumkwaliteittoetse gereguleer en geoordeel. Voedselveiligheid vereis dat daar egter op meer as kwaliteittoetse gesteun word. Dit maak dit noodsaaklik dat die laboratoriumanalises van melk in alle gevalle aan die hand van evaluering van higiëniese en sanitêre toestande tydens melkproduksie geharmoniseer word. Die manier waarmee die probleemsituasie met betrekking tot melkkwaliteit benader word, definieer die implementering van strategie. Die Omgewingsgesondheidspraktisyns (OGPs) van die WDM het die voorskrifte van die toepaslike wetgewing geïmplementeer, maar elkeen volgens ‘n eie benadering. ‘n Sodanige modus operandi maak voorsiening vir die inkonsekwente uitvoering van enige beleid. Die implementering van beleid moet relevant, spesifiek, geskik en moet doelgerig wees. Gevolglik waar voornoemde kriteria, strategie of beleid wat melkveiligheid aangaan nie nagekom word nie, noodsaak dit derhalwe ‘n aanpassing van beleid vir soverre die benadering tot strategie vir melkveiligheid geraak word. Die WDM kan meer doen om probleme wat verband hou met die bakteriologiese kwaliteit van melk by melkstalle te bestuur. Die beheerstrategie van die bestuur word as onvanpas beskou en dit gaan gepaard met ‘n gebrek aan ywer om die essensie van die probleem aan te spreek. Die inwerkingstelling van wetgewing noodsaak ’n objektiewe benadering gepaardgaande met ’n geskikte strategie vir die uitvoering van beleid. Die bevindinge van die empiriese analise tesame met ’n bespreking aangaande die implementering van die WDM melkbeheerstrategie bied ’n verduideliking van die tekortkominge in daardie verband aan. Derhalwe word ’n verbeterde beleidsraamwerk voorgestel om die melkveiligheid by melkstalle te beheer. Daarbenewens word ’n strategiese raamwerk om die melkveiligheidbeleid deur middel van programme te uit te voer, ook voorgestel.

Submitted by (edna.saturno@ufrpe.br) on 2016-07-25T14:39:33Z No. of bitstreams: 1 Pedro Paulo Feitosa de Albuquerque.pdf: 705656 bytes, checksum: 3b387f372f7375364f614a52bba0c4df (MD5) / Made available in DSpace on 2016-07-25T14:39:33Z (GMT). No. of bitstreams: 1 Pedro Paulo Feitosa de Albuquerque.pdf: 705656 bytes, checksum: 3b387f372f7375364f614a52bba0c4df (MD5) Previous issue date: 2014-02-25 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The aim of the present study was to detect the IS900 region of MAP in bovine milk samples using the Real Time Polymerase Chain Reaction (qPCR) and conventional PCR, as well as to study the agreement between the tests used. In total, 121 bovine milk samples were collected from properties considered as a focus of MAP in the micro-region of Garanhuns, Pernambuco, Brazil. Of the 121 milk samples analyzed, it was possible to detect the DNA of MAP in 20 samples (16.52%) using conventional PCR and in 34 samples (28.09%) using qPCR. The DNA of the agent was detected on all of the six properties studied, ranging from 10.00% to 23.81% in the conventional PCR and from 10.00% to 36.84% in the qPCR. The agreement between qPCR and conventional PCR was moderate (Kappa =0.53; χ2 = 38.08; p<0.000). The sensitivity and specificity of conventional PCR in relation to qPCR was 50% and 96.6%, respectively. Based on these results, it was possible to conclude that the IS900 region of MAP is present in bovine milk in the micro-region of Garanhuns, Pernambuco. It was also possible to conclude that the qPCR technique used in the present study was more sensitive than conventional PCR in terms of detecting these bacteria in milk samples. / Objetivou-se com este estudo detectar a região IS900 do MAP em amostras de leite bovino utilizando-se as técnicas de Reação em Cadeia da Polimerase em Tempo Real (qPCR) e a PCR convencional, e realizar um estudo de concordância entre os testes utilizados. Foram coletadas 121 amostras de leite bovino de propriedades consideradas foco para o MAP pertencentes à microrregião de Garanhuns, Pernambuco, Brasil. Das 121 amostras de leite analisadas foi possível detectar o DNA do MAP em 20 (16,52%) amostras na PCR convencional e em 34 (28,09%) na qPCR. O DNA do agente foi detectado em todas as seis propriedades estudadas, variando de 10,00% a 23,81% na PCR convencional e de 10,00% a 36,84% na qPCR. A concordância entre a qPCR e PCR foi moderada (Kappa =0,53; χ2 = 38,08; p<0,000) e a sensibilidade e especificidade da PCR convencional com relação a qPCR foi de 50% e 96,6%, respectivamente. Conclui-se que o DNA da região IS900 do MAP está presente no leite bovino na região estudada e que a qPCR é uma técnica sensível e rápida para detecção desse agente em amostras de leite.

Three experiments were conducted to determine the relationship of milk fat secretion to concentration and ratio of rumen fermentation products, blood metabolic parameters, ration characteristics, and certain enzymic activity involved in fatty acid synthesis. In Experiment I, 21 cows in mid-lactation were randomized according to milk production to (1) roughage ration (~22% CF) restricted to NRC; (2) concentrate ration (~12% CF) ad libitum; or (3) concentrate ration (~12% CF) restricted to NRC and fed for 30 days. Ad libitum concentrate showed higher dry matter intake (DMI), body weight (BW), milk production than restricted concentrate. The opposite was true for fat test. Concentrate rations (ad libitum and restricted) when compared to roughage showed higher values for DMI, BW, milk production, serum glucose, molar proportion propionate and valerate and lower values for fat test and molar proportion acetate. In Experiment II, 24 cows 180 d. post parturition, were assigned to four rations based on anticipated parturition. Rations were: (1) roughage ad libitum (~20% CF); (2) concentrate ad libitum (~10% CF); (3) concentrate restricted (~10% CF) to NRC; and (4) normal ration (~14% CF) ad libitum. Cows on ad libitum and restricted concentrate consumed less dry matter, acid detergent fiber, neutral detergent fiber and crude protein than control cows (roughage and normal). Cows fed control rations gained more weight than concentrate fed cows. No difference was found for milk production. Milk protein was higher for control cows. Concentrate cows had lower fat test than control cows. Cows on all rations decreased fat test up to week three or four but by week eight or nine, concentrate fed cows had decreased to their lowest fat test. Energy restriction improved milk fat secretion compared to ad libitum intake after the eighth week. Molar proportion VFA favored propionate for concentrate cows and was similar for restricted and ad libitum concentrate. Total ruminal VFA concentration was higher for ad libitum concentrate cows and those cows showed slightly higher glucose and smaller blood acetate compared to control. Twelve cows in mid-lactation were fed the rations of Experiment II to determine the effect of available energy intake on activity of key enzymes of fatty acid synthesis. Activity of 6-phosphogluconate dehydrogenase was not different among rations. Mammary tissue showed from five to six times higher activity than adipose tissue. Fat tissue activity tended to be higher for concentrate rations compared to roughage. No differences among rations were found for glucose 6-phosphate dehydrogenase nor isocitrate dehydrogenase. Mammary tissue was 21 to 28 times more active than adipose tissue for latter enzyme. Fatty acid synthetase showed no difference in activity due to rations, but mammary tissue was seven to ten times more active than adipose tissue. / Ph. D.

Orientador: Guilherme de Paula Nogueira / Coorientadora:Elisa Helena Giglio Ponsano / Banca: Carlos Antonio de Miranda Bomfim / Banca:Maria Carolina Villani Miguel / Resumo: No Brasil, a produção de leite tem importância social, bem como econômica, pois está presente em número significativo de propriedades com mão-de-obra familiar. Entretanto, baixas produtividade e qualidade ainda são características do leite brasileiro. O objetivo do trabalho foi identificar fatores que influenciam a produtividade, a contagem bacteriana total (CBT), a contagem de células somáticas (CCS) do leite e verificar se o produto está adequado às normas da legislação brasileira. A pesquisa se baseou em entrevista estruturada e coleta de leite das propriedades participantes (n=45), pertencentes à microrregião de Birigui-SP, para análise. Foram realizadas regressão multivariada, análise de variância multivariada, com pós-teste de Tukey, e determinou-se coeficiente de correlação entre variáveis. A produtividade média observada foi de 6.274,9±4.981,4 L/ha/ano. Observou-se que a produtividade foi influenciada positivamente pela base da alimentação volumosa e produção de leite por dia de intervalo entre partos. Não houve diferença significativa entre a produtividade das propriedades que utilizavam pastejo rotativo e fornecimento de volumoso no cocho, mas estas foram superiores àquelas que utilizavam pastejo contínuo ou pastejo alternado. O fato de se possuir tanque refrigerador individual favoreceu a menor CBT. Observou- se mais alta CCS no leite de produtores que realizavam o "California Mastitis Test" (CMT) com maior frequência. O leite de apenas quatro produtores (8,9%) apr... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: In Brazil, dairy production has social, as well as economic, importance, because it is present in significant number of farms with family labor. However, low productivity and low quality still are features of the Brazilian product. The aim of this study was to evaluate the influence of factors on milk productivity, total bacterial count (TBC) and somatic cell count (SCC) and verify if the product is suited to the requirements of Brazilian law. The research was based on data from structured interview and analyses of milk samples collected from each farm (n=45), which belongs to Birigui-SP microregion. Multivariate regression and multivariate analysis of variance, with Tukey's post-test, was performed and correlation coefficient was determined. The observed average productivity was 6,274.9±4,981.4 L/ha/year. Roughage feeding base and milk production per day of calving interval influenced positively the productivity. There was no difference in productivity between farms that used short-duration grazing system and roughage feeding in the trough, both had higher productivity than farms that used continuous grazing system and alternated grazing system. Milk of producers that had individual cooler tank had a lower TBC. It was observed that milk of producers that performed California Mastitis Test (CMT) frequently had a higher SCC. Milk from four farms (8.9%) was suitable for all analyzed parameters. / Mestre

Submitted by João Lucas Cânovas Delfino null (jlcdelfino@gmail.com) on 2016-08-01T19:51:03Z No. of bitstreams: 1 Delfino, João Lucas Cânovas - Dissertação.pdf: 1039434 bytes, checksum: 341be358ddbbff25bf44e43f0a660f28 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-08-03T14:46:19Z (GMT) No. of bitstreams: 1 delfino_jlc_me_araca.pdf: 1039434 bytes, checksum: 341be358ddbbff25bf44e43f0a660f28 (MD5) / Made available in DSpace on 2016-08-03T14:46:19Z (GMT). No. of bitstreams: 1 delfino_jlc_me_araca.pdf: 1039434 bytes, checksum: 341be358ddbbff25bf44e43f0a660f28 (MD5) Previous issue date: 2016-07-14 / No Brasil, a produção de leite tem importância social, bem como econômica, pois está presente em número significativo de propriedades com mão-de-obra familiar. Entretanto, baixas produtividade e qualidade ainda são características do leite brasileiro. O objetivo do trabalho foi identificar fatores que influenciam a produtividade, a contagem bacteriana total (CBT), a contagem de células somáticas (CCS) do leite e verificar se o produto está adequado às normas da legislação brasileira. A pesquisa se baseou em entrevista estruturada e coleta de leite das propriedades participantes (n=45), pertencentes à microrregião de Birigui-SP, para análise. Foram realizadas regressão multivariada, análise de variância multivariada, com pós-teste de Tukey, e determinou-se coeficiente de correlação entre variáveis. A produtividade média observada foi de 6.274,9±4.981,4 L/ha/ano. Observou-se que a produtividade foi influenciada positivamente pela base da alimentação volumosa e produção de leite por dia de intervalo entre partos. Não houve diferença significativa entre a produtividade das propriedades que utilizavam pastejo rotativo e fornecimento de volumoso no cocho, mas estas foram superiores àquelas que utilizavam pastejo contínuo ou pastejo alternado. O fato de se possuir tanque refrigerador individual favoreceu a menor CBT. Observou- se mais alta CCS no leite de produtores que realizavam o “California Mastitis Test” (CMT) com maior frequência. O leite de apenas quatro produtores (8,9%) apresentou-se adequado para todos os parâmetros analisados. / In Brazil, dairy production has social, as well as economic, importance, because it is present in significant number of farms with family labor. However, low productivity and low quality still are features of the Brazilian product. The aim of this study was to evaluate the influence of factors on milk productivity, total bacterial count (TBC) and somatic cell count (SCC) and verify if the product is suited to the requirements of Brazilian law. The research was based on data from structured interview and analyses of milk samples collected from each farm (n=45), which belongs to Birigui-SP microregion. Multivariate regression and multivariate analysis of variance, with Tukey’s post-test, was performed and correlation coefficient was determined. The observed average productivity was 6,274.9±4,981.4 L/ha/year. Roughage feeding base and milk production per day of calving interval influenced positively the productivity. There was no difference in productivity between farms that used short-duration grazing system and roughage feeding in the trough, both had higher productivity than farms that used continuous grazing system and alternated grazing system. Milk of producers that had individual cooler tank had a lower TBC. It was observed that milk of producers that performed California Mastitis Test (CMT) frequently had a higher SCC. Milk from four farms (8.9%) was suitable for all analyzed parameters.