The effect of monolaurin (0.35 mM) and lauric acid (5.0 mM) alone and in combination has been tested on growth of three cheesebome strains of Listeria monocytogenes and two strains of L. innocna. The Listeria spp. were grown in Tryptose Soya Yeast Glucose Broth (TSGYB) in shake culture at 10 °C and an initial pH of 7.0. The additives were dissolved in butter oil 10 % (w/v). Lauric acid (5.0 mM) increased the doubling time of the five Listeria spp. by 3 - 8 h at 10 °C. Monolaurin by itself was found to slightly increase or decrease the doubling time depending on the microbial strain. Monolaurin had an augmentative effect when combined with lauric acid in the presence of butter oil, where the doubling time increased between 10 to 15 h depending on the strain. Inhibition of Listeria spp. was seen in the model bi-phasic broth system. A model food system was developed to test the antimicrobial properties of lauric acid and monolaurin, where the fat soluble additives were dissolved in cream and milk with 3.6% (w/v) fat. The milk was reconstituted from skim milk and cream 40 % (w/v) fat containing lauric acid or monolaurin. This milk was used to make a soft-ripened cheese of the Brie Camembert type. Two strains of L. innocna were added to the reconstituted milk. During production of the soft-ripened cheese, a draining table was designed to comply with COSHH regulations so that the whey containing L. innocua could be removed and disposed of by heating for 30 min at 121 °C.In cheeses without lauric acid or monolaurin the population of L. innocua increased from 10[3] g[-1] to 10[7] cfu g[-1] on the surface of the cheese. The counts in the centre of the cheese were less at 10[5] cfu g[-1] after ripening for 28 d at 10 °C. Addition of 0.9 mM monolaurin reduced the count to 10[5] cfu g[-1] after 28 d ripening at 10 °C on the surface of the cheese. The effect of increasing the initial draining time at ambient temperature from 24 h to 48 h reduced the population to 0 after 28 d ripening at 10 °C. Unlike experiments in broth culture, addition of lauric acid changed the aroma of the Camembert-type cheese to give a blue cheese aroma. This was due to the conversion of lauric acid to a methyl ketone (2-undecanone) by the starter fungus Penicillium camembertii. Due to lack of stability of lauric acid in this system, lauric acid was omitted from the reconstituted milk in further experiments. During production of cheese, lactose was converted to mainly lactic acid by metabolism of the lactic acid starter. The presence of lactic acid combined with the added monolaurin resulted in a significant reduction in the population of L. innocua particularly when the draining time was increased from 24 h to 48 h. The unusual approach in this study was to dissolve the biocide in the non-aqueous phase, butter oil in the experiments in broth culture and in cream in the model cheese experiments. An untrained taste panel detected monolaurin (0.9 mM) in soft-ripened cheese. Some respondents liked the 'mature' taste whilst others described it as 'farmyard like'. In food systems the use of antimicrobials which result in an increase in the lag phase or a reduction in the overall population of pathogens, has a significant role in promoting the microbiological safety of a product which is eaten without further heat treatment.

Tesis por compendio / [ES] Los residuos de antibióticos en la leche constituyen un aspecto de gran importancia para la salud pública y pueden tener un efecto negativo en la industria láctea. Para proteger al consumidor se han establecido los Límites Máximos de Residuos (LMR) para diferentes antibióticos en la leche cruda. Sin embargo, la transferencia de estas sustancias de la leche al queso ha sido poco estudiada y, por lo tanto, se desconoce el impacto de la presencia de antibióticos sobre el proceso de elaboración y la seguridad del queso. El objetivo de la tesis ha sido evaluar la transferencia de los antibióticos más empleados en el ganado caprino lechero, desde la leche a quesos curados y frescos, así como estudiar el efecto sobre el proceso de fabricación y la calidad de los quesos durante la maduración. Para alcanzar el objetivo se han realizado elaboraciones de quesos curados (60 días) a partir de leche en presencia de diversos antibióticos (amoxicilina, bencilpenicilina, cloxacilina, eritromicina, ciprofloxacina, enrofloxacina y oxitetraciclina) a niveles admisibles (LMR) y fabricaciones de leche libre de antibiótico como referencia. Los resultados obtenidos sugieren que la mayor parte de los antibióticos en la leche no afectaron al proceso de fabricación del queso. Solamente la eritromicina y oxitetraciclina incrementaron el tiempo requerido en el proceso de elaboración. Respecto a las características del queso evaluadas a diferentes tiempos de maduración (0, 30 y 60 días), las pocas diferencias encontradas se refirieron a la concentración de ácidos grasos libres, el color y las propiedades de textura, pero en su mayoría no fueron detectadas en la evaluación sensorial. Sin embargo, cantidades variables de antibióticos, se transfirieron de la leche al queso (entre 7,4 y 68%). Las quinolonas y oxitetraciclina presentaron las más elevadas tasas de retención, así como persistencia a lo largo de la maduración, con altas concentraciones de quinolonas (enrofloxacina: 148±12 µg/kg; ciprofloxacina: 253±24 µg/kg) y de oxitetraciclina (20±5,7 µg/kg) después de los 60 días de curado. En el estudio de quesos frescos, se evaluó la presencia de residuos de medicamentos en la leche pasteurizada y en los quesos frescos a partir de leche de cabra con antibióticos (amoxicilina, bencilpenicilina, cloxacilina, neomicina, eritromicina, ciprofloxacina, enrofloxacina y oxitetraciclina) a niveles de seguridad (LMR). Los resultados mostraron que altas cantidades de antibióticos, entre el 71 y el 100% de la concentración inicial en la leche cruda, permanecieron en la leche pasteurizada y que fueron transferidas al queso en gran medida, con porcentajes de retención que oscilaron entre 37,5 y 75%. Con respecto a los márgenes de seguridad de estos productos, se calcularon teniendo en cuenta diferentes grupos de edades (niños, adolescentes y adultos), y los efectos negativos de estos antibióticos sobre la salud. Los resultados indicaron que el mínimo margen de seguridad en la leche pasteurizada lo presentaron la ciprofloxacina, enrofloxacina, y eritromicina para el grupo de niños. En relación al queso fresco, se obtuvo un elevado margen de seguridad para todos los antibióticos y grupos de edad considerados, lo que sugiere que este producto probablemente no tenga efectos negativos en la salud del consumidor. Es importante enfatizar que concentraciones relativamente altas de antibióticos podrían permanecer en la leche de cabra pasteurizada y productos relacionados, como los quesos frescos y de corta maduración. La presencia de estos antibióticos podría contribuir al desarrollo y propagación de la resistencia a los antimicrobianos que actualmente constituye un grave problema a nivel mundial. Los resultados de este trabajo podrían servir a las autoridades de salud pública para evaluar si los sistemas actuales de control de antibióticos en la leche y los productos lácteos son adecuados o deberían ser revisados. / [CA] Els residus d'antibiòtics en la llet animal constitueixen un aspecte de gran importància per a la salut pública, i poden tindre un efecte negatiu en la indústria làctia. Per a protegir al consumidor s'han establit els Límits Màxims de Residus (LMR) per a diferents medicaments veterinaris en la llet crua. No obstant això, la transferència d'estes substàncies de la llet al formatge ha sigut poc estudiada i, per tant, es desconeix el impacte de la presència de antibiòtics sobre el procés d'elaboració i la seguretat del formatge. L'objectiu de la tesi ha sigut avaluar la transferència dels antibiòtics més empleats en el bestiar caprí lleter, des de la llet a formatges curats i frescos, així com l'efecte sobre el procés de fabricació i la qualitat dels formatges durant la maduració. Per a aconseguir l'objectiu s'han realitzat elaboracions de formatges curats (60 dies) a partir de llet en presència de diversos antibiòtics (amoxicilina, bencilpenicilina, cloxacilina, eritromicina, ciprofloxacina, enrofloxacina i oxitetraciclina) a nivells admissibles (LMR) i fabricacions de llet lliure d'antibiòtic (control) com a referència. Els resultats obtinguts suggerixen que la major part dels antibiòtics en la llet no van afectar el procés de fabricació del formatge. Només l'eritromicina i l'oxitetraciclina van incrementar el temps requerit en el procés d'elaboració del formatge. Respecte a les característiques del formatge avaluades a diferents temps de maduració (0, 30 i 60 dies), les poques diferències trobades es van referir a la concentració d'àcids greixos lliures, el color i les propietats de textura, però majoritàriament no van ser detectades en l'avaluació sensorial. No obstant això, quantitats variables d'antibiòtics, es van transferir de la llet al formatge (entre 7,4 i 68%). Les quinolones i l'oxitetraciclina van presentar les més elevades taxes de retenció, així com persistència al llarg de la maduració, amb altes concentracions de quinolones (enrofloxacina: 148±12 µg/kg; ciprofloxacina: 253±24 µg/kg) i d'oxitetraciclina (20±5,7 µg / kg) després dels 60 dies de curació. En l'estudi de formatges frescos, es va avaluar la presència de residus de medicaments en la llet pasteuritzada i en els formatges frescos a partir de llet de cabra amb antibiòtics (amoxicilina, bencilpenicilina, cloxacilina, neomicina, eritromicina, ciprofloxacina, enrofloxacina i oxitetraciclina) a nivells de seguretat (LMR). Els resultats van mostrar que altes quantitats d'antibiòtics, entre el 71 i el 100% de la concentració inicial en la llet crua, van romandre en la llet pasteuritzada i que van ser transferides al formatge en gran manera, amb percentatges de retenció que van oscil·lar entre 37,5 i 75%. Respecte als marges de seguretat d'estos productes, es van calcular tenint en compte diferent grups d'edats (xiquets, adolescents i adults), i els efectes negatius d'estos antibiòtics sobre la salut. Els resultats van indicar que el mínim marge en la llet pasteuritzada ho van presentar la ciprofloxacina, la enrofloxacina, i l'eritromicina per al grup de xiquets. En relació al formatge fresc, es va obtindre un elevat marge de seguretat per a tots els antibiòtics i grups d'edat considerats, la qual cosa suggereix que este producte probablement no tinga efectes negatius en la salut del consumidor. És important emfatitzar que concentracions relativament altes d'antibiòtics podrien romandre en la llet de cabra pasteuritzada i productes relacionats, com els formatges frescos i de curta maduració. La presència d'estos antibiòtics podria contribuir al desenrotllament i propagació de la resistència als antimicrobians que actualment constitueix un greu problema a nivell mundial. Els resultats d'este treball podrien servir les autoritats de salut pública per a avaluar si els sistemes actuals de control d'antibiòtics en la llet i els productes lactis són adequats o haurien de ser revisats. / [EN] Antibiotic residues in milk are a great concern for public health, and they have negative implications for the dairy industry. To guarantee the safety of milk and derivates, Maximum Residues Limits (MRL) for different veterinary drugs in raw milk have been established. However, the transfer of these substances from milk to cheese, has been poorly studied and, therefore, the impact of the use of raw milk containing antibiotics on the cheese-making and the cheese safety is thus far unknown. The aim of this thesis was to evaluate the transfer of the most widely used antibiotics used in dairy goats, from milk to matured and fresh cheese, as well as their effect on the cheese-making process and the quality characteristics of the cheeses during ripening. To this end, antibiotic-free milk was spiked individually with seven veterinary drugs (amoxicillin, benzylpenicillin, cloxacillin, erythromycin, ciprofloxacin, enrofloxacin and oxytetracycline) and used to make mature Tronchón cheeses, which were analyzed at different ripening times (0, 30, and 60 days) to determine antibiotic residues, pH, chemical composition, proteolytic and lipolytic activities, color, and textural properties. The cheese-making process was unaffected by the presence of most antibiotics in milk. Only erythromycin and oxytetracycline significantly increased the time required for cheese production. Regarding cheese characteristics, the few differences found were related to the free fatty acid concentration, color and textural properties, which remained mostly undetected by the sensory analysis panelists. However, variable amounts of antibiotics, ranging from 7.4 to 68 %, were transferred from milk spiked with antibiotics, to cheese. Oxytetracycline and quinolones presented the highest retention rates as well as persistence along maturation, with high concentrations of quinolones (enrofloxacin: 148±12 µg/kg, ciprofloxacin: 253±24 µg/kg) and oxytetracycline residues (20±5.7 µg/kg) in cheeses after 60 days of ripening. In the study of fresh cheeses was evaluated the presence of drug residues in pasteurized fluid milk and fresh cheeses obtained from goat milk containing antibiotics (amoxicillin, benzylpenicillin, cloxacillin, neomycin, erythromycin, ciprofloxacin, enrofloxacin and oxytetracycline) at safety levels (MRLs). The safety margin of these dairy products for consumers was also evaluated. Results showed that high amounts of antibiotics, between 71-100% of the initial concentration in raw milk, remained in pasteurized goat milk and were transferred to cheese to a high extent, with retention percentages ranging from 37.5 to 75%. Regarding the safety margin of goat milk products, calculated taking into account different age groups (children, teenagers and adults), and the published negative effects of such antibiotics on consumer health, results indicate that the minimum safety margin of pasteurized milk was obtained for ciprofloxacin, enrofloxacin and erythromycin in the group of children. Regarding fresh cheese, an elevated safety margin was obtained for all antibiotics and age groups considered. It is important to emphasize that relatively high concentrations of antibiotics could remain in pasteurized fluid goat milk and related products such as fresh cheese and cheeses of a short ripening period. The presence of these antibiotics in dairy products might contribute to the development and spread of antimicrobial resistance, which is considered an important public health concern worldwide. The result of this research could serve the public health authorities to assess if current control systems of antibiotics in milk and dairy products are adequate or have to be revised. / Por último, quiero dar las gracias a la educación pública que permite que la economía no sea una barrera para la formación y al “Programa de ayudas a la investigación” (FPI-2014) de la Universitat Politècnica de Valencia, que me concedió la oportunidad de realizar está investigación durante los 4 años de trabajo. / Quintanilla Vázquez, PG. (2019). Effect of antibiotics in goat milk on the manufacture and characteristics of cheese [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/125701 / TESIS / Compendio

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-02-09T11:58:42Z No. of bitstreams: 1 arquivo total.pdf: 1438788 bytes, checksum: 9590b3fb5151d31119e2920baea83d58 (MD5) / Made available in DSpace on 2017-02-09T11:58:42Z (GMT). No. of bitstreams: 1 arquivo total.pdf: 1438788 bytes, checksum: 9590b3fb5151d31119e2920baea83d58 (MD5) Previous issue date: 2016-06-20 / Minimally ripened cheeses, a category that includes the coalho cheese, have physicochemical characteristics that facilitate the survival and growth of pathogenic bacteria associated with foodborne illness outbreaks. Use of mathematical models in the predictive microbiology allows to estimate the effect of biotic and abiotic conditions on microbial growth, and hence the possible risk associated with the presence and increase of the population of a specific pathogen in a particular food. Considering these aspects, the present study aimed to: (i) assess the coalho cheese as a potential substrate for the growth of the pathogenic bacteria Escherichia coli, Listeria monocytogenes, Staphylococcus aureus and Salmonella spp. in function of temperature, pH and water activity (aw) found in commercial samples of this type of product; and (ii) performing primary modeling growth of different strains of E. coli in coalho cheese samples stored under low temperature. The estimates of the growth kinetics parameter (maximum growth rate - Grmax, log UFC/g/h) in function of the combination of the values (percentile 20, 50, 75 and 90) for temperature, pH and aw were generated by ComBase Predictor. The growth kinetics (Grmax) of E. coli strains in coalho cheese (10 °C) was assessed by using the primary model of Baranyi and Roberts (1994), available in spreadsheet DMFit 3.5 (ComBase). The largest Grmax values as a function of different combinations of temperature, pH and aw were observed for L. monocytogenes (Grmax 0.01 to 0.07 log CFU/g/h), Salmonella (Grmax 0.01 to 0.04 log CFU/g/h) and S. aureus (Grmax <0.01 to 0.05 log CFU/g/h). E. coli Grmax values varied from 0.01 to 0.03 log CFU/g/h. Overall, the Grmax values increased as the temperature increased in cheese samples, regardless of the aw and pH values. The estimated Grmax values for the E. coli strains when inoculated into coalho cheese ranged from 0.01 to 0.03 log CFU/g/h, respectively. High R2 values (≥ 0.97) were obtained to the growth curves of all E. coli strains tested. The estimates of the growth kinetics parameters in function of the values of temperature, aw and pH tested, as well as those observed in artificially contaminaed cheese samples showed that the coalho cheese is characterized as favorable substrate for the survival and growth of E. coli, L. monocytogenes, Salmonella spp. and S. aureus, which may pose a risk to consumers’ health if contaminated with these bacteria during production, transportation or marketing. / Os queijos de baixa maturação, categoria que inclui o queijo de coalho, possuem características físico-químicas que podem facilitar a sobrevivência e crescimento de bactérias patogênicas associadas a surtos de doenças transmitidas por alimentos. O emprego de modelos matemáticos na microbiologia preditiva permite estimar o efeito das condições bióticas e abióticas sobre o crescimento microbiano, e, consequentemente, o possível risco associado à presença e evolução da população de um patógeno em um alimento determinado. Considerando estes aspectos, o presente estudo teve como objetivos: (i) avaliar o queijo de coalho como substrato potencial para o crescimento das bactérias patogênicas Escherichia coli, Listeria monocytogenes, Staphylococcus aureus e Salmonella spp. em função de valores de temperatura, pH e atividade de água (aa) encontrados em amostras comerciais deste tipo de produto; e (ii) realizar modelagem primária do crescimento de diferentes cepas de E. coli em amostras de queijo de coalho armazenadas sob baixa temperatura. As estimativas dos parâmetros de cinética de crescimento (taxa máxima de crescimento – Grmax, log UFC/g/) em função da combinação dos valores (Percentil 20, 50, 75 e 90) de temperatura, pH e aa foram geradas pelo ComBase Predictor. A cinética de crescimento das cepas de E. coli em queijo de coalho (10 °C) foi avaliada por meio do Grmax utilizando o modelo primário de Baranyi e Roberts (1994), disponível na planilha do DMFit 3.5 (ComBase). Os maiores valores de Grmax em função das diferentes combinações de temperatura, pH e aa foram verificados para L. monocytogenes (Grmax 0,01 – 0,07 log UFC/g/h), Salmonella spp. (Grmax 0,01 – 0,04 log UFC/g/h) e S. aureus (Grmax 0,01 – 0,05 log UFC/g/h). Os valores de Grmax para E. coli variaram de 0,01 a 0,02 log UFC/g/h. De forma geral, os valores de Grmax aumentaram, proporcionalmente a temperatura nas amostras de queijo, a despeito dos valores de aa e pH. Os valores estimados de Grmax das cepas de E. coli quando inoculadas em queijo de coalho variaram de 0,01 a 0,03 log UFC/g/h. Foram obtidos elevados valores de R2 (≥ 0,97) para as curvas de crescimento de todas as cepas de E. coli testadas. As estimativas dos parâmetros de cinética de crescimento em função dos valores de temperatura, aa e pH testados, bem como àqueles verificados em amostras artificialmente contaminadas, mostram que o queijo de coalho se caracteriza como substrato favorável para a sobrevivência e crescimento de E. coli, L. monocytogenes, Salmonella spp. e S. aureus, podendo representar um risco a saúde aos consumidores caso seja contaminado com estas bactérias durante a sua produção, transporte ou comercialização.