Physiological and Technological Properties of Probiotic Lacticaseibacillus rhamnosus GG Encapsulated with Alginate-Chitosan Mixture and Its Incorporation in Whole Milk.

This study developed and evaluated chitosan-sodium alginate capsules containing the probiotic Lacticaseibacillus rhamnosus GG using extrusion and emulsification techniques. The encapsulated L. rhamnosus GG cells were also evaluated for technological and probiotic-related physiological functionalities, as well as when incorporated in UHT and powdered milk. Extrusion (86.01 ± 1.26%) and emulsification (74.43 ± 1.41%) encapsulation techniques showed high encapsulation efficiency and high survival rates of L. rhamnosus GG during 28 days of refrigeration and room temperature storage, especially emulsification capsules (> 81%). The encapsulated L. rhamnosus GG cells showed high survival rates during exposure to simulated gastrointestinal conditions (72.65 ± 1.09-114.15 ± 0.44%). L. rhamnosus GG encapsulated by extrusion and emulsification performed satisfactorily in probiotic-related physiological (pH and bile salts tolerance) and technological properties (positive proteolytic activity, diacetyl and exopolysaccharides production, high NaCl tolerance (> 91%), besides having high heat tolerance (> 76%)). L. rhamnosus GG in extrusion and emulsification capsules had high survival rates (> 89%) and did not significantly affect physicochemical parameters in Ultra-High Temperature (UHT) and powdered milk during storage. The results demonstrate that L. rhamnosus GG can be successfully encapsulated with alginate-chitosan as a protective material through extrusion and emulsification techniques. UHT and powdered milk could serve as appropriate delivery systems to increase the intake of this encapsulated probiotic by consumers.

The effect of homogenisation pressure on the microstructure of milk during evaporation and drying: particle-size distribution, electronic scanning microscopy, water activity and isotherm.

Homogenisation is a widely used technique in manufacturing powdered milk with a direct impact on product solubility, and the homogenisation pressure is a central attribute of this process. We aimed to understand the effect of increasing homogenisation pressures (0/0, 15/5, and 75/5 MPa, 1st/2nd stages) on particle-size distribution during homogenised whole milk powder manufacture and rehydration of the final product. The fluid milk was thermally treated, homogenised, concentrated by rotary evaporation, and then dried using a spray dryer. Particle size (Dv90) was monitored at all stages of the manufacturing process. The final product (milk powder) was analysed using particle-size distribution, electronic scanning microscopy, water activity, and isotherms. The results demonstrated that increasing the homogenisation pressure leads to milk powder with smaller particle size when rehydrated (Dv90 values: 6.08, 1.48 and 0.64 µm for 0, 20 and 80 MPa, respectively). Furthermore, the volume (%) of the particles in the 'sub-micro' region (smaller than 1.0 µm) presented an inversely proportional profile to the homogenisation pressure (homogenised fluid milk: 86.1, 29.3 and 2.4%; concentrated milk: 86.1, 26.5 and 5.7%, and reconstituted milk powder: 84.2, 31.8 and 10.9%). Surprisingly, this pattern was not observed in the SPAN value (which corresponds to the width or range of the size distribution based on the volume). Additionally, the increase in the homogenisation pressure did not affect the sorption isotherm pattern. These results demonstrate that increasing the homogenisation pressure decreases the particle size of the reconstituted powdered milk, indicating the potential for future studies on how this phenomenon affects its physicochemical and final product properties.

Infrared spectroscopy combined with random forest to determine tylosin residues in powdered milk.

The contamination of milk by antibiotic residues is a worldwide health and food safety problem. There is a need to develop new methods for the rapid determination of antibiotic residues in milk. A method has been developed for determining tylosin residues directly in powdered milk using Fourier Transformed Infrared spectroscopy (FTIR). Tylosin is a broad-spectrum macrolide antibiotic. The spectra obtained were submitted to chemometric analysis to obtain a prediction model for tylosin concentration in powdered milk. Using the Boruta algorithm, the absorption bands related to the milk contamination by the antibiotic were identified. Random forest was shown to be adequate for the prediction of tylosin residues in milk at low concentrations (≤ 100 µg L-1) and the prediction model generated showed high correlation and determination coefficients (greater than 0.95). The proposed methodology proved to be efficient for the investigation of antibiotic residues in powdered milk.

Genetic and toxigenic diversity of Bacillus cereus group isolated from powdered foods.

Bacillus cereus is a human pathogenic bacterium that produces emetic and diarrheal foodborne diseases. This study evaluated the genetic and toxigenic diversity in B. cereus group isolates from powdered foods collected in public educational institutions, bakeries and powdered food companies located in Medellín, Colombia. B. cereus was detected in 35 of 305 (11%) powdered food samples and 52 B. cereus were isolated. The presence of ten toxin genes, hblCDAB, nheABC, cytK2, entFM and cesB, was evaluated in the isolates by multiplex PCR. The nheABC operon was found in all isolates (100%), hblCDAB in 22 (42%), hblCDA in 8 (15%) and hblCD in 3 (6%); the cytK2 gene was detected in 32 isolates (62%) and entFM in 32 (62%). Notably, the cesB gene was not detected. According to the presence of toxin genes, fifteen profiles were identified. The predominant toxigenic profile contained all toxin genes but cesB. A large genetic diversity was observed by GTG5 fingerprinting with 46 isolates grouped in seven clusters and the remaining six clustering individually. There was no relationship between toxigenic profiles and genetic clusters, but some genetic clusters seemed to be related to particular powdered food types. In general, the results evidenced high genetic and enterotoxigenic diversity among the B. cereus group isolates.

Industrial milk powder in bioassays for evaluation of cytotoxicity and genotoxicity / Leites industrializados, tipo em pó, frente a bioensaios de avaliação de citotoxicidade e genotoxicidade

Considering the widespread consumption of milk powder by the general population as well as the lack of studies on the toxicity of such industrialized foods, this study evaluated the cytotoxic and genotoxic potential of powdered milk from four reputed companies in the food market of Brazil and other South American countries. Milk samples were evaluated in root meristem cells of Allium cepa L., at concentrations of 0.065 and 0.13 g/mL, for 24 and 48 hours of exposure; and by means of cell viability in culture of cells of normal lineage, via MTT test, for 24 hours, at concentrations of 0.016; 0.032; 0.065 and 0.13g/mL. The concentration 0.13 g/mL was the one suggested for consumption in all milk packages evaluated in this study. In A. cepa, we observed that the milks, at both concentrations and at the two exposure times investigated, reduced the cellular proliferation of root meristems demonstrating a significant cytotoxicity. Furthermore, 0.13g/mL milks at the exposure time of 24h induced an expressive frequency of cellular alterations in the plant tissue, showing to be genotoxic. In the in vitro evaluation, three milks at 0.065 g/mL and all milks at 0.13 g/mL have significantly reduced cell viability, proving to be cytotoxic to the analyzed cell culture. Therefore, under the studied conditions, the powdered milks evaluated caused significant genetic instability to the cells of the test systems used.

Devido o amplo consumo de leite em pó pela população em geral, bem como, a carência de estudos sobre a toxicidade de tais alimentos industrializados, objetivou-se na presente pesquisa avaliar o potencial citotóxico e genotóxico de leites em pó provenientes de quatro empresas de reconhecida atuação no mercado de alimentos brasileiro e de outros países da América do sul. As amostras de leite foram avaliadas em células meristemáticas de raízes de Allium cepa L., nas concentrações 0,065 e 0,13g/mL, por 24 e 48 horas de exposição; e por meio da viabilidade celular em cultura de células de linhagem normal, via teste MTT, por 24 horas, nas concentrações 0,016; 0,032; 0,065 e 0,13g/mL. A concentração 0,13 mL/kg foi a sugerida para consumo em todas embalagens de leites avaliados neste estudo. Em A. cepa, verificou-se que os leites, nas duas concentrações e nos dois tempos de análise considerados, reduziram a proliferação celular dos meristemas de raízes demonstrando citotoxicidade significativa. Ainda, os leites na concentração 0,13g/mL induziram, no tempo de exposição 24h, frequência expressiva de alterações celulares ao tecido vegetal, mostrando-se genotóxicas. Na avaliação in vitro, três leites na concentração 0,065g/mL e todos na concentração 0,13g/mL reduziram significativamente a viabilidade celular mostrando-se citotóxicos a cultura de células analisada. Portanto, nas condições de estudo estabelecidas, os leites em pó avaliados causaram significativa instabilidade genética as células dos sistemas testes utilizados.

Calibration strategies for the direct determination of Ca, K, and Mg in commercial samples of powdered milk and solid dietary supplements using laser-induced breakdown spectroscopy (LIBS).

This study describes the application of laser-induced breakdown spectroscopy (LIBS) for the direct determination of Ca, K and Mg in powdered milk and solid dietary supplements. The following two calibration strategies were applied: (i) use of the samples to calculate calibration models (milk) and (ii) use of sample mixtures (supplements) to obtain a calibration curve. In both cases, reference values obtained from inductively coupled plasma optical emission spectroscopy (ICP OES) after acid digestion were used. The emission line selection from LIBS spectra was accomplished by analysing the regression coefficients of partial least squares (PLS) regression models, and wavelengths of 534.947, 766.490 and 285.213nm were chosen for Ca, K and Mg, respectively. In the case of the determination of Ca in supplements, it was necessary to perform a dilution (10-fold) of the standards and samples to minimize matrix interference. The average accuracy for powdered milk ranged from 60% to 168% for Ca, 77% to 152% for K and 76% to 131% for Mg. In the case of dietary supplements, standard error of prediction (SEP) varied from 295 (Mg) to 3782mgkg-1 (Ca). The proposed method presented an analytical frequency of around 60 samples per hour and the step of sample manipulation was drastically reduced, with no generation of toxic chemical residues.

Presencia de los genes de toxigenicidad nheA, nheBy nheC en cepas de Bacillus cereus aisladas de leches deshidratadas en Costa Rica / Detection of toxigenic genes nheA, nheB and nheC in Bacillus cereus strains isolated from powdered milk samples in Costa Rica

La leche en polvo es un producto de alto consumo humano que no precisa de ser conservado en frío, no obstante, diversos microorganismos pueden deteriorarlo. En la población costarricense, también se observa este alto consumo, por la facilidad del alimento para transporte, preparación y su costo competitivo. Bacillus cereus es una bacteria potencialmente patógena asociada a este tipo de producto, capaz de desarrollar toxinas dependiendo de la presencia o ausencia de los respectivos genes codificantes. En este estudio se determinó la presencia de los genes toxigénicos nheA, nheB y nheC en cepas de B. cereus aisladas de leche deshidratada vendida en el mercado nacional costarricense.Se examinaron cinco lotes diferentes, de diez marcas comerciales de leche en polvo distribuidos en el área metropolitana de San José Costa Rica. Se procedió a cuantificar B. cereus en las muestras de leche en polvo mediante la técnica de Número Más Probable (NMP) e identificar los aislamientos utilizando el equipo automatizado Vitek®. Adicionalmente, se determinó la presencia de los genes nheA, nheB y nheC mediante la técnica de PCR. La frecuencia de aislamiento de Bacillus cereus en las muestras de leche en polvo analizadas alcanzó un 50%, con cantidades que oscilaron entre 3 y >100 NMP/g. Se recuperaron 19 cepas de B. cereus aisladas, cinco fueron positivas para los tres genes toxigénicos, lo cual revela la presencia de B. cereus potencialmente toxigénico en leches deshidratadas del mercado nacional, lo que representa un riesgo para la salud pública.

Powdered milk is a frequently consumed product that does not need to be kept under cold conditions. Nevertheless, different microorganisms may contaminate it. Powdered milk is a highly consumed product by Costa Rican population, and Bacillus cereus is a potentially pathogenic bacteria associated to it, with the ability to develop toxins depending on the presence of the respective codifying genes. The aim of this study was to determine the presence of the toxigenic genes nheA, nheB and nheC from B. cereus strains, found in powdered milk sold at the Costa Rican national market. Five different lots of ten brands of powdered milk, distributed in the metropolitan area of San José, Costa Rica were analyzed. B cereus load was quantified using the Most Probable Number technique and identified using the Vitek® system. The presence of the toxigenic genes was determined using the PCR technique. The isolation frequency of this bacteria in the powdered milk samples analyzed reached 50%, with populations ranging from 3 to >100 MPN/g. Five out from nineteen strains were found positive for the three toxigenic genes, indicating contamination with potentially toxigenic B. cereus in powdered milk distributed in the national market, and an important risk for public health.

Uso de diluentes alternativos a baixas temperaturas na manutenção da qualidade espermática do sêmen suíno

Inúmeros trabalhos tem sido realizados com o propósito de melhorar a conservação do sêmen suíno, seja com o uso de diluentes ou faixas de temperaturas alternativas, ou ainda adicionando antioxidantes ou aminoácidos ao diluente. O presente estudo teve por objetivo definir um diluente e temperatura alternativa de conservação do sêmen suíno e avaliar os efeitos da inclusão de trolox ou betaína na manutenção da qualidade espermática. O sêmen de oito reprodutores foram coletados semanalmente através da técnica da mão enluvada e no laboratório foram submetidos às análises de vigor e motilidade no dia da coleta e durante o período de conservação (D0 a D4), avaliados quanto a vitalidade, integridade de acrossoma e teste hiposmótico no D0 e D4. Inicialmente os ejaculados foram conservados em três diluentes: Beltsville Thawing Solution (BTS), água de coco em pó (ACP) e leite em pó desnatado (LPD); e submetidos a duas temperaturas de armazenamento 17 e 10 °C, totalizando seis tratamentos. Definido o melhor tratamento, adicionou-se três diferentes concentrações de trolox (1000, 2000 e 3000 ?M/ 100 mL) e de betaína (1, 2 e 3g/ 100 mL). Dos diluentes testados, o LPD a 10 °C proporcionou uma melhor conservação da motilidade e vigor espermáticos, além de maior taxa de vitalidade e maior percentual de células com acrossoma íntegro. Diante desses resultados, o LPD a 10 °C apresentou-se como melhor diluente para a realização das etapas seguintes. As amostras acrescidas de diferentes concentrações de trolox não foram melhores quando comparadas ao tratamento controle em nenhuma das análises realizadas. Concentrações elevadas de trolox (2000 e 3000 ?M) apresentaram efeitos negativos sobre o vigor e a motilidade espermática. Quanto às amostras acrescidas de betaína, também não foram observados efeitos benéficos. A betaína provavelmente devido a seus efeitos osmóticos, reduziu o vigor e motilidade das amostras, esse efeito foi maior quando se elevou a concentração desse aminoácido. O LPD pode ser utilizado como diluente para o sêmen suíno acondicionado a 10 °C para ser utilizado por até 24 horas após a diluição. Não se pode comprovar os efeitos benéficos do trolox na conservação do sêmen suíno em meio à base de LPD. E a betaína apresentou efeitos negativos sobre a qualidade espermática quando adicionada ao diluente à base de LPD, em concentrações acima de 1% em temperatura de conservação a 10 °C.

Reacción en cadena de la polimerasa para la detección de Salmonella sp. en leche en polvo: optimización del método en 12 horas / Polimerase chain reaction technique for detecting Salmonella sp. in powdered milk: optimization of the method in 12 hours

Los métodos tradicionales para identificar Salmonella sp. se basan en el empleo de medios de cultivo que permiten la recuperación del microorganismo, el aislamiento en medios selectivos, la identificación bioquímica y caracterización serológica. Estos métodos son dispendiosos, tienen baja especificidad, baja sensibilidad y consumen mucho tiempo. El principal objetivo de este trabajo fue estandarizar y optimizar la técnica de PCR para detectar Salmonella sp. en 12 horas, a partir de ADN de cultivos puros y en muestras de leche en polvo, inoculadas intencionalmente con 200, 20 y 2 UFC/mL. Para la extracción del ADN se estudió la conveniencia de fenol:cloroformo:alcohol isoamílico y Chelex® 100. La temperatura de hibridización y las concentraciones de cloruro de magnesio, empleando un diseño factorial incompleto 6x7, permitieron establecer un límite de detección de hasta 10 pg de ADN en cultivos puros de Salmonella typhi. La PCR se basó en la exclusividad de los oligonucleótidos 139-141, los cuales amplificaron una banda de 284 pb para la identificación de género. Los resultados muestran que: (I) la adición de Novobiacina (45 mg/L) o de verde brillante (10 mg/L) como inhibidores de flora acompañante, después de las primeras tres horas del pre-enriquecimiento no selectivo de 6 horas, no influye significativamente en la recuperación de las células bacterianas; (II) obtener biomasa de la primera dilución en base 10 y emplear la técnica de fenol:cloroformo:alcohol isoamílico para la obtención de ADN, se pueden detectar 2 UFC/mL de Salmonella sp. en leche en polvo y que el tiempo de detección se reduce considerablemente...

The traditional methods to identify Salmonella sp. are based on the culture medium use that allows the recovery of the micro organism, isolation in selective media, biochemical and serologic characterization. These methods are tedious, have a low specificity and sensitivity and they generally consume a long time. The main objective of this study was to standardize and to optimize the PCR technique to detect Salmonella sp. in 12 hours, from DNA of pure cultures and from powdered milk samples, intentionally inoculated with 200, 20 and 2 CFU/mL. For the extraction of DNA, two methods were used: phenol:chloroform:isoamyl alcohol and chelex® 100. The optimization of the temperature of hibridización and the concentrations of Magnesium Chloride, using an incomplete factorial desing 6x7 allowed to establish a detection limit of up to 10 pg of DNA from pure cultures of Salmonella typhi. The PCR was based on the specificity of oligonucleotidos the 139-141, that amplified a band of 284 pb for the gender identification. The results show that: (I) the inhibitor addition of accompanying flora like Novobiocin (45 mg/L) or brilliant green (10 mg/L) as inhibitors of accompanying flora, after the first three hours in the nonselective pre-enrichment of 6 hours, does not significantly influence in the recovery of the bacterial cells, (II) when obtaining biomass of the first dilution in base 10 and using the phenol:chloroform:isoamyl alcohol technique for the extraction of DNA; can be detected 2 CFU/mL Salmonella s.p. from powdered milk and that the PCR technique reduces the time of test considerably...

Ocorrência de bactérias da espécie Bacillus cereus em amostras de leite em pó integral obtidas no comércio varejista da cidade de São Luís, MA / Occurrence of bacteria from the sort Bacillus cereus in samples of milk in integral powder obtained in the commerce retailer from the city of São Luís, MA

Com o objetivo de pesquisar bactérias da espécie Bacillus cereus em amostras de leite em pó obtidas no comércio varejista da cidade de São Luís-MA, foram analisadas 75 amostras do produto, conforme as metodologias recomendadas por Mossel et. Al. (1967) e Uboldi Eiroa (1982). Os resultados evidenciaram que 5 (6,66 por cento) das amostras apresentaram células vegetativas e/ou esporos e 16 (21,33 por cento) apresentaram somente esporos, sugestivas de estarem contaminadas por bactérias do grupo Bacillus cereus. Após a realização dos testes bioquímicos verificou-se que 13 (17, 33 por cento) das amostras forma positivas para bactérias do grupo B. cereus e 3 (4 por cento) do total das amostras analisadas apresentaram contaminação acima do valor máximo permitido pela legislação, portanto inadequadas para o consumo. Estes achados sugerem um produto que apresenta riscos para a Saúde Pública.(AU)

Occurrence of Bacillus cereus in powdered milk samples obtained in the retail trade of São Luís-MA city In order to research Bacillus cereus in powdered milk samples obtained at markets of São Luis city, 75 samples were analyzed according to Mossel et al. (1967) and Uboldi Eiroa (1982). The results showed that 5 (6.66%) of the samples presented vegetative cells and/or spores and 16 (21.33%) only spores, suggesting contamination by bacterias of the Bacillus cereus group. The bioquimical analyses demonstrated that 13 (17.33%) were positive for Bacillus cereus group and 3(4%) from all samples presented contamination above the maximum limit alIowed by legislation, therefore inadequated for consumption. Theses findings suggest that the product represent a risk I for public health. (AU)