Rapid microbiological detection method based on ultrasonic instrumentation

Los métodos de detección rápida de microorganismos se están convirtiendo en una herramienta esencial para el control de calidad en el área de la biotecnología, como es el caso de las industrias de alimentos y productos farmacéuticos y bioquímicos. En este escenario, el objetivo de esta tesis doctoral es desarrollar una técnica de inspección rápida de microoganismos basada en ultrasonidos. La hipótesis propuesta es que la combinación de un dispositivo ultrasónico de medida y un medio líquido diseñado específicamente para producir y atrapar burbujas, pueden constituir la base de un método sensible y rápido de detección de contaminaciones microbianas. La técnica presentada es efectiva para bacterias catalasa-positivas y se basa en la hidrólisis del peróxido de hidrógeno inducida por la catalasa. El resultado de esta reacción es un medio con una creciente concentración de burbujas. Tal medio ha sido estudiado y modelado desde el punto de vista de la propagación ultrasónica. Las propiedades deducidas a partir del análisis cinemático de la enzima se han utilizado para evaluar el método como técnica de inspección microbiana. En esta tesis, se han investigado aspectos teóricos y experimentales de la hidrólisis del peróxido de hidrógeno. Ello ha permitido describir cuantitativamente y comprender el fenómeno de la detección de microorganismos catalasa-positivos mediante la medida de parámetros ultrasónicos. Más concretamente, los experimentos realizados muestran cómo el oxígeno que aparece en forma de burbujas queda atrapado mediante el uso de un gel sobre base de agar. Este gel fue diseñado y preparado especialmente para esta aplicación. A lo largo del proceso de hidrólisis del peróxido de hidrógeno, se midió la atenuación de la onda y el “backscattering” producidos por las burbujas, utilizando una técnica de pulso-eco. Ha sido posible detectar una actividad de la catalasa de hasta 0.001 unidades/ml. Por otra parte, este estudio muestra que por medio del método propuesto, se puede lograr una detección microbiana para concentraciones de 105 células/ml en un periodo de tiempo corto, del orden de unos pocos minutos. Estos resultados suponen una mejora significativa de tres órdenes de magnitud en comparación con otros métodos de detección por ultrasonidos. Además, la sensibilidad es competitiva con modernos y rápidos métodos microbiológicos como la detección de ATP por bioluminiscencia. Pero sobre todo, este trabajo muestra una metodología para el desarrollo de nuevas técnicas de detección rápida de bacterias basadas en ultrasonidos. ABSTRACT In an industrial scenario where rapid microbiological methods are becoming essential tools for quality control in the biotechnological area such as food, pharmaceutical and biochemical; the objective of the work presented in this doctoral thesis is to develop a rapid microorganism inspection technique based on ultrasounds. It is proposed that the combination of an ultrasonic measuring device with a specially designed liquid medium, able to produce and trap bubbles could constitute the basis of a sensitive and rapid detection method for microbial contaminations. The proposed technique is effective on catalase positive microorganisms. Well-known catalase induced hydrogen peroxide hydrolysis is the fundamental of the developed method. The physical consequence of the catalase induced hydrogen peroxide hydrolysis is an increasingly bubbly liquid medium. Such medium has been studied and modeled from the point of view of ultrasonic propagation. Properties deduced from enzyme kinematics analysis have been extrapolated to investigate the method as a microbial inspection technique. In this thesis, theoretical and experimental aspects of the hydrogen peroxide hydrolysis were analyzed in order to quantitatively describe and understand the catalase positive microorganism detection by means of ultrasonic measurements. More concretely, experiments performed show how the produced oxygen in form of bubbles is trapped using the new gel medium based on agar, which was specially designed for this application. Ultrasonic attenuation and backscattering is measured in this medium using a pulse-echo technique along the hydrogen peroxide hydrolysis process. Catalase enzymatic activity was detected down to 0.001 units/ml. Moreover, this study shows that by means of the proposed method, microbial detection can be achieved down to 105 cells/ml in a short time period of the order of few minutes. These results suppose a significant improvement of three orders of magnitude compared to other ultrasonic detection methods for microorganisms. In addition, the sensitivity reached is competitive with modern rapid microbiological methods such as ATP detection by bioluminescence. But above all, this work points out a way to proceed for developing new rapid microbial detection techniques based on ultrasound.

Effects of the correction of particle microbial contamination and particle transit model in the rumen on in situ protein evaluation of grass hays.

Effects of considering the particle comminution rate -kc- in addition to particle rumen outflow -kp- and the ruminal microbial contamination on estimates of by-pass and intestinal digestibility of DM, organic matter and crude protein were examined in perennial ryegrass and oat hays. By-pass kc-kp-based values of amino acids were also determined. This study was performed using particle transit, in situ and 15N techniques on three rumen and duodenum-cannulated wethers. The above estimates were determined using composite samples from rumen-incubated residues representative of feed by-pass. Considering the comminution rate, kc, modified the contribution of the incubated residues to these samples in both hays and revealed a higher microbial contamination, consistently in oat hay and only as a tendency for crude protein in ryegrass hay. Not considering kc or rumen microbial contamination overvalued by-pass and intestinal digestibility in both hays. Therefore, non-microbial-corrected kp-based values of intestinal digested crude protein were overestimated as compared with corrected and kc-kp-based values in ryegrass hay -17.4 vs 4.40%- and in oat hay -5.73 vs 0.19%-. Both factors should be considered to obtain accurate in situ estimates in grasses, as the protein value of grasses is very conditioned by the microbial synthesis derived from their ruminal fermentation. Consistent overvaluations of amino acid by-pass due to not correcting microbial contamination were detected in both hays, with large variable errors among amino acids. A similar degradation pattern of amino acids was recorded in both hays. Cysteine, methionine, leucine and valine were the most degradation-resistant amino acids.

Effects of the ruminal comminution rate and microbial contamination of particles on accuracy of in situ estimates of ruminal degradability and intestinal digestibility of feedstuffs.

Effects of considering the comminution rate -kc- and the correction of microbial contamination -using 15N techniques- of particles in the rumen on estimates of ruminally undegraded fractions and their intestinal digestibility were examined generating composite samples -from rumen-incubated residues- representative of the undegraded feed rumen outflow. The study used sunflower meal -SFM- and Italian ryegrass hay -RGH- and three rumen and duodenum cannulated wethers fed with a 40:60 RGH to concentrate diet -75 g DM/kgBW0.75-. Transit studies up to the duodenum with Yb-SFM and Eu-RGH marked samples showed higher kc values -/h- in SFM than in RGH -0.577 vs. 0.0892, p = 0.034-, whereas similar values occurred for the rumen passage rate -kp-. Estimates of ruminally undegraded and intestinal digestibility of all tested fractions decreased when kc was considered and also applying microbial correction. Thus, microbial uncorrected kp-based proportions of intestinal digested undegraded crude protein overestimated those corrected and kc-kp-based by 39% in SFM -0.146 vs. 0.105- and 761% in RGH -0.373 vs. 0.0433-. Results show that both kc and microbial contamination correction should be considered to obtain accurate in situ estimates in grasses, whereas in protein concentrates not considering kc is an important source of error.

VIS/NIR spectral signature for the identification of peanut contamination of powder foods

Visible-near infrared reflectance spectra are proposed for the characterization of IRMM 481 peanuts variety in comparison to powder food materials: wheat flour, milk and cocoa. Multidimensional analysis of reflectance spectra of powder samples shows a specific NIR band centred at 1200 nm that identifies peanut compared to the rest of food ingredients, regardless compaction level and temperature. Spectral range of 400-1000 nm is not robust for identification of blanched peanut. The visible range has shown to be reliable for the identification of pre-treatment and processing of unknown commercial peanut samples. A spectral index is proposed based on the combination of three wavelengths around 1200 nm that is 100% robust against pre-treatment (raw or blanched) and roasting (various temperatures and treatment duration).

NIR hyperspectral imaging for detection of nut contamination

NIR hyperspectral imaging for detection of nut contamination