In vivo assay to identify bacteria with ß-glucosidase activity

Background: ß-Glucosidase assay is performed with purified or semipurified enzymes extracted from cell lysis. However, in screening studies, to find bacteria with ß-glucosidase activity among many tested bacteria, a fast method without cell lysis is desirable. In that objective, we report an in vivo ß-glucosidase assay as a fast method to find a ß-glucosidase producer strain. Results: The method consists in growing the strains for testing in a medium supplemented with the artificial substrate p-nitrophenyl-ß-glucopyranoside (pNPG). The presence of ß-glucosidases converts the substrate to p-nitrophenol (pNP), a molecule that can be easily measured in the supernatant spectrophotometrically at 405 nm. The assay was evaluated using two Bifidobacterium strains: Bifidobacterium longum B7254 strain that lacks ß-glucosidase activity and Bifidobacterium pseudocatenulatum B7003 strain that shows ß-glucosidase activity. The addition of sodium carbonate during pNP measurement increases the sensitivity of pNP detection and avoids the masking of absorbance by the culture medium. Furthermore, we show that pNP is a stable enzymatic product, not metabolized by bacteria, but with an inhibitory effect on cell growth. The ß-glucosidase activity was measured as units of enzyme per gram per minute per dry cell weight. This method also allowed the identification of Lactobacillus strains with higher ß-glucosidase activity among several lactobacillus species. Conclusion: This in vivo ß-glucosidase assay can be used as an enzymatic test on living cells without cell disruption. The method is simple, quantitative, and recommended, especially in studies screening for bacteria not only with ß-glucosidase activity but also with high ß-glucosidase activity.

El efecto antihipertensivo de las leches fermentadas / The antihypertensive effect of fermented milks

Existe una gran variedad de leches fermentadas con bacterias lácticas, con propiedades que promueven la salud. Recientemente se ha comunicado que las proteínas de los alimentos pueden, además, ejercer otras funciones in vivo, por medio de sus péptidos con actividad biológica. Estos péptidos se encuentran encriptados dentro de la estructura primaria de las proteínas y pueden ser liberados por fermentación de la leche, hidrólisis enzimática, o bien durante el tránsito gastrointestinal. Las funciones que presentan son diversas, ya que pueden actuar en diferentes sistemas del cuerpo humano: el cardiovascular, el digestivo, el endocrino, el inmune y el nervioso. Los péptidos bioactivos que presentan un efecto en el sistema cardiovascular (antihipertensivo, antitrombótico, antioxidante o hipocolesterolémico) pueden reducir los factores de riesgo para la manifestación de enfermedades crónicas y ayudar a mejorar la salud humana. Los péptidos bioactivos más estudiados son aquellos que ejercen un efecto antihipertensivo a través de la inhibición de la enzima convertidora de angiotensina (ACE). Este documento se enfoca en la producción de péptidos antihipertensivos inhibidores de la ACE en leches fermentadas, en su historia, y en las pruebas in vivo realizadas en ratas y en humanos, donde se ha demostrado su efecto hipotensor.

There is a great variety of fermented milks containing lactic acid bacteria that present health-promoting properties. Milk proteins are hydrolyzed by the proteolytic system of these microorganisms producing peptides which may also perform other functions in vivo. These peptides are encrypted within the primary structure of proteins and can be released through food processing, either by milk fermentation or enzymatic hydrolysis during gastrointestinal transit. They perform different activities, since they act in the cardiovascular, digestive, endocrine, immune and nervous systems. Bioactive peptides that have an antihypertensive, antithrombotic, antioxidant and hypocholesterolemic effect on the cardiovascular system can reduce the risk factors for chronic disease manifestation and help improve human health. Most studied bioactive peptides are those which exert an antihypertensive effect by inhibiting the angiotensin-converting enzyme (ACE). Recently, the study of these peptides has focused on the implementation of tests to prove that they have an effect on health. This paper focuses on the production of ACEinhibitory antihypertensive peptides from fermented milks, its history, production and in vivo tests on rats and humans, on which its hypotensive effect has been shown.