Screening of anti-inflammatory milk proteins and peptides using luminol-enhanced chemiluminescence assay

vokBE

Chemiluminescence of non-differentiated THP-1 promocytes

Human leukemic THP-1 promonocytes are widely used as a model for peripheral blood monocytes. However, superoxide production during respiratory burst (RB) of non-differentiated THP-1 (nd-THP-1) cells is very low. Here we present a rapid and low-cost method for measuring the chemiluminescence (CL) of opsonized zymosan (OZ) induced RB which allows detection of Escherichia coli lipopolysaccharide (LPS) induced priming of nd-THP-1 cells on the basis of CL reaction kinetics. Maximum CL intensity obtained was 2.20+- 0.25 and 1.30+- 0.11 relative light units, while CL peak time was achieved at 18.1+- 2.6 and 28.7+- 1.3 min in primed and non-primed cells, respectively. The priming of nd-THP-1 cells with LPS evoked tzpical TNF-[alfa] and IL-6 production. We tested the effects of bovine lactoferrin and protein fractions from Lactobacillus helveticus BGRA43 fermented milk for potential anti-inflammatory effects on LPS primed nd-THP-1 cells. Four fractions were found to inhibit the OZ-induced CL in a dose-dependent manner (IC50 3-30 g/mL), while lactoferrin inhibited CL to a lesser extent (IC50 270? g/mL). These results suggest that measuring CL response of nd-THP-1 cells can serve as a method for screening anti-inflammatory compounds which could be used in reducing the risk of phagocyte-mediated inflammatory diseases

Chemiluminescence of non-differentiated THP-1 promonocytes : developing an assay for screening anti-inflammatory milk proteins and peptides

v2011o

Colostrum of Healthy Slovenian Mothers: Microbiota Composition and Bacteriocin Gene Prevalence

<div><p>Microbial communities inhabiting the breast milk microenvironment are essential in supporting mammary gland health in lactating women and in providing gut-colonizing bacterial 'inoculum' for their infants’ gastro-intestinal development. Bacterial DNA was extracted from colostrum samples of 45 healthy Slovenian mothers. Characteristics of the communities in the samples were assessed by polymerase chain reaction (PCR) coupled with denaturing gradient gel electrophoresis (DGGE) and by quantitative real-time PCR (qPCR). PCR screening for the prevalence of bacteriocin genes was performed on DNA of culturable and total colostrum bacteria. DGGE profiling revealed the presence of <i>Staphylococcus</i> and <i>Gemella</i> in most of the samples and exposed 4 clusters based on the abundance of 3 bands: <i>Staphylococcus epidermidis/Gemella</i>, <i>Streptococcus oralis/pneumonia</i> and <i>Streptococcus salivarius</i>. <i>Bacilli</i> represented the largest proportion of the communities. High prevalence in samples at relatively low quantities was confirmed by qPCR for enterobacteria (100%), <i>Clostridia</i> (95.6%), <i>Bacteroides-Prevotella</i> group (62.2%) and bifidobacteria (53.3%). Bacterial quantities (genome equivalents ml^-1) varied greatly among the samples; <i>Staphylococcus epidermidis</i> and staphylococci varied in the range of 4 logs, streptococci and all bacteria varied in the range of 2 logs, and other researched groups varied in the range of 1 log. The quantity of most bacterial groups was correlated with the amount of all bacteria. The majority of the genus <i>Staphylococcus</i> was represented by the species <i>Staphylococcus epidermidis</i> (on average 61%), and their abundances were linearly correlated. Determinants of salivaricin A, salivaricin B, streptin and cytolysin were found in single samples. This work provides knowledge on the colostrum microbial community composition of healthy lactating Slovenian mothers and reports bacteriocin gene prevalence.</p></div

Correlation of <i>Staphylococcus</i> and <i>Staphylococcus epidermidis</i> in 29 colostrum samples in which both groups were detected.

<p>The data are presented as copies (GE) ml^-1.</p