The Covalent Binding of Genistein to the Non-prosthetic-heme-moiety of Bovine Lactoperoxidase Leads to Enzymatic Inactivation

Objective Genistein, a major soy isoflavone metabolite （SIF）, inactivates oxidation activity of bovine lactoperoxidase （LPO）. Modification of the heme moiety of LPO by nitrogen-containing compounds has been shown to inactivate LPO. In contrast, SIF mediated inactivation of LPO does not involve a heme modification and the mechanism of SIF inhibition is poorly understood. Methods After inactivation of LPO by genistein in the presence of H202, trypsin-digested LPO peptide fragments were collected and analyzed by MALDI-TOF-MS to characterize the chemical binding of genistein（s） to LPO. Results The heme moiety of LPO was not modified by genistein. A covalent binding study showed that 3H-genistein bound to LPO with a ratio of ~12 to 1. After HPLC analysis and peak collection, trypsin-digested peptide fragments were analyzed by MALDI-TOF-MS. The 3H-genistein co-eluted peptide fragments （RT=24 min） were putatively identified as 1991VGYLDEEGVLDQNR214 with two bound genistein molecules or a genistein dimer （2 259 Da）, 486TPDNIDIWlGGNAEPMVER504 with two bound genistein molecules or a genistein dimer （2 663 Da）, and 161ARWLPAEYEDGLALPFGWTQR182 with three bound genistein molecules or a genistein trimer （3 060 Da）. The fragment with a mass of 2 792 Da （RT=36 min） was identified as 132CDENSPYR139 with three genistein molecules or a genistein trimer. Conclusions The results suggest that LPO was inactivated by irreversible covalent binding of genistein or genistein polymers to particular peptide fragments constituting regions of the outward domain. No genistein interaction with the prosthetic heme moiety of LPO was observed.

A Novel and Reliable Spectrophotometric Method for Determination of Lactoperoxidase Activity in Raw Milk

With 3, 3’5, 5’-tetramethylbenzidine（TMB） as the detection substrate, a reliable and highly selective method was established and optimized for the determination of Lactoperoxidase（LP） activity in raw milk. The method was based on the enzymatic reaction principle, where hydrogen peroxide oxidated TMB in the presence of LP. The optimized conditions of this assay system were obtained, consisting of 20 mmol · L-1 TMB solution, 0.6 mmol · L-1 hydrogen peroxide and 0.1 mol · L-1 Citric Acid（CA）/0.2 mol · L-1 disodium hydrogen phosphate（Na P） buffer（pH 4.8）. TMB detection method was applied to the analysis of LP in milk samples with a practical working concentration range from 2 to 14 mg · L-1. The intra-and inter-batch variation coefficients were all below 5%, indicating a good repeatability. Confirmation test between TMB method and 2, 2-azinobi（3-ethylbenzothiazoline-6-sulphonate） diammonium salt（ABTS） method was carried out, and the results of TMB assay were in accordance with that of ABTS method.

Preservation of Raw Camel Milk by Lactoperoxidase System Using Hydrogen Peroxide Producing Lactic Acid Bacteria

This study was conducted to investigate the effect of lactic acid bacteria (LAB) activated lactoperoxidase system (LPs) on keeping quality of raw camel milk at room temperature. Camel milk samples were collected from Errer valley, Babile district of eastern Ethiopia. The level of hydrogen peroxide (H2O2) for activation of LPs was optimized using different levels of exogenous H2O2. Strains of LAB (Lactococcus lactis 22333, Weissella confusa 22308, W. confusa 22282, W. confusa 22296, S. Infatarius 22279 and S. lutetiensis 22319) with H2O2 producing properties were evaluated, and W. confusa 22282 was selected as the best strain to produce H2O2. Storage stability of the milk samples was evaluated through the acidification curves, titratable acidity (TA), total bacterial count (TBC) and coliform counts (CC) at storage times of 0, 6, 12, 18, 24 and 48 hours. The LP activity and the inhibitory effect of activated LPs were evaluated by growing E. coli in pasteurized and boiled camel milk samples as contaminating agent. Results indicated that the W. confusa 22282 activated LPs generally showed significantly (P < 0.05) slower rates of acidification, lactic acid production and lower TBC and CC during the storage time compared to the non-activated sample. The H2O2 producing LAB and exogenous H2O2 activated LPs in pasteurized camel milk significantly reduced the growth of E. coli population compared to non-activated pasteurized milk. Overall, the result of acid production and microbial analysis indicated that the activation of LPs by H2O2 producing LAB (i.e. W. confusa 22282) maintained the storage stability of raw camel milk. Therefore, it can be concluded that the activation of LPs by biological method using H2O2 producing LAB can substitute the chemical activation method of LPs in camel milk.

Effects of Lactoperoxidase System Activation-Oregano Essential Oilor Chlorine on the Quality of Modified Atmosphere Packaged Fresh-Cut Iceberg Lettuce

The present work focused on the effects of the Modified Atmosphere Packaging (MAP) 1 (5% O2 and 10% CO2) or 2 (2% O2 and 5% CO2) and the previous addition of Lactoperoxidase System (LPS) and Oregano essential oil or chlorine washing on the quality of fresh-cut lettuce during refrigerated storage at +4?C. Our results showed the significant effect of this combined treatment on quality improvement during storage. Thus, mesophilic bacteria was reduced in treated samples compared to those untreated with number which not exceeded the critical of 5 × 107 UFC?g-1 (p 2 and CO2 levels created by both atmosphere were not significantly different between the two treatments (p > 0.05). Brightness of lettuce samples was significantly reduced during storage. Thereafter, the PCA data showed the effect of combined treatment on the preservation of hygienic, physico-chemical and sensory quality up to the 7th day of refrigerated storage of these treated samples. The results obtained draw attention to modified atmosphere packaging lettuce and the addition of bio-preservatives which could be an alternative of choice to replace chlorine to preserve the sanitary quality of green products.

Evaluation of the Use of Sodium Thiocyanate and Sodium Percarbonate in the Activation of the Lactoperoxidase System in the Conservation of Raw Milk without Refrigeration in the Ecuadorian Tropics

Milk production in Ecuador has enormous economic importance and large-, medium- and small-scale producers all participate in the market. There are multiple climatic regions, and dairy production is present in every one of them. High ambient temperatures in the Ecuadorian tropics represent a key challenge to the conservation of milk in the custody of smallholders. The objective of the present study was to evaluate the efficiency of the application of a chemical activator of the Lactoperoxidase System (LP-s) in the conservation of raw milk, at room temperature, in the Ecuadorian tropics. In the present study, sodium thiocyanate—0.36 g&#183L-1 of milk—and sodium percarbonate—1.36 g&#183L-1 of milk—as an activator of LP-s were used and the pH and microbiological characteristics (total coliforms, Staphylococcus aureus, total aerobes, molds and yeasts) of the milk at different storage times (0, 4 and 8 hours). The results obtained in the present study showed a significant difference between the two groups under study at 8 hours of storage at room temperature in all parameters (except yeasts where there was no growth in the two treatments), being relevant the significant decrease of the bacterial content. Thus the present study shows that the use of sodium thiocyanate and sodium percarbonate in the above described concentrations could be modulating the activation of LP-s that provides an efficient alternative for the conservation of the raw milk without refrigeration, improving the income for losses of the product and obtaining a raw material of good quality for sale or for further processing, mainly for small producers who do not have the economic resources to have refrigeration means for their product and who must transport their milk for considerably longer distances until they arrive at the collection centers or the processing plants for sale, thus showing that the method used in the present study is not only effective but also has a relatively low cost and easy application.

Development of a test strip for rapid detection of lactoperoxidase in raw milk

Traditional methods for detecting lactoperoxidase （LP） are complex and time-consuming, so a test strip was made based on the enzymatic reaction principle to enable quick and convenient detection of LP in raw milk. In this study 0.1 mol/L citric acid （CA）/0.2 mol/L disodium hydrogen phosphate （NAP） buffer solution （pH 5.0）, 22 mmol/L 3,3＇,5,5＇-tetramethylbenzidine （TMB）, 0.6 mmol/L hydrogen peroxide （H202）, and 0.5% Tween-20 or 0.3% cetyltri- methyl ammonium bromide （CTAB） were optimal for preparing a quick, sensitive, and accurate LP test strip. The coefficient of variation （CV） of the estimated LP concentrations ranged from 2.47% to 6.72% and the minimum LP concentration detected by the test strip was 1-2 mg/L. Estimates of active LP in sixteen raw milk samples obtained using the test strip or the TMB method showed a good correlation （c=0.9776）. So the test strip provides a quick, convenient, and accurate method for detecting the LP concentration of raw milk.

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