Etienne Dako

Autolytic properties and aminopeptidase activities of lactic acid bacteria

Abstract The ability of nine lactic acid bacteria ( Lactococcus , Lactobacillus and Pediococcus ) with potential in cheese maturation to lysis under various physicochemical conditions was studied. The lysis efficiency under various pH and NaCl concentrations was evaluated by measuring aminopeptidase and dipeptidylaminopeptidase activity released in cell supernatant. Two Lb. casei (L2A, 137) exhibited high peptidase activities and were more sensitive to lysis. Our results indicate clearly that autolysis is optimal with Lactobacillus casei casei (L2A) and Lactobacillus casei pseudoplantarum (137) at pH5.2 with 2–4% NaCl concentration and at 30 °C.

Autolysis of lactic acid bacteria: Impact on flavour development in cheese

Abstract Early autolysis of cheese related microorganisms in cheese should potentially release the intracellular enzymes into the cheese matrix. Such an event should lead to a more rapid development of flavour in cheese. The autolytic properties of several cheese related bacteria as well as the different factors affecting the process are discussed. Enzyme release during cell autolysis is also considered.

The Problems Associated with Enzyme Purification

This chapter aims to highlight the difficulties encountered during the purification of native cellular and membrane-bound enzymes from whole cell extracts. There are many reasons for wanting to purify enzymes, such as to fully characterize them or to mass produce them for commercial purposes. Regardless of the reason for wanting to purify an enzyme, the general extraction and purification procedures are essentially the same. However, depending on the properties of the enzyme, certain modifications of the methods must be considered regarding specific problems that can be encountered throughout the process, such as enzyme insolubility and loss of enzyme activity. Although several classic and more modern methods are available to solve these kinds of challenges, the enzyme purification step remains a major challenge for any method of extraction used.

A new approach for the purification and characterisation of PA49.5, the main prebiotic of Lactococcus lactis subsp. cremoris

The main autolysin PA49.5, an enzyme that hydrolyzes or destroys the components of a biological endogenous cell or a tissue, was purified 3045 times from the homogenate of a whole cell extract of Lactococcus lactis subsp. cremoris ATCC 9596 (Mc5), with a recovery yield of 52%. The purification of the protein was carried out through a micro-purification technique using SDS-BigCHAP polyacrylamide gel electrophoresis and concentrated with a Microcon-10 filtration system. SDS-polyacrylamide gel electrophoresis of the purified enzyme confirmed the presence of only one band having a molecular weight of 49.5 kDa. In view of its insolubility, PA49.5 contained in the cell extract precipitate was solubilized in the presence of 0.1% (w/v) of BigCHAP, a non-ionic detergent. Higher concentrations of this detergent completely inhibited the activity of solubilized PA49.5 or prevented its solubilization. The optimal pH and temperature for PA49.5 enzymatic activity are 7.5 and 45 degrees C respectively. In addition 0.1% or less of PA49.5 significantly increased Mc5 lysis. We observed 55% more lysis with 0.25 mug of purified PA49.5 compared to the control. Gas chromatography analysis of the components of the crude cell extract, of the precipitate and of the supernatant indicates the presence of at least 6 fatty acids. The long-chained fatty acids (e.g. C18:0 and C18:3) detected represent 81.65% of the precipitate from which PA49.5 was purified. Of these two acids, the C18:0 (stearic acid) alone represents 47.40% of the precipitate. Mc5 releases proteins at the beginning (major peak) and at the end (moderate peak) of the exponential stage of growth. Analysis by denaturing polyacrylamide gel electrophoresis with Mc5 cell walls incorporated as the autolysins substrate identified a band corresponding to PA49.5 in the second peak of protein secretion.

Virulence, serotype and phylogenetic groups of diarrhoeagenic Escherichia coli isolated during digestive infections in Abidjan, Côte d'Ivoire

The virulence, serotype and phylogenetic traits of diarrhoeagenic Escherichia coli were detected in 502 strains isolated during digestive infections. Molecular detection of the target virulence genes, rfb gene of operon O and phylogenetic grouping genes Chua, yjaA and TSPE4.C2 was performed. Prevalence of strains harbouring virulent genes was 7.8%. The virulent genes eaeA, bfp, stx2, st1, lt, aggA, east1, ipaH, ial, cnf1 and afa were detected. EAEC (36%) and both EPEC and ATEC (25.6%) are the most detected pathovars (p<0.05). STEC (5.1%), NFEC (7.7) and DAEC (7.7) are less represented. Serogroups are overall diversified (89%), however, serogroups O157, O103 and O86, previously known to be associated with virulence were revealed. Most of the E. coli pathovars (53%) belonged to phylogenetic group A and in decreasing importance order, to D (23.5%), B1 (11.7%) and B2 (11.7%) groups. The study shows a diversified population of intestinal strains (84.6%), with a low phenotypic and phylogenetic link lower (p<0.05). Due to the great diversity of pathotypes, continuous monitoring should be implemented to identify risk factors and major pathways of contamination that help defining strategies to reduce infections associated with E. coli. Keywords: Escherichia coli , virulence gene, serogroup, phylogenetic group, diversity African Journal of Biotechnology , Vol. 13(9), pp. 998-1008, 26 February, 2014

Characterization of virulence potential of Pseudomonas aeruginosa isolated from bovine meat, fresh fish, and smoked fish

Pseudomonas aeruginosa owns a variability of virulence factors. These factors can increase bacterial pathogenicity and infection severity. Despite the importance of knowledge about them, these factors are not more characterized at level of strains derived from local food products. This study aimed to characterize the virulence potential of P. aeruginosa isolated from various animal products. Several structural and virulence genes of P. aeruginosa including lasB, exoS, algD, plcH, pilB, exoU, and nan1 were detected by polymerase chain reaction (PCR) on 204 strains of P. aeruginosa. They were isolated from bovine meat (122), fresh fish (49), and smoked fish (33). The 16S rRNA gene was detected on 91.1% of the presumptive strains as Pseudomonas. The rpoB gene showed that 99.5% of the strains were P. aeruginosa. The lasB gene (89.2%) was the most frequently detected (p < 0.05). In decreasing importance order, exoS (86.8%), algD (72.1%), plcH (72.1%), pilB (40.2%), and exoU (2.5%) were detected. The lasB gene was detected in all strains of P. aeruginosa serogroups O11 and O16. The prevalence of algD, exoS, and exoU genes in these strains varied from 51.2% to 87.4%. The simultaneous determination of serogroups and virulence factors is of interest for the efficacy of surveillance of infections associated with P. aeruginosa.

Factors associated with cessation of exclusive breastfeeding before six months among mothers in the Miramichi region (Canada)

The study was designed to investigate the cessation factors associated with the exclusive breastfeeding in the Miramichi region. It was a descriptive cross-sectional and analytical study, which took place from 2011 to 2014. A total of 294 mothers and their 294 children aged from 0 to 42 months constituted the study population.The data collection concerning the breastfeeding in infant was done by questionnaire addressed to the parents. A multivariate statistical analysis was realized to identify the determinants of the exclusive breastfeeding cessation. The adjusted Odds Ratio (ORa) and their interval were calculated. The average age of mothers and the duration of breastfeeding were respectively 26.86 ± 6.44 years and 19.22 ± 3.28 months. In multivariate analysis, the early cessation of exclusive breastfeeding is associated with the maternal age (adjusted odds ratio (ORa) = 1.2), the level of education of the mother (ORa) = 1.6), the mode of feeding (ORa) = 1.9), and the duration of breastfeeding (ORa) = 2.2); which are predictors of childhood obesity in Miramichi. We have identified modifiable factors in order to improve the practice of exclusive breastfeeding in Miramichi Factors associated with cessation of exclusive breastfeeding before six months among mothers in the Miramichi region (Canada)

Extraction and Solubilization Methods Suitable for Proteomic Analysis of Common Bean Seed of Ivory Coast

The extraction and solubilization of proteins from seeds of Phaseolus vulgarisms for two-dimension polyacrylamide gel electrophoresis (2D-PAGE) and analysis by mass spectrometry are very sensitive procedures. In this study, we used two methods of extraction and solubilization of proteins, the urea/thiourea method and the trichloroacetic acid (TCA)/acetone precipitation method, in order to determine their effectiveness in separating proteins from bean seeds by 2D-PAGE. In both methods, proteins were well separated by 2D PAGE with minor variations in the protein pattern. These two extraction methods showed that it was possible to separate hundreds of very resolvent proteins by 2D electrophoresis. A protein spot was selected on the 2D-PAGE gel, digested with trypsin and analyzed by mass spectrometry (LCMS/ MS). The results suggest that thiourea/urea and TCA methods were effective and reliable for the extraction and solubilization for 2D analysis of proteins from seeds of Phaseolus vulgaris .

Antimicrobial effects of capsaicinoids and lecithin on the growth of food borne pathogenic bacteria

impact of the addition of solid CO2 on olive oil (EVOO/VOO) production in terms of extraction yield and chemical quality was studied at pilot scale in an industrial oil-press in two different crop seasons. The addition of CO2,s to the fruits induces the intracellular water freezing and the consequent laceration of cellular membranes could induce the diffusion of many cellular compounds. In the experimental conditions adopted, the addition of the cryogen to the olives during pre-milling phase greatly increased the extraction yield (ranging from ≅ 1 to ≅ 21 %), with respect to the related control. Furthermore, the use of solid CO2 had no negative effects on chemical composition of the olive oil, maintaining the highest quality according to EU legal standards showed by control. Therefore, the utilization of solid CO2 as cryogen could be an appropriate technology to improve olive oil yield and quality and increase its shelf-life. The utilization of solid carbon dioxide in the extraction of extra-virgin olive oil VOO/EVOO yield and quality as a function of extraction conditions adoptedA about negative environmental impacts of conventional farming techniques, coupled with positive consumer perceptions of the safety and quality of organic foods have contributed to growth in their demand. It has been demonstrated that organically produced foods have lower levels of pesticides and veterinary drug residues, and in many cases, lower nitrate contents. Organic production methods lead to increases in nutrient content, particularly organic acids and polyphenolic compounds, many of which are considered to have potential human health benefits as antioxidants. Organic crops tend to have more vitamin C, sugars and phenolics and fewer nitrates than conventionally grown produce. It has been estimated that organic crops, overall, contain 21% more iron and 29% more magnesium than their conventional counterparts. Organic crops showed higher phosphorus and dry matter content and lower nitrogen content than conventional crops. The quality of amino acids was higher in organic products indicating that more essential amino acids were available. Increased amounts of omega-3 fatty acids and conjugated linoleic acid in organic dairy were noted. Consumption of organic foods provides protection against exposure to organophosphorus pesticides commonly used in agricultural practices. Organic fruits and vegetables appear to have the potential to diminish the mutagenic action of toxic compounds and inhibit the proliferation of certain cancer cell lines. For prevention of allergic dermatitis, the consumption of organic dairy and meats can make a significant difference in health outcomes. This review discusses the differences between organic foods and conventional foods on the parameters of food safety and nutritional composition.Defatted ground nut, soybean and palm kernel cakes were stored for three months at ambient, refrigeration and display conditions. Samples were assayed for nutritive, aflatoxins and microbiological properties. Proximate parameters significantly (p less than 0.05) reduced with storage. Mineral composition reductions of 3.0-35.00%, 1.2-18.75% and 6.2-64.25% were observed for ambient, refrigeration and display condition storage respectively. There was a significant (p less than 0.05) increase in both bacterial and fungal counts with increased storage duration. Aflatoxin B1 contents of 21.65-49.26, 14.57-27.24 and 29.14-61.32 μg/kg were detected in cakes stored at room, refrigeration and display condition respectively while the Aflatoxin B2 contents were 9.85-14.13, 3.24-10.02 and 12.96-17.04 μg/kg respectively. Proper storage/handling of feed ingredients should form a core component of Good Manufacturing Practice (GMP) of feed production in tropical countries. The regulatory agencies in these countries should enforce a revised GMP in order to guarantee a more healthy and productive populace.B is providing us with a wide range of options for how we can use agricultural and commercial forestry lands. The cultivation of genetically modified (GM) crops on millions of hectares of lands and their injection into our food chain is a huge global genetic experiment involving all living beings. The introduction of genetically modified (GM) products to the food market resulted in them becoming a controversial topic, with their proponents and contestants. GM foods are useful in controlling the occurrence of certain diseases and furthermore there is a growing body of research which has identified that GM food is reported to be high in nutrients and overall suggest that GM foods contain more minerals and vitamins than those found in traditionally grown foods. Moreover GM foods are known to taste better. Another reason for people opting for GM foods is that they have an increased shelf life and hence there is less fear of foods getting spoiled quickly, which is an important issue for those who are impoverished. However in contrast, it is believed that consumption of these genetically engineered foods can cause the development of diseases which are immune to antibiotics. More importantly, despite ever advancing technology in this field, as these foods are new inventions, not much is known about their long term effects on human beings. This presentation aims to review the potential benefits and risks resulting from the consumption of transgenic food in order to help health professionals and the public to decide if it is indeed safe to consume GM foods.F laws and regulations provide food producers and food equipment manufacturers with the requirements to manage potential food safety risks. The globally influential international food standards Codex Alimentarius, published by the Food and Agriculture Organization of the United Nation and the World Health Organization, sets up a global framework of general principles of food hygiene. Supporting this target, the European Commission has developed several European regulations and directives that primarily address the food processor and require that food is produced in a safe manner. However despite all joint efforts over the last decades and the fact that most Food Business Operators are following one or more recognized certifications schemes, it still goes wrong. To achieve good design that takes into account regulatory and clean ability requirements, the equipment manufacturer will need a solid foundation of experienced people, the willingness to strive for innovative solutions, and commitment to ongoing education and learning. Establishing these foundational elements is vitally important for food equipment manufacturers who aim to achieve the ultimate goals food safety.I resistance (IR) is a critical metabolic abnormality requiring attention. In addition to type 2 diabetes mellitus (T2D), its roots are found in cardiovascular diseases, stroke, Alzheimer disease and longevity. The association of obesity with T2D has been recognized for decades, and the major basis for this link is the ability of obesity to engender IR. The growing obesity epidemic in the United States continues to escalate the number of individuals at risk for developing IR. In the United States, an estimated ~78 million individuals are affected by IR. Both genetic and environmental factors are involved; however, only 5–10% of patients exhibit specific genetic mutations responsible for IR. Therefore, an outstanding percentage is due to environmental factors, many of which are modifiable, such as the diet. Although the exact mechanisms of IR have not been described fully, cellular events sensitive to oxidative stress and inflammation have been described and suggested as causative factors of IR. Fruits and vegetables, and particularly those with higher polyphenolic content are suggested to have favorable effects on human health due to their ability to modulate oxidative and/or inflammatory stress in peripheral and central tissues. Therefore, this presentation will be focus on the studies that has demonstrated the effects of polyphenolic compounds on oxidativeinflammatorystress and further, the relationship of these responses to insulin action in humans.In this study, mustard seeds as exogenous source of myrosinase were added to cooked broccoli as a condiment with a view to intensify the formation of sulforaphane. Thermal inactivation of myrosinases from broccoli and mustard seeds was studied. Thermal degradation of broccoli glucoraphanin was also investigated. In addition, the effect of mustard seeds addition on sensory profiling and consumer acceptability was assessed.W flour was replaced with breadfruit flour (0 to 40%) to produce bread. Composition and pasting properties of the flours and their composite flour blends were determined. Baking and sensory qualities of the resulting bread samples were evaluated. Protein starch and fiber contents of the breadfruit and wheat flours were 2.6; 61.30; 7.79% and 10.9; 69.89; 2.81% respectively. These significantly influenced the rheological (pasting, farinograph and alveograph) and baking properties of the dough of the composite blends and the bread made from it. Peak, holding, breakdown and final viscosities of the blends ranges from 122.3 to 147; 85.4 to 98.6; 28.74 to 49.18 and 128.6 to 147.4 RVU respectively. Dough tolerance to mixing was significantly affected as dough stability, breakdown time and dough development time decreased from 9.30 to 0.77min., 10.8 to 2.50min. and 8.20 to 1.60min while water absorption increased from58.63 to 65.96% respectively. Peak height of the alveograph increased from 90 to 226mm while length and max. inflation attained decreased from 80 to 26mm and 199 to 114. Specific volume and total quality score decreased from 3.00 to 1.32cm3/g and from 80 to 41.6% respectively. Bread from the blends was acceptable to customers within 10% substitution level.P compounds play a key role in the quality and health benefits of a wine so that, in the last twenty years, many devices have been created to assist the winemaker in obtaining products characterized by a good structure and color intensity and strongly linked to its territory of origin. In this experimental work a versatile innovative tank was set up to allow the automation of the processes of fermentation, maturation and storage of wines, including the mixing and retention of suspended solids (maturation on lees). The work has been carried out according to the following steps:S alcohol (Polyols), are currently used as a bulk sweetener in reduced calorie foods. It has been part of the human diet for thousands of years as it is present in fruits such as pears, melons and grapes, as well as foods such as mushrooms and fermentation –derived foods (wine, soy sauce and cheese). Sugars alcohol is believed to be good sugar substitute for people with diabetes plus they do not contribute to dental caries (cavities). The most common sugars alcohol available in the market is sorbitol, mannitol, xyletol and erythretol. Manufacturing, benefits and applications in food and drinks for these common sugars alcohol and others will be presented.

Modeling of cytochrome P450 (Cyt P450, CYP) channels

The precise location of a substrate in cytochrome P450 (CYP) governs the orientation of the oxidation position. Such information is generally obtained from biochemical data, but modeling approaches have also been used to explain these locations. We used X-ray data and modeling techniques to distinguish between the series of putative linear or curved channels which lead the substrate from the outer side of the protein to the inner, and then into the heme pocket; these techniques were also used to identify the largest such channels. Two new methods for precisely determining the 3-D structure of proteins using X-ray crystallography were proposed in order to identify these channels: first, the use of both straight and curved channels, and second, the sphere method. These data are compared with Poulos channels, and with Caver (or Mol on line) modeling methodologies. Our methods were developed from studies of the interaction between cytochrome P450CAM (CYP101) from Pseudomonas putida (as expressed in Escherichia coli) and the indolic base β-carboline. Apart from the identification of potential access channels leading to the heme-containing active site, a new explanation was advanced for the substrates hydroxylation position. The sphere method seems to have potential to become a general and direct method for prediction of substrate access channels from reduced- or low-resolution crystallographic data.