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This research was funded as part of the ELDERMET project, one of the four projects funded by the DAFF / HRB under the Food for Health Research Initiative, 2007.
1Hopkins, M.J., and Macfarlane, G.T. (2002). J. Med. Microbiology. 51: 448-454.
2Flint, H. J., Duncan, S., H., Scott, K. P., and Louis, P. (2007). Environmental Microbiology. 9 (5): 1101-1111.
3Franchimont, D., Vermeire, S., El Housni, H., Pierik, M., Van Steen, K, Gustot, T., Quertinmont, E., Abramowicz, M., Van Gossum, A., Devie`re, J., and Rutgerts, P. (2004). Gut. 53: 987-992.
4Kassinen, A., Krogius-Kurikka, L., Mäkivuokko, H., Rinttilä, T., Paulin, L., Corander, J., Malinen, E., Apajalahi, J., and Palva, A. (2007). Gastroenterology. 133 (1): 24-33.
DETERMINATION OF GENOPROTECTIVE AND COX-2 MODULATORY EFFECTS OF PHYTOSTEROLS IN CACO-2 CELLS
T.J. Daly, S.A. Aherne, T.P. O’Connor and N.M. O’Brien
Department of Food & Nutritional Sciences, University College Cork, Cork, Ireland
Phytosterols are bioactive plant-derived compounds with similar chemical structure and biological functions as cholesterol. The major dietary phytosterols include β-sitosterol, campesterol and stigmasterol. Previously, we investigated the effects of phytosterols on the viability of human intestinal Caco-2 cells. In the present study we determined if supplementation with campesterol, -sitosterol or -sitostanol (25 μM) for 48 h exerted genoprotective effects and/or COX-2 modulation in Caco-2 cells. DNA damage was determined using the comet assay after pre-incubation with the phytosterols followed by exposure to either a food mutagen, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), or hydrogen peroxide (H2O2) for 30 min. COX-2 production was measured by ELISA after treatment with or without the phytosterols in the presence or absence of a COX-2 promoter interleukin-1β (IL-1β). Incubation with MNNG or H2O2 significantly induced DNA damage (P<0.01). Supplementation with the phytosterols did not protect against MNNG- or H2O2-induced DNA damage. Furthermore, the phytosterols did not significantly affect basal or IL-1β-enhanced COX-2 production. To conclude, campesterol, β-sitosterol or β-sitostanol, at a concentration of 25 μM, did not exert genoprotective or COX-2 modulatory effects in Caco-2 cells.
It is commonly believed that diet can contribute to, or help lower, cancer risk. Some dietary components may reduce the risk of cancer by decreasing the effects of toxins, such as food mutagens, which may contribute to cancers in the body especially along the route of exposure1. Dietary components may also reduce the toxic effects of oxidative stress which has been identified in the development of human chronic diseases such as certain cancers and cardiovascular diseases2,3.
DNA damage is a source of genetic mutations and can therefore be responsible for the onset of cancers. Furthermore, numerous studies have proposed a relationship between cyclooxygenase-2 (COX-2) expression and carcinogenesis, in particular, colon cancer. Interleukin-1β (IL-1β) is one of the main cytokines involved in the inflammatory process. It has been shown to induce COX-2 expression and can initiate a cascade of signalling events involving the biosynthesis of prostaglandins, which are implicated in inflammation and carcinogenesis. In the present study we determined if the phytosterols campesterol, β-sitosterol or β-sitostanol exerted genoprotective effects and/or COX-2 modulation in human intestinal Caco-2 cells.
MATERIALS AND METHODS
Human colon adenocarcinoma Caco-2 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with foetal bovine serum (10% v/v FBS) non-essential amino acids (1% v/v) and in the absence of antibiotics. Caco-2 cells were seeded at 3 x 104 cells/cm2 into 96-well culture plates and allowed adhere for 24 h in complete medium. After 24 h, the culture medium was removed and replaced with growth medium containing the phytosterols campesterol, β-sitosterol or β-sitostanol (25 µM). Cells were allowed to grow for 48 h. After incubation, DNA damage was determined using the comet assay4 following exposure to either the food mutagen, N-methyl-N′-nitro-N-nitrosoguanidine (MNNG, 25 µM), or hydrogen peroxide (H2O2, 50 µM) for 30 min. COX-2 production was measured by ELISA following incubation with or without the phytosterols in the presence or absence of the COX-2 promoter IL-1β (10 ng/ml).
RESULTS AND DISCUSSION
Treatment with MNNG or H2O2 caused a significant increase in DNA damage (P<0.01) when compared with untreated cells (Table 1). Pre-incubation for 48 h with either campesterol, β-sitosterol or β-sitostanol did not protect against MNNG- or H2O2-induced DNA damage. IL-1β was found to significantly increase COX-2 production. The phytosterols did not significantly modulate basal or IL-1β-induced COX-2 levels in Caco-2 cells.
Table 1. Effects of phytosterols on DNA damage and COX-2 production.
n4 independent experiments. *P<0.01 compared with positive control: ANOVA, Dunnett’s test.
Our present findings show that, at 25 μM, campesterol, β-sitosterol or β-sitostanol did not exert genoprotective or COX-2 modulatory effects in Caco-2 cells.
This work was funded by the Department of Agriculture and Food under Food Institutional Research Measure as administered by the National Development Plan 2000-2006.
1 Kelloff, G.J., Crowell, J.A., Steele, V.E., Lubet, R.A., Malone, W.A., Boone, C.W., Kopelovich, L., Hawk, E.T., Lieberman, R., Lawrence, J.A., Ali, I., Viner, J.L. and Sigman, C.C. (2000). J. Nutr. 130: 467-471.
2 Federico, A., Morgillo, F., Tuccillo, C., Ciardiello, F. and Loguercio, C. (2007). Int. J. Cancer, 121: 2381-2386.
3 Siekmeier, R., Steffen, C. and Marz, W. (2007). J. Cardiovasc. Pharmacol. Ther. 12: 265-282.
4 Tice, R.R., Andrews, P.W., Hirai, O. and Singh, N.P. (1990). In Biological and Reactive Intermediates IV (p. 157-164). Witmer, C. M. (Ed.) The single cell gel (SCG) assay: an electrophoretic technique for the detection of DNA damage in individual cells (p. 157-164). Plenum Press: New York.
TO ASSESS THE ANTIOXIDANT POTENTIAL OF PEPTIDES DERIVED FROM BOVINE MUSCLE PROTEINS
R. Di Bernardini1, P. Harnedy1, D. Bolton1, N. Brunton1, J. Kerry2 and A.M. Mullen1
1 Ashtown Food Research Centre, Teagasc, Ashtown, Dublin 15
2 Departments of Food & Nutritional Sciences, University College Cork, Cork
In recent years it has become widely recognised that antioxidants are important in the prevention of food deterioration and human diseases. With some artificial antioxidants posing potential health risks the search is on for isolation and exploitation of natural antioxidant agents. The objective of this study was to investigate the antioxidant activity of bovine cytoplasmic protein hydrolysates generated using a commercial proteolytic enzyme. Results of the study show that bovine cytoplasmic protein hydrolysates had antioxidant activity, with activity varying depending on the degree to which the protein extract was hydrolysed.
Free radicals and active oxygen species, such as superoxide, are produced in the body as part of normal cellular metabolism. While the body has its own defence system against these compounds, such as antioxidant enzymes and endogenous antioxidants, excess production can result in oxidative damage of vital cellular components1, and lead to many degenerative diseases, such as cancer2. Oxidation can also affect food, causing rancidity and/or deterioration of the nutritional quality, colour, flavour, texture and safety of food1. Reduction of such damage in the body may be brought about by dietary intake3. In the case of food products artificial preservatives are utilised, however because of negative health implications their use in foodstuff is a cause of concern in many countries, leading to the search for safer antioxidants from natural sources4. Protein hydrolysates with antioxidant activity, generated from the enzymatic hydrolysis of various proteins sources, including meat, have been reported 5-7. Some meat by-products, which are considered to possess low economic value by the meat industry, possess high protein content. Considering the large amount of by-products produced by the meat industry, it would be greatly beneficial for these low-value by-products to act as sources of antioxidant products (e.g. peptides or hydrolysates). The objective of this work was to study the antioxidant activity of enzymatic hydrolysates generated from bovine liver cytoplasmic protein extracts.
MATERIALS AND METHODS
Cytoplasmic proteins from bovine liver tissue samples were extracted with a low salt buffer by homogenisation. Protein hydrolysates were prepared by digestion of the protein fraction with a commercial enzyme for seven time periods. Analysis of the cytoplasmic protein extract and hydrolysates were carried out by 1D SDS-PAGE. The antioxidant activity of all seven hydrolysates was determined using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay with results expressed in terms of their IC50 (g/L) values.
Results and Discussion
Preliminary results of radical scavenging antioxidant activity assays indicated that all seven protein hydrolysates had antioxidant activity, with the lowest activity reported for the first time period with varying scavenging activity detected for each of the other time periods. Different levels of activity observed at different degrees of protein hydrolysis suggest that the change in antioxidant activity may be as a result of polypeptide/peptide formation. These findings are the initial stages in mining for bioactive peptides with antioxidant activity form bovine protein sources.
The results obtained from this study indicate that low value bovine offal (liver) cytoplasmic protein hydrolysates generated with a commercial enzyme have antioxidant activity. These hydrolysates have the potential to act as the starting point for fractionation and purification of natural antioxidant peptides (or hydrolysates) that could be used as components to inhibit oxidation in the body or use as a natural preservatives in foodstuff.
Funding for this research was provided under the National Development Plan, through the Food Institutional Research Measure, administered by the Department of Agriculture, Fisheries & Food, Ireland and by TEAGASC under the Walsh Fellowship Scheme.
1Antolovich M., Prenzler P. D., Patsalides E., McDonald S. and Robards K. Analyst. 2002, 127, 183-198
2 Pihlanto A., Akkanen S. and Korhonen H. Food Chemistry. 2008, 109, 104-112
3Hernardez-Ledesma B., Dávalos A., Bartolomé B. and Amigo L. Journal of Agriculture and Food Chemistry. 2005, 53, 588-593
4Chiue H., Kusano T. and Iwami K. Journal of Nutritional Science and Vitaminology. 1997, 43, 145-154
5Jun S.-Y., Park P.-J., Jung W.-K and Kim S.-K. European Food Research and Technology. 2004, 219, 20-26
6Chen H.-M., Moramoto K. and Yamauchi F. Journal of Agriculture and Food Chemistry. 1995, 43, 574-578
7Saiga A., Tanabe S. and Nishimura T. Journal of Agriculture and Food Chemistry. 2003, 51, 3661-3667
APPLICATION OF FLOW CYTOMETRY FOR PROBIOTIC VIABILITY ANALYSIS IN PROTEIN SYSTEMS
S. Doherty1, P. R. Ross1,2, C. Stanton1,2, G. F. Fitzgerald2,3 and A. Brodkorb1
1 Moorepark Food Research Centre, Teagasc, Moorepark, Fermoy, Co. Cork, 2Alimentary Pharmabiotic Centre, Cork and 3Department of Microbiology, University College Cork, Cork, Ireland
The viability of probiotic bacteria is of paramount importance for their application in functional foods. The aim of this study was to demonstrate proof of the principle that flow cytometry (FACS) could be employed for viability assessment of probiotics immobilized in protein networks; however, FACS analysis is hampered by the presence of protein particles. Thus, a rapid FACS assay based on enzymatic digestion of whey proteins was developed to reduce the protein background without affecting cell viability. Dual-staining of samples with fluorescent dyes that indicate live (thiazole orange, TO) and dead (propidium iodide, PI) cells enabled determination of viable cell populations. A good correlation (r >0.99) between the FACS assay and the conventional plate counting technique was achieved. Three functional probiotic populations could be distinguished: live, dead and compromised cells. Thus, FACS has the potential to assess the functionality of probiotic populations in milk protein systems.
Probiotics are described as ‘living microorganisms, which upon ingestion in certain numbers exert health benefits beyond inherent basic nutrition’1. Species are selected mainly on the basis of their potential health-associated properties; however, further criteria should also be fulfilled such as resistance to technological processes and gastric acid. In survival studies the quantitative assessment of cell viability is crucial; thus bacterial viability must be demonstrated by replication in a validated laboratory system. The conventional method for quantitative survival studies is the plate count technique; however, there are a number of disadvantages associated with this approach since plate counting requires 2/3 days of incubation and bacteria may occur in chains, resulting in underestimation of the true bacterial count. Previous work investigated the influence of cell immobilization in heated whey protein products on in vitro survival of Lb. rhamnosus GG (LGG) at elevated temperatures. Traditional plate counts advocated whey protein networks as a safeguard for LGG during heat stress; however, a rapid and reliable alternative technique for viability assessment is desired to confirm these findings. Thus, flow cytometry is an appealing candidate for fast viability assessment. Preliminary work verified that pure populations of LGG (free cells) were easily detected by the flow cytometer; however, no distinct separation of live, dead and injured cell populations appeared for cells immobilized in whey protein. This implied that sample analysis without protein digestion was insufficient to allow definition of probiotic viability. For this reason, the inclusion of an enzymatic treatment was evaluated during this study in order to remove or modify proteins and thereby enable distinction of probiotics by FACS.
MATERIALS AND METHODS
The probiotic strain LGG was propagated from 1% (v/v) anaerobic inoculations for 18 h at 37°C. These stationary phase cells were harvested and the resuspended cell suspension was either immobilized in whey protein or utilized (as a control) in a free-cell condition. Following homogenisation, samples were diluted to an approximate cell density of 107 CFU/mL. Each sample was treated with (10%, v/v) Proteinase K, incubated at 37°C for 30 min, dual-stained with TO and PI, and incubated according to the manufacturer’s instructions. Data acquisition was performed on a BD FACS Canto II flow cytometer with BD FACS Diva software. For each sample, 10,000 events were captured and photomultiplier tube (PMT) voltages and threshold values were adjusted using adequate controls.
RESULTS AND DISCUSSION
After the application of protease in association with fluorescent dyes, LGG immobilized in whey protein emerged in exactly the same position on bivariate dot plots as control (free) bacteria. The success of this approach may be attributable to the heat treatment applied to whey protein prior to cell immobilization, which ultimately enhanced its sensitivity to proteolysis by Proteinase K. As a result, whey protein was digested adequately and counts of particles that were similar in size to or larger than LGG were decreased significantly. Furthermore, the probiotic cell-membrane integrity was not affected by protein digestion as evidenced by the high cell viability (> 99%) acquired during FACS analysis. The key criterion for accepting this FACS methodology was that the regression between FACS results and plate counts closely matched the guidelines of Feldsine et al 2. FACS results exhibited a very strong correlation with plate counts across the cell concentration range tested due to the incorporation of a homogenisation step, which eliminated the possibility for bacterial aggregates or chains being enumerated as one unit. A correlation coefficient of 0.99 demonstrated that underestimation of total cell counts was avoided, indicating that almost all probiotic cells counted by FACS formed colonies on agar plates. High cell viability was well-documented by plate enumeration and the high fluorescence intensity from TO-stained cell populations concurred with these findings. However, following 14 days storage at 37°C, a general physiological heterogeneity was revealed within immobilized samples relating to membrane integrity, with the emergence of a double-stained cell population. The presence of such double-stained populations indicated that probiotic cell-membranes were irreversibly damaged during extended storage, under which PI penetration was permitted, but intracellular accumulated TO could still be retained. It is also noteworthy that free LGG cells were permeabilized and thus intensely stained with PI after only 24 h storage, indicating complete cell loss. Thus, fluorescent dot plot patterns characterized different magnitudes of membrane degradation in immobilized samples compared to the free-cell reference and clearly portrayed the succession of cell changes that occurred during storage at elevated temperatures.
Traditionally, viability in bacteria is synonymous with the ability to form colonies on solid growth media and to proliferate in liquid nutrient broths. These traditional methodologies lack the resolving power to provide real-time results. However, this FACS assay was performed within 40 min; thus advocating FACS as a rapid detection method. This research provides impetus for this technology to be applied to a broad range of microbiological assays for determining cell viability within milk protein systems.
The work was funded by the Dairy Research Trust, the Irish Government under the National Development Plan 2000-2008. S. Doherty is supported by the Walsh Fellowship Scheme.
1 Guarner, F., and Schaafsma, G. J. (1998). Int. J. Food Microbiol. 39:237-8.
2 Feldsine, P., Abeyta, C., Andrews, W. H. (2002). J. AOAC Int. 85:1187-200.
MOLECULAR ANALYSIS OF TENDERSTRETCH ON LOW VOLTAGE ELECTRICALLY STIMULATED POSTMORTEM BOVINE SKELETAL MUSCLE PROTEIN COMPOSITION USING 2- DIMENSIONAL ELECTROPHORESIS BASED PROTEOMICS
E. Downeyl, S.R. Pennington2, K. Brandon1, A. White1 and A.M. Mullen1
lTeagasc, Ashtown Food Research Centre, Castleknock, Dublin 15, Ireland and 2Conway Institute Proteome Research Centre, University College Dublin, Belfield, Dublin 4, Ireland.
Using 2-dimensional electrophoresis (2-DE) based proteomics we have investigated the protein expression profile of bovine M. longissimus thoracis et lumborum at day 2 (n=3) and day 14 (n=3) in ‘tenderstretched’ and conventionally hung carcasses. From 1248 protein features detected by image analysis, 191 manually curated spots were subjected to statistical analysis. From this initial analysis, at day 2 postmortem; 33 spots were up regulated and 10 were down regulated (P.<0.01); whilst at day 14 postmortem, 60 up regulated and 20 down regulated protein spots (P.<0.01) were found in the tenderstretched samples. Mass spectrometry (MALDI TOF-TOF and LTQ linear ion trap) has been used to identify some of these proteins.
‘Tenderstretch’ has been shown to help prevent sarcomere shortening in postmortem skeletal muscle and subsequently decrease shear force and improve meat tenderness1. It has been suggested that ‘tenderstretch’ may not only affect the structural integrity of the sarcomere2 but that it may increase the rate of proteolysis by elevation of intracellular Ca2+ levels3, activation of calcium dependant proteases4, and stretch induced exposure, within the muscle fibre, of potential proteolytic substrates5.
MATERIALS AND METHODS
Cross bred heifers (n=3) were captive bolt stunned and exanguinated conventionally. The carcasses were electrically stimulated at 60V for 20 secs. Half of each carcasses (n=3) was conventionally hung while the other half (n=3) was ‘tenderstretch’ hung. M. longissimus thoracis et lumborum samples were taken from the excised muscle for protein analysis at day 2 and day 14 postmortem. Myofibrillar proteins were extracted6 and quantified7 before 2-DE was carried out over a pH range 4-78 using 100ug (analytical)/250ug (mass spectrometry) protein load. All gels were stained with analytical (InsightBio) or mass spectrometry suitable silver stain (GE Healthcare). The 2-DE gel images were analysed using Progenesis software (Nonlinear Dynamics). Peptides were extracted from the spots of interest by trypsin. All samples were analysed on a 4800 MALDI ToF-ToF (Applied Biosystems) and/or a LTQ Linear Ion Trap (Thermo Finnegan).
RESULTS AND DISCUSSION
Proteins were extracted from bovine M. longissimus thoracis et lumborum and visualised after 2-DE with a silver staining procedure. The proteolytic patterns of electrically stimulated bovine carcasses what were either conventionally hung or ‘tenderstretched’ were monitored at both day 2 and day 14 postmortem as illustrated by representative gel images in figure 1 and figure 2. A total of 1248 protein features were detected by image analysis software across the two treatment groups. Initial analysis revealed that at day 2 postmortem: 33 spots were up regulated and 10 were down regulated (P.<0.01) in tenderstretched samples compared to conventionally hung carcasses. After aging to 14 days postmortem, 60 up regulated and 20 down regulated protein spots (P.<0.01) were detected in the tenderstretched samples. Fifteen protein spots of interest were identified by MALDI ToF-ToF and/or LTQ Linear ion trap mass spectrometry (table 1).
Figure 1: 2-DE of electrically stimulated, conventionally hung bovine M. longissimus thoracis et lumborum at day 14 postmortem over a pH range 4-7 and on 12% polyacrylamide separating gels.
Figure 2: 2-DE of electrically stimulated, tenderstretched hung bovine M. longissimus thoracis et lumborum at day 14 postmortem over a pH range 4-7 and on 12% polyacrylamide separating gels.
Table 1: Mass spectrometry results from 15 protein spots of interest as identified by MALDI ToF-ToF and/or LTQ linear ion trap
The protein expression profile of bovine M. longissimus thoracis et lumborum has been observed to be changing between conventionally hung and ‘tenderstretch’ hung animals at both day 2 and day 14 postmortem. Mass spectrometry has identified some of these changes although further verification and validation work is required. It is anticipated that this work will lead to a better understanding at the molecular level of the biological basis of meat quality and the molecular processes through which carcass interventions impact meat quality.
The Food Institutional Research Measure (FIRM). The Teagasc Walsh Fellowship Scheme. The Conway Institute Mass Spectrometry Resource, UCD.
1Sorheim, O and Hildrum, K.I. (2002). Trends in Food Science & Technology, 13(4), 127-135.
2Hopkins, D.L. and Thompson, J.M. (2001). Meat Science, 57(1), 1-12.
3Armstrong, R.B., Duan, C., et al. (1993).. J. Appl. Physiol., 74(6), 2990-2997.
4Wrogemann, K., Hayward, W.A.K., et al. (1979). Annals of the New York academy of Sciences, 317(1), 30-45.
5Hwang, I.H., Devine, C.E., et al. (2003). Meat Science, 65(2), 677-691.
6Hwang, I.H., Park, B.Y., et al. (2005). Meat Science, 69, 79-91.
7Ramagli, L.S. & Rodriguez, L.V. (1985). Electrophoresis, 6, 559.
8Focking, M., Boersema, P.J., et al. (2006). Proteomics, 6, 4914-4931.
MICELLARISATION (BIOAVAILABILITY) OF CAROTENOIDS FROM ORGANIC BABY MEALS
P. Duane, O. O’Connell, L. Ryan, S.A. Aherne-Bruce, N.M. O’Brien
Department of Food and Nutritional Sciences, University College Cork, Ireland
To increase our understanding of the potential health benefits of carotenoids it is important to obtain insight into their bioavailability from foods. Carotenoid micellarisation refers to the transfer of carotenoids from the digested food (digestate) to the micelles in the gut before absorption. Therefore monitoring this transfer of carotenoids can be used as an effective tool for the initial screening of carotenoid bioavailability. The objectives of the present study were to assess the micellarisation of carotenoids from three baby meals and to determine the effects of cooking (hob and microwave methods). Both raw and cooked meals were subjected to an in vitro digestion procedure. Carotenoid micellarisation ranged from 0 - 91% depending on the meal, carotenoid, and the cooking method tested. Cooking had no major impact on the bioavailability of carotenoids from the meals which suggests that the ingredients used to prepare baby meals play an important role on the availability of carotenoids from foods.
Between the ages of four and six months babies’ nutrient requirements increase dramatically and, consequently, consumption of baby food products in addition to milk is required1. Organic baby food now accounts for almost half of baby food bought worldwide, even though research data on the nutritional benefits to babies of consuming organic foods compared with their non-organic counterparts are scarce and/or conflicting. Commercially available vegetable-containing baby foods are a source of both carotene (-carotene, β-carotene, lycopene) and xanthophyll (β-cryptoxanthin, lutein, zeaxanthin) carotenoids, which have been shown to possess various bioactive properties2,3. To increase our knowledge about the amounts of carotenoids that are potentially available for absorption by babies from commercially available organic baby foods, carotenoid bioavailability from these food products should be investigated.
Bioavailability is termed as the fraction of an ingested nutrient available for use in normal physiological functions and storage in the body. Carotenoid bioaccessibility, which is defined as the amounts of carotenoid(s) that are available for absorption in the gut after digestion4,5, has been of recent research interest. In addition, the efficiency of carotenoid micellarization, i.e. the transfer of carotenoids from the digestate (digested food) to the micelle (aqueous) fraction, is of particular importance as it is a good indicator of carotenoid bioavailability6,7. Therefore, the objectives of the present study were first, to determine the micellarisation of carotenoids from commercially available organic baby food products; and, second, to determine the effects of cooking (hob and microwave methods) on these foods.
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