A Simple and Fast HPLC Method to Determine Lycopene in Foods

Carotenoids, among which lycopene—the principal pigment found in tomatoes—are lipophilic compounds which play a very important role in human health and nutrition. They are also recognised as strong antioxidants due to their ability to trap singlet oxygen and eliminate the peroxyl radical. The availability of reliable information on lycopene content of foods is essential both for the evaluation of diet and for epidemiological research relating the intake of lycopene. This paper describes a simple and fast HPLC/UV method for lycopene determination in a wide range of food products. All-E-lycopene together with its Z isomers were eluted isocratically using a carotenoid C30 reversed-phase column. The in-house validated HPLC method had a limit of quantification of 60 ng lycopene/g product and high precision and accuracy. The analytical method was successfully applied to several food products such as raw vegetables and fruits and also processed foods. Tomato and tomato-containing products contained the highest amounts of lycopene. While raw foods and minimally processed foods contained above 94% of all-E-lycopene, processed foods (such as soups, pasta sauces, pizza and cheese) contained from 76% to 87% of all-E-lycopene.     

Antiviral activity of donkey milk protein fractions on echovirus type 5

Antiviral activity of Ragusano donkeys' milk proteins was investigated for the effect on echovirus type 5, known to infect the gastrointestinal tract of humans. Three protein fractions were tested; casein (CN), whey protein (WP) and a low molecular whey protein fraction (LWP; <30,000 Da). The antiviral activity of WP and LWP was tested on echovirus type 5 at three concentrations (1, 5 and 10 mg mL^?1); CN was assessed only at the lower concentration. All donkey milk protein fractions showed significant inhibition on virus replication at the concentration of 1 mg mL^?1, and both WP and LWP fractions showed significant inhibition on the virus replication at all concentrations tested. The strongest antiviral effect was observed for the WP fraction. These findings show that the different whey proteins in donkey milk, probably acting in synergy, exert antiviral activity on echovirus 5 and might contribute to prevent gastrointestinal virus infections in humans.     

Quantitative PCR for the specific quantification of Lactococcus lactis and Lactobacillus paracasei and its interest for Lactococcus lactis in cheese samples

The first objective of this work was to develop real-time quantitative PCR (qPCR) assays to quantify two species of mesophilic lactic acid bacteria technologically active in food fermentation, including cheese making: Lactococcus lactis and Lactobacillus paracasei. The second objective was to compare qPCR and plate counts of these two species in cheese samples. Newly designed primers efficiently amplified a region of the tuf gene from the target species. Sixty-three DNA samples from twenty different bacterial species, phylogenetically related or commonly found in raw milk and dairy products, were selected as positive and negative controls. Target DNA was successfully amplified showing a single peak on the amplicon melting curve; non-target DNA was not amplified. Quantification was linear over 5 log units (R² > 0.990), down to 22 gene copies/μL per well for Lc. lactis and 73 gene copies/μL per well for Lb. paracasei. qPCR efficiency ranged from 82.9% to 93.7% for Lc. lactis and from 81.1% to 99.5% for Lb. paracasei. At two stages of growth, Lc. lactis was quantified in 12 soft cheeses and Lb. paracasei in 24 hard cooked cheeses. qPCR proved to be useful for quantifying Lc. lactis, but not Lb. paracasei.     

Photobleaching with phloxine B sensitizer to reduce food matrix interference for detection of Escherichia coli serotype O157:H7 in fresh spinach by flow cytometry

A flow cytometric method (RAPID-B™) with detection sensitivity of one viable cell of Escherichia coli serotype O157:H7 in fresh spinach (Spinacia oleracea) was developed and evaluated. The major impediment to achieving this performance was mistaking autofluorescing spinach particles for tagged target cells. Following a 5 h non-selective enrichment, artificially inoculated samples were photobleached, using phloxine B as a photosensitizer. Samples were centrifuged at high speed to concentrate target cells, then gradient centrifuged to separate them from matrix debris. In external laboratory experiments, RAPID-B and the reference method both correctly detected E. coli O157:H7 at inoculations of ca. 15 cells. In a follow-up study, after 4 cell inoculations of positives and 6 h enrichment, RAPID-B correctly identified 92% of 25 samples. The RAPID-B method limit of detection (LOD) was one cell in 25 g. It proved superior to the reference method (which incorporated real time-PCR, selective enrichment, and culture plating elements) in accuracy and speed.     

Assessment of two enzyme-linked immunosorbent assay tests marketed for detection of ruminant proteins in finished feed

The performance characteristics of two enzyme-linked immunosorbent assay (ELISA) test kits, ELISA Technologies' MELISA-Tek test and Tepnel BioSystems' BioKit for (Cooked) Species Identification test, designed to detect ruminant proteins in animal feed, were evaluated. The test kits were evaluated by using acceptance criteria developed by the U.S. Food and Drug Administration's Center for Veterinary Medicine Office of Research for evaluating selectivity, sensitivity, ruggedness, and specificity. The acceptance criteria for determining success used a statistical approach requiring a 90% probability of achieving the correct response within a 95% confidence interval. In practice, this measure requires the test to achieve the correct response 58 times for every 60 samples evaluated, or a 96.7% accuracy rate. A minimum detection level of 0.1% bovine meat and bone meal (BMBM) was required, consistent with the sensitivity of the analytical methods presently used by the U.S. Food and Drug Administration. Selectivity was assessed by testing 60 dairy feed samples that contained no added animal proteins; sensitivity was determined by evaluating 60 samples (per level of fortification) of this same feed that contained 0.025, 0.05, 0.1, 0.25, 0.5, 1, or 2% BMBM. The MELISA-Tek test passed the acceptance set-point criteria for selectivity assessment but failed the sensitivity assessment at all levels except at the 2% level. The MELISA-Tek test came close to passing at the 1% level, detecting true-positive findings at a rate of 93%, but failed at lower levels, in spite of the label claim of 0.5% sensitivity. The BioKit for (Cooked) Species Identification test detected only 2 of 17 samples fortified at the 2% BMBM level and failed to detect any other BMBM-fortified samples. The results of this evaluation indicate that neither test is adequate for regulatory use.     

Aging-induced changes in microstructure and water distribution in fresh and cooked pork in relation to water-holding capacity and cooking loss - A combined confocal laser scanning microscopy (CLSM) and low-field nuclear magnetic resonance relaxation study

Confocal laser scanning microscopy (CLSM) and low-field nuclear magnetic resonance (LF-NMR) relaxometry were combined to characterize microstructural changes and water distribution in fresh and cooked pork during an aging period of 14 days. At day 1 (24 h postmortem) a few muscle fibres, which appear swollen, were observed in both fresh and cooked meat. An identical microstructure was still apparent after 14 days, however, the number of muscle fibres showing distinguished characteristics was found to increase throughout the aging period. Hence, it was apparent that during aging the individual fibres swell and disintegrate at different rates. Development in water-holding capacity (WHC) was followed during the aging period using gravimetric methods, and an increase in the WHC in the fresh meat was observed, which resembled the amount of extramyofibrillar water measured by NMR relaxometry (T₂₂ population). This was consistent with the CLSM images, as a substantial increase in the number of myofibrils that appeared swollen, capable of holding more water, was observed during aging. In the cooked meat the width of the T_21c population, reflecting the myofibrillar water in the cooked meat, was seen to decrease during the entire storage period, which corresponds to the development of a more homogeneous structure. In the CLSM data a continuous degradation during the storage period was observed, which could resemble a shift to a more homogeneous structure. Comparison of CLSM of transverse sections of fresh and cooked pork revealed a pronounced shrinkage of muscle fibres upon cooking. This resulted in large gaps between the cooked muscle fibres, which also was visible as shrinkage at the level of the individual myofibrils. This pattern was also reflected in the NMR relaxation data. The cooking-induced shrinkage of the myofibrils occurred concomitantly with a decrease in the amount of intermyofibrillar water within the individual fibre and an increase in the larger extramyofibrillar spaces between fibres, i.e. water is expelled from the myofibrillar matrix upon cooking. Accordingly, the present study demonstrated that the use of CLSM together with NMR relaxometry can provide further information on the relationship between structural characteristics of meat and resultant water distribution.     

Pelvic suspension and fast post-mortem chilling: Effects on technological and sensory quality of pork - A combined NMR and sensory study

The effects of pre-rigor excision, which results in a fast post-mortem chilling; pelvic suspension, which results in a muscular stretching; control treatment of m. longissimus dorsi on water distribution measured by (1)H NMR relaxometry and on sensory properties were investigated in two sub-studies including a total of 16 pigs. Determination of sarcomere length revealed significant effects of the treatments on the degree of contraction, as pre-rigor excision and pelvic suspension resulted in shorter and longer sarcomere length, respectively. In addition, an effect of treatment on pH measured at 24h post-mortem was found, as pre-rigor excision was associated with a higher ultimate pH. NMR measurements revealed that water distribution in the meat was affected to a minor degree by the various treatments. However, an interaction between treatment and ageing period was observed, as the data demonstrated an effect of pre-rigor excision on water distribution in the cooked samples when meat was only aged for 2 days, but this effect was eliminated when meat was aged for 5 days. The effect of pre-rigor excision on water distribution in cooked meat after 2 days of ageing is suggested to reflect structural constraints in the meat that are eliminated during ageing. Sensory analyses demonstrated strong interactions between treatment and position on the muscle and between treatment and ageing period. However, in general pre-rigor-excised meat was sensed as significantly juicier compared with the other two treatments. Accordingly, the study demonstrates that under the present conditions the ultimate pH is more important for juiciness than the sarcomere length.