Thin layer-chromatographic separation of fat soluble and water soluble food dyes

Summary The use of fat soluble and water soluble food dyes in food technology requires reliable proofs and precise identification of these substances in foodstuffs. This problematic has been studied more detailed in our Institute. During these studies some suitable procedures for thin layer chromatographic separation of these substances have been developed.For the separation of fat soluble food dyes the chromatography on thin layers of aluminium oxide and the chromatography on thin layers of starch with reversed phases were used. In the first case good separation was achieved with a solvent system containing petroleum ether and carbon tetrachlorid and their mixtures. In the second case the starch plates were impregnated with paraffin or vegetable oil, and as mobile phases a solvent system containing methanol-water-acetic adic (16 : 3 : 1) and others solvent mixtures currently used in paper chromatography were used.Water soluble food dyes were separated on thin layers of polyamide powder, which was used also for their quantitative isolation from foodstuffs. The best results were achieved with basic solvent system, for example, with a mixture containing ammonia-methanol water (5 : 15 : 80).The developed analytical procedures are suitable for the proof and the separation of fat soluble and water soluble food dyes in all foodstuffs.

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Zusammenfassung Die Verwendung von wasser- und fettlöslichen Lebensmittelfarben erfordert verläßlichen Nachweis und genaue Identifikation dieser Stoffe in den Lebensmitteln. Wir haben diese Problematik in unserem Institut eingehend studiert und einige Verfahren der chromatographischen Trennung dieser Stoffe mittels DC erarbeitet.Zur Trennung der fettlöslichen Farbstoffe verwendeten wir einerseits DC-Platten mit Aluminiumoxid, andererseits führten wir die Trennung dieser Stoffe auf einer dünnen Stärkeschicht mit umgekehrter Phase durch. Im ersten Falle wurde eine gute Trennung mit Laufmitteln erreicht, die Petroläther und Tetrachlormethan und deren Gemische enthalten. Im zweiten Falle wurde als stationäre Phase Paraffinöl, als mobile Phase eine Gemisch von Methanol, Wasser und Essigsäure im Verhältnis 16 : 3 : 1 und andere Gemische in gleichen Verhältnissen verwendet, die sich bei der Papier-Verteilungschromatographie bewährt haben.Wasserlösliche Farbstoffe wurden auf einer dünnen Schicht von Polyamidpulver getrennt, das auch zu deren quantitativer Isolation aus den Lebensmitteln verwendet wurde. Die besten Ergebnisse wurden mit alkalischen Laufmitteln erzielt, z.B. mit dem Gemisch von Ammoniak, Methanol und Wasser im Verhältnis 5 : 15 : 80.Die erarbeiteten analytischen Verfahren für Nachweis und Trennung fett- und wasserlöslicher Lebensmittelfarben bewährten sich für alle Lebensmitteltypen.

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Résumé L'utilisation des colorants alimentaires solubles dans l'eau et dans la graisse exige des preuves sûres et l'identification exacte de ces substances dans les aliments. Nous avons étudié ces problèmes en détail à notre institut et élaboré quelques procédés de séparation chromatographique de ces substances par chromatographie sur couche mince.A la séparation des colorants solubles dans la graisse, nous avons utilisé d'une part des couches versées sur l'alumine, d'autre part la séparation de ces substances sur une couche mince de fécule à phase inverse. Au premier cas une bonne séparation fut atteinte aux systèmes comprenant l'éther de pétrole et le tétrachlore carbonique, ou leurs mélanges. A l'autre cas on applique, comme une phase ancrée, de l'huile paraffinique, comme une phase mobile, du mélange de méthanol, d'eau et d'acide acétique en proportion 16 : 3 : 1 et d'autres en mêmes proportions qui avaient fait leurs preuves à la chromatographie de séparation sur le papier.Les colorants solubles dans l'eau furent séparés sur une couche mince de poudre polyamidé qui avait été de même appliqué à leur isolement quantitatif de l'aliment. Les meilleurs résultats furent atteints avec les systèmes alcalins p.e. avec le mélange de méthanol, d'ammoniac et d'eau en proportions 5 : 15 : 80.Les procédés analytiques élaborés pour des preuves et les séparations des colorants solubles dans la graisse et dans l'eau prouvèrent leur attestation avec tous les types de matières alimentaires.

     

Leukocytic responses and intestinal mucin dynamics of broilers protected with Enterococcus faecium EF55 and challenged with Salmonella Enteritidis

The protective effect of Enterococcus faecium EF55 in chickens challenged with Salmonella enterica serovar Enteritidis phage type 4 (SE PT4) was assessed. The antibacterial effect on the bacterial microflora in the small intestine in relation to white blood cell count, phenotyping of peripheral blood and intestinal lymphocytes, functional activity of lymphocytes and phagocytes and mucin quantitation were investigated. Day-old chicks (85) were randomly divided into four groups. The probiotic group (EF) and Salmonella + probiotic group (EFSE) received E. faecium EF55 (10⁹ CFU – 3 g/group/day) for 21 days. The Salmonella group (SE) and EFSE group were infected with Salmonella Enteritidis (10⁸ CFU in 0.2 ml PBS) in a single dose per os on day four of the experiment. The control group chicks (C) were fed a commercial diet without added bacteria. Supplementation of EF55 in the diet of the chickens in the EFSE group, challenged with S. Enteritidis, caused the density of the intestinal mucin layer to increase significantly in non-specific regions (duodenum and jejunum), but decrease significantly in target regions (caeca) for S. Enteritidis. Probiotic treatment also appeared to result in a significantly higher number of lymphocytes in peripheral blood and a tendency to increase CD3, CD4, CD8, and IgM positive cells 3 days post-infection with S. Enteritidis. The results demonstrated an antibacterial effect and suggested that EF55 had a moderating effect on intestinal mucin production and leukocytic response in the early phase of S. Enteritidis infection.     

Correlation of Different Biochemical Parameters in Blood Sera of Healthy and Sick Cows

Metabolic diseases of cows represent the leading internal pathology in Lithuania in terms of incidence and economic impact. This paper summarizes the mineral metabolic state of milk cows, and details the influence of feeding on serum levels of calcium, nonorganic phosphorus, magnesium, potassium, sodium, glucose, total protein, and urea quantity, and correlationd among them, in healthy dry or post-calving cows as well as in cows with osteomalacia and milk fever after calving. There was less pronounced hypocalcaemia and there were only minor changes in phosphorus, magnesium, potassium and sodium in the serum of healthy dry and post-calving cows that had silage and mineral-vitamin supplements, compared to cows that did not have supplements and silage. There was a fall in calcium and phosphorus (to 1.89 +/- 0.12 mmol/L and 0.71 +/- 0.06 mmol/L, respectively) in the blood of cows sick with milk fever after calving, while levels of magnesium and potassium were increased. The correlation between calcium and phosphorus was r = 0.6993, p < 0.001 in the serum of sick cows. There was a fall in calcium, phosphorus and magnesium (to 1.86 +/- 0.46 mmol/L, 0.75 +/- 0.37 mmol/L, and 0.60 +/- 0.19 mmol/L, respectively) and an increase in sodium level (to 158.90 +/- 19.30 mmol/L) in the blood of cows with osteomalacia in comparison with healthy cows.     

Effects of feeding buffering mineral mixture on subacute rumen acidosis and some production traits in dairy cows

This trial was designed in order to evaluate the incidence of subacute rumen acidosis (SARA) during early lactation and to investigate the possibilities for its prevention by use of a buffering mineral mixture. On the beginning of the trial it was found that the pH value of rumen fluid in 4 animals was lower than normal (pH < 6.0) and that 20% of animals have had SARA. The control and the experimental group of cows were fed the same meal with exception of concentrated feed which in the experimental group contained the mineral mix with buffering activity in amount of 1%. Continuous addition of buffering mineral mixture in the amount of 1% in concentrated feed for early lactation cows successfully prevents SARA formation and leads to increased milk production, as well as increased milk fat and protein content.     

N. femoralis, N. obturatorius and N. ischiadicus: deviation in creation in the dogs

Two hundred and twelve dog cadavers belonging to different breeds were examined, to investigate the formation of the femoral, obturator and the sciatic nerve. Besides the commonly described formation patterns of the mentioned nerves, some variations were also found. These variations were not gender-related, but on the other hand we discovered a certain correlation between the variations appearing in the formation of the femoral, obturator and the sciatic nerve. In 74.05% of cases, the femoral nerve was formed from ventral branches of the 4th, 5th and 6th lumbar nerve, and 16.98% of the dogs had the nerve formed from ventral branches of the 3rd, 4th and 5th lumbar nerve. Many dogs (i.e. 2.30%) had the femoral nerve formed from the ventral branches of the 5th, 6th and 7th, the 3rd, 4th, 5th and 6th or the 4th, 5th, 6th and 7th lumbar nerve, respectively. In 1.88% of dogs in particular, the femoral nerve was formed from ventral rami of the 4th and 5th lumbar nerve. In 66.98% of the examined dogs, the obturator nerve was formed from the ventral branches of the 4th, 5th and 6th lumbar nerve, followed by 16.59% of the dogs with the obturator nerve formed from the ventral rami of the 4th and 5th lumbar nerve and 9.43% of dogs in which the nerve was formed from the ventral branches of the 5th and 6th lumbar nerve. In 4.71% of dogs, the obturator nerve was formed from the ventral rami of the 4th, 5th, 6th and 7th lumbar nerve, while only 2.30% of the examined dogs had the same nerve formed from the ventral branches of the 5th, 6th and 7th lumbar nerve. The sciatic nerve was formed from ventral branches of the 6th and 7th lumbar nerve and the 1st sacral nerve in 86.79% of the dogs. In 5.18% of cases, the same nerve was formed from a junction of the ventral branches of the 7th lumbar and the 1st and 2nd sacral nerve, and, in the same percentage of cases, it was formed from a junction of ventral branches of the 6th and 7th lumbar nerve and the 1st and 2nd sacral nerve. In 2.83% of the dogs, it was formed from a junction of the ventral branches of the 6th and 7th lumbar and the 1st sacral nerve. The correlation of variations established in the formation of the femoral, obturator and the sciatic nerve was not statistically significant.     

Antimutagenic effects of extracts from sage (Salvia officinalis) in mammalian system in vivo

Naturally occurring antimutagenic factors, especially those of plant origin, have recently become a subject of intensive research. Antimutagenic properties of terpenoid fractions of sage (Salvia officinalis) were tested in mammalian system in vivo through examining the ability of sage to decrease the frequency of aberrant cells induced by a potent mutagen. First, groups of mice were treated with three concentrations of sage alone and it was established that the frequency of aberrant cells after treatment with a concentration of 25 microL/kg was not significantly different from the negative control (olive oil), while that found after treatment with the 50 microL/kg concentration differed significantly (chi2(1) = 4.05, p < 0.05). Sage used at a concentration of 100 microL/kg was cytotoxic. Mitomycin C (MMC), known as a potent mutagen, was used for induction of chromosome aberrations. Post-treatment with sage suppressed the effects of MMC significantly. Both concentrations (25 microL/kg and 50 microL/kg) produced a significant decrease in the frequency of aberrations relative to MMC (chi2(1) = 5.42, p < 0.02, chi2(1) = 14.93, p < 0.001, respectively). The percent of aberrations decreased with increasing concentrations of sage. Only nontoxic concentrations of sage without mutagenic effects can be recommended for use as inhibitors of mutagenesis or carcinogenesis.     

Genotyping of Bacillus anthracis Isolated from Croatia and Bosnia and Herzegovina

Anthrax is a serious disease caused by Bacillus anthracis. Humans can become infected by handling products from infected animals, by breathing spores and rarely by eating undercooked meat from infected animals. The genome of B. anthracis is highly monomorphic and thus shows very low DNA sequence variation. We analysed the molecular characteristics of 12 B. anthracis isolates from outbreaks in Croatia and Bosnia and Herzegovina, which have occurred during the past 10 years along with two vaccine strains. Genotyping system based on variable‐number tandem repeat analysis at six loci revealed that six isolates belong to genotype from the A1.a cluster whilst six isolates relate to the B2 cluster, compared to 89 previously described genotypes. The distribution of two evolutionarily distant clusters suggests an introduction of B. anthracis to this area in at least two separate events.     

Fructose 1,6-biphosphate aldolase in the sera of Simmental young bulls Connection with the fattening capacity, muscle quantity and protein content

SUMMARY: The activity of enzyme aldolase (ALD) was determined in the sera of Simmental young bulls, with the purpose to see whether there exists a relationship between the enzyme activity and fattening capacity and whether the ALD could be an indicator of the muscle quantity and protein content. Early ALD activity was correlated with slaughter weight and ADG in the last fattening month. The regression analysis suggests that young bulls with higher serum ALD acitivity at the start of the experiment had more total muscles and less bone in the analysed rib part. On the basis of ALD activity in the first fattening month it was possible to estimate protein and fat content in the MLD. ZUSAMMENFASSUNG: Fructose-1,6-biphosphat Aldolase beim Serum Simmentaler Jungbullen. Beziehung zwischen Mastf?higkeit, Muskelmenge und Proteingehalt Aldolase (ALD) Enzymt?tigkeit im Serum von Simmentaler Jungbullen wurden in Hinblick auf eine Beziehung mit Mastf?higkeit, Muskelmenge und Proteingehalt untersucht. Es zeigt sich eine Korrelation zwischen früher ALD Aktivit?t und Schlachtgewicht und Tageszuwachs im Endmastmonat. Regressionsanalyse ergab, da? Jungbullen mit h?herer ALD Aktivit?t bei Mastbeginn mehr Muskelanteil im m. longissimus dorsi und weniger Knochen besitzen. ALD Aktivit?t im ersten Mastmonat erlaubt Sch?tzung des Protein- und Fettgehalts im m. long, dorsi. RESUMEN: Fructosa 1,6-bifosfato aldolasa en el suero de los terneros simentales. Relatión con sus capacidades de ser cebados, su cantidad de músculos y el contenido de proteínas Con el fin de establecer la relación entre la actividad de enzimas y las capacidades durante el proceso de la ceba, asf como para precisar si el ALD puede servir como indicador de la cantidad de músculos y el contenido de proteinas, intentaba determinarse la actividad de los enzimas de aldolasa (ALD) en el suero de los terneros simentales. A base de la actividad indicial del ALD, es posible estimar el peso final de matadero y la crecencia diaria media durante el último mes de la ceba. Tras una análisis regresiva se ha probado que los terneros con una actividad mayor del ALD en el suero a principios del proceso de su ceba tienen más músculos en total en la parte de costillas y m. longissimus dorsi mientras que tienen menos huesos. A base de la actividad del ALD durante el primer mes de la ceba es posible estimar el contenido de proteinas y grasa en m. long, dorsi. RéSUMé: Fructose 1.6-biphosphate aldolase dans le sérum des taurillons de Simmental. Le rapport entre l'aptitude à l'angraissement, la proportion des muscles et la teneur en protéines On a déterminé l'activité de l'enzyme aldolase (ALD) dans le sérum des taurillons de Simmental afin de vérifier s'il existait un rapport entre l'activité des enzymes et l'aptitude à l'engraissement et si l'ALD pouvait être indicatrice de la proportion de muscles et de la teneur en protéines. A la base de l'activité initiale de l'ALD, il est possible d'évaluer le poids de la carcasse et le gain quotidien moyen au cours du dernier mois d'engrais. Par analyse régressive, il a été établi que les taurillons à l'activité supérieure de l'ALD dans le sérum avaient au début de l'engrais plus de muscles au total dans la région costale et le m. longissimus dorsi et moins d'os. A la base de l'activité de l'ALD au cours du premier mois d'engrais il est possible d'évaluer la teneur en protéines et en graisse dans le muscle longissimus dorsi.     

Distribution and Genotypic Characterization of Campylobacter jejuni Isolated from Poultry in Split and Dalmatia County,Croatia

Consumption of poultry contaminated with Campylobacter jejuni has been recognized worldwide as the leading cause of campylobacteriosis. Therefore, the aim of our study was to investigate the prevalence and genotype diversity of Campylobacter jejuni in poultry meat intended for consumption in Split and Dalmatia County, which is the second biggest County in Croatia. Furthermore, we also wanted to discover possibly stable clones of C. jejuni appearing in different samples and periods of time, which would indicate their ability to persist in or adapt to poultry. In the period from March 2008 until June 2010, 834 samples of poultry from various sources were examined using a surface swab technique. Isolation of C. jejuni was performed by Preston broth and Karmali agar. Identification of the isolates was carried out using biochemical tests. C. jejuni was found in 84 of 574 chicken samples (14.6%) and in nine of 260 samples of turkey (3.5%). Pulse‐field gel electrophoresis (PFGE) was used to analyse 61 obtained isolates using SmaI and KpnI. Of 22 different macrorestriction profiles (MRP) that were found, five were detected in poultry from both different locations and periods of time. Samples from 11 locations were found to be contaminated with more than two different genotypes of C. jejuni. Interestingly, the same MRP were found both in poultry declared to be of domestic origin and in the poultry imported from abroad. The prevalence of C. jejuni in poultry samples was in accordance with previously reported results. Genotypic analysis indicated that the population of C. jejuni in Split and Dalmatia County was diverse and that multiple strains of C. jejuni could be found in the same poultry samples. Furthermore, the same genotypes were identified from the samples obtained from different locations and periods of time, which could support the theory of a global existence of certain MRP that are able to persist in or adapt to poultry.     

Microbiological identification and analysis of swine tonsils collected from carcasses at slaughter

The primary objective of this 7-month study was to determine the prevalence of porcine pathogens of the tonsil of the soft palate of swine at slaughter. Additional objectives were to determine if sampling the carcasses of normal or abnormal hogs provided different microbiological profiles and if the slaughter plant provides a feasible sampling frame and environment for detecting and monitoring important pathogens in tonsils that have health implications for both swine and humans. A total of 395 samples were collected from 264 farms. Of these, 180 tonsils were collected from normal carcasses and 215 tonsils were collected from carcasses that were diverted to the hold rail. Laboratory testing included bacteriological culture and identification as well as real time-polymerase chain reaction (PCR) testing for porcine reproductive and respiratory syndrome virus (PPRSV) and immunohistochemistry (IHC) for porcine circovirus-2 (PCV-2). The most commonly isolated bacteria included: Streptococcus suis (53.7%), Arcanobacterium pyogenes (29.9%), Pasteurella multocida (27.3%), and Streptococcus porcinus (19.5%). Virus screening revealed evidence of PRRSV and PCV-2 in 22.0% and 11.9% of the samples, respectively. Salmonella Typhimurium and Yersinia enterocolitica were isolated in 0.5% and 1.8% of the samples, respectively. Tonsils collected from the hold rail were more likely to be positive for Staphylococcus hyicus [odds ratio (OR) = 7.51, confidence interval (CI) = 2.89 to 19.54], Streptococcus porcinus (OR = 9.93, CI = 4.27 to 23.10), and Streptococcus suis (OR = 2.16, CI = 1.45 to 3.24). Tonsils collected from abnormal carcasses were less likely to be positive for Staphylococcus aureus (OR = 0.05, CI = 0.005 to 0.482).