Simultaneous determination of sorbic acid, benzoic acid and parabens in foods: a new gas chromatography-mass spectrometry technique adopted in a survey on Italian foods and beverages.

A gas chromatographic-mass spectrometric technique is described for the simultaneous determination of sorbates, benzoates and other lipophilic preservatives in foods and beverages. The selected ions monitoring (SIM) technique allowed unambiguous identification of the compounds under study. This methodology eliminated all kinds of interferences from the complex food matrices which affect most routinely-used techniques, HPLC included. A very simple and time-saving extraction procedure was therefore employed, since subsequent purification steps were unnecessary, even for detection of trace levels of preservatives. The detection limits fell within the range of 100-200 pg. With this analytical technique, we have conducted a survey on sorbic acid, benzoic acid, methyl, ethyl, and propyl 4-hydroxybenzoate levels in 249 samples of foods and beverages on sale in markets in the Rome area. Samples were chosen from among those currently preserved by these additives. All compounds were also determined by a routinely-used HPLC technique for method comparison.

Effect of some food preservatives on aflatoxin production.

The effect of some food preservatives, such as sorbic (SA) and propionic (PA) acids, on aflatoxin production in synthetic media or in moistened (20%) wheat seeds, was investigated. The preservatives tested, added to synthetic media at sublethal concentrations both at the inoculum and after 5 days of incubation, stimulated aflatoxin production by Aspergillus parasiticus. Sorbic and propionic acids are metabolized by the fungus in vivo and in vitro. Lower concentrations of PA and SA (0.05 to 0.1% w/w) in wheat seeds are ineffective against both fungal growth and aflatoxin production, whilst the combined use of butylated hydroxy toluene (BHT), and PA or SA was more effective in controlling aflatoxin production than their use as single components.

Nutritional aspects of food preservatives.

Despite the benefits attributed to food preservatives, some concern still remains regarding their safety and possible influence on nutrients. Surprisingly, there is quite a lack of scientific knowledge in this field. In order to describe a few examples, the effects of the extensively used sulphite on thiamine, folates, pyridoxal and other nutrients have been reported. Among its antibrowning effects, inhibition of ascorbic acid browning is also considered. As far as sorbic acid is concerned, notwithstanding its easy reaction with protein, probably the acid environment of the stomach determines the breakdown of the sorbic-protein adducts. Detoxication of nitrite by tocopherol and ascorbic acid leads, in the last case, to dehydroascorbic acid and its oxidative products with loss of vitamin activity. Any oxidizing substance destroys ascorbic acid, vitamin E and free vitamin A. Phosphates are largely used with different aims, including preservation, in food processing. Their antimicrobial activity is due to both a direct effect and an interaction with other antimicrobials. Sequestering capacity of phosphates and its nutritional implications are discussed. Also mechanisms of action of organic acids are reported, focusing on sorbic acid effects on single amino acids and proteins. Finally, the little information available about the potential impact of food preservatives on nutritional functions is presented.

In vitro model for the evaluation of toxicity and antinutritional effects of sulphites.

The food preservatives, sulphur dioxide and its salts, are known to present some toxic, mutagenic and antinutritional effects; in fact they interact with a number of nutrients, e.g. some vitamins, notably thiamine (Th) and folic acid (FA). The effect of different concentrations of sodium bisulphite in cell culture media has been studied in vitro on a human cell line, HEp-2, deriving from a carcinoma of the larynx. Moreover, the sulphites have been tested with different levels of Th and FA with the aim of elucidating how much the cellular response depended on either the anti-nutritional effect or the toxicity of sulphites. Cell growth has been taken as an index of cytotoxicity and measured both as total protein content and as colony-forming ability. With no Th and FA in the culture medium, a clear decrease of cell growth was observed either with or without addition of sodium bisulphite. A dose-dependent reduction of protein content was detected in cells treated with 10, 50, 100, 200, 250 or 500 microM sodium bisulphite. Moreover, when the cells were treated with 10 or 100 microM of this compound, the colony-forming ability was reduced both in number and colony size. As far as the interaction of the two vitamins with sodium bisulphite is concerned, when these nutrients were present in the medium at 0.5, 1.0, 1.5, 2.0 or 2.5 mg/l, a similar growth profile, determined from their concentration, was observed in treated and control cells, the growth levels being affected by the sodium bisulphite contents. At higher levels of Th and FA, the growth index was still increasing only in treated cells, this phenomenon being particularly evident in cultures treated with 200 microM sodium bisulphite. The colony-forming ability was reduced in controls but still increased in treated cells at the highest concentration of vitamins.

Evaluation of an HPLC method for the determination of sulphiting agents in foods.

A method based upon a combination of a modified Monier-Williams procedure and an HPLC separation and quantitation of sulphite, has been developed. Its efficiency has been tested, in comparison with the Monier-Williams method, for the SO2 recovery in various foods. Desorption of SO2 from different matrices is obtained by distillation in strongly acid solution. Gaseous SO2, collected and oxidized to sulphate with hydrogen peroxide, is neutralized with sodium hydroxide. The neutralized solutions are diluted and injected by an autosampling injector into an HPLC apparatus consisting of a strong anion exchange column, eluted with a potassium hydrogen phthalate solution (0.15 g/l, pH 5.7) at a 3 ml/min flow rate and detected at 280 nm by an UV spectrophotometer. The combined method shows a good detection limit as well as high chromatographic resolution, avoiding potential interference of other volatile compounds. It is also time-saving and utilized basic laboratory equipment.

Effect of dietary lipids on hepatic and intestinal monooxygenases in mice.

The effect of dietary lipids on hepatic and intestinal monooxygenases was studied by feeding C57BL/6N mice (for 2 wks) diets containing 5% and 23.5% (wt/wt) olive oil or corn oil. At the end of the feeding period, we measured arylhydrocarbon hydroxylase (AHH) activity in S9 preparations from liver, small intestine, and colon; and, using the same S9 preparations from the liver, we observed the activation of the following three dietary promutagens: 2-amino-3-methylimidazo(4,5-f)quinoline, 2-amino-3,8-dimethylimidazo(4,5-f) quinoxaline, and 2-amino-6-methyldipyrido(1,2-a:3',2'-d)imidazole. The results showed that high-fat diets increased hepatic AHH activity both in corn oil and olive oil diets compared with the low-fat diets; also, a 5% corn oil diet had significantly higher AHH activity compared with the 5% olive oil diet. AHH activity was, respectively, 48.6 +/- 5.1 and 79.5 +/- 11.4 pmol 3OH-benzo[a]pyrene formed/mg/min in the 5% and 23.5% olive oil diets and 66.1 +/- 5.1 and 83.9 +/- 12.2 in the 5% and 23.5% corn oil diets; values are means +/- SE, n = 16. The results also showed a significant increase in the ability of hepatic S9 fractions from animals on high-fat diets to activate promutagens in the Salmonella/plate test. On the contrary, AHH activity in the small intestine and colon was not affected by the fat content of the diet.

Acute toxicity of 3-deoxy-4-sulphohexosulose in rats and mice, and in vitro mutagenicity in the Ames test.

3-Deoxy-4-sulphohexosulose (DSH) is formed in sulphited foods by the interaction of SO2 and intermediates of the Maillard reaction. The acute intragastric toxicity of DSH has been studied in rats and mice, and the LD50 was found to exceed 5 g/kg body weight in both species. The only adverse effect seen in a 14-day post-dosing period was a transient diarrhoea in the first 24 hr. DSH was shown to be non-mutagenic in four strains of Salmonella typhimurium in the Ames test, with and without metabolic activation by S-9 mix from Aroclor-treated rats.

Metabolism of 3-deoxy-4-sulphohexosulose, a reaction product of sulphite in foods, by rat and mouse.

The excretion of single intragastric doses of 14C-labelled 3-deoxy-4-sulphohexosulose (DSH) was studied in male CF1 mice and male and female Wistar albino rats. Urine and faeces were collected 6, 12, 24, (36), 48 and 72 hr after administration of 2100 mg [14C]DSH/kg body weight (to mice), 1700 mg/kg (to male rats) and 100 and 500 mg/kg (to male and female rats). After 72 hr, plasma and total carcass levels were determined in some experiments. In mice 29% of the administered radioactivity was excreted in the urine, 50% in the faeces and some 13% in cage washings. In rats, faecal excretion varied between 58.5 and 73%. Urinary excretion varied between 16.5 and 31% and was slightly higher in male than in female rats. No radioactivity was detected in expired air of rats, and carcass levels in rats and mice after 72 hr were less than 0.1% of the dose. TLC analysis of urine extracts revealed only unchanged [14C]DSH. In similar studies, male rats and mice were given 35S-labelled DSH in a dose of 6500 mg/kg or 10,700 mg/kg, respectively. Urinary activity accounted for 19.5% of the dose in rats and 27.5% in mice by 72 hr and no 35S-labelled sulphate was detectable in the urine. Organ analyses at nine intervals from 0.25 to 24 hr after intragastric administration of 1600 and 1800 mg [14C]DSH/kg to male rats and mice, respectively, showed that at all times most of the 14C activity was associated with the gastro-intestinal tract in both species. Maximum tissue levels were 2.16% of the dose in the rat liver 0.5 hr after dosing and 1.57% in the mouse kidney after 0.25 hr. Significant amounts of activity (greater than 0.25% of the dose) occurred transiently also in the pancreas and lungs of both species, in the rat testes and in the mouse bladder. Maximum plasma levels were 0.09% of the dose/ml in rats 0.5 and 1 hr after dosing and 0.34%/ml in mice at 0.25 hr.

[Chemical and nutritional evaluation of commercial bakery products for children (author's transl)]. / Studi sul valore nutritivo di alcuni prodotti da forno destinati prevalentemente all'infanzia

Various bakery products, differing in either composition or processing, all widely consumed in Italy by school children, have been studied. Protein, lipid, reducing and total sugars, amino acid and available lysine contents have been determined, as well as biological parameters have been calculated to verify hypotheses about growth factors. The products examined have low protein biological value and the proteins are considerably demaged by technological processing. Lysine, always the limiting amino-acid in these proteins, is shown to be unavailable in percentages varying from 20 to 60% of the total lysine. Upon describing growth as a function of protein content, the correlation improves considerably. Furthermore, when this variable, protein content, is associated with the corrected chemical score in a linear model, the correlation reaches even higher values.

[Investigation on wheat varieties of better protein quantity and quality (author's transl)]. / Ricerca di varietà di frumento migliori per quantità e qualità proteiche

A research has been conducted on several hundreds of cultivars and selection lines of bread wheat to find out new varieties of better nutritive characteristics. For a coarse evaluation of protein quantity and quality, a screening method (dyebinding capacity, DBC) was utilized. On the same samples, nitrogen was determined also by the Kjeldahl method; in some cases aminoacids analysis was carried out. A very high correlation was found between protein content and DBC and, even higher in this case, between DBC and lysine content (in samples with a high DBC). In this way it has been possible to sort out a certain number of varieties of interest from a nutritive standpoint and in relation to their protein and essential amino acids contents. The importance of these findings is discussed.

Genetic control of esterases in common wheat.

A refined technique of gel electrofocusing revealed the esterases in caryopses of Triticum aestivum, T. durum, and Triticum timopheevi. In T. aestivum, 17 isoenzymatic bands were ascertained in the pH 5-8 range: 11 were of higher intensity, 4 were weak and two very weak. Using Chinese Spring nulli-tetrasomic lines it was possible to locate the genetic control of several isoenzymes in the chromosomes of the homoeologous group 3. In chromosome 3A three bands are coded; in 3B four bands are coded; and in 3D two bands out of the eleven of higher intensity. T. durum, as expected, lacks bands coded in T. aestivum by chromosome 3D. T. timopheevi presents a quite distinct isoenzyme pattern, thus confirming its different speciation. Two major bands do not disappear in any of the nulli-tetra lines analyzed: it is supposed that these isoenzymes could be coded by at least two of the chromosomes of the group three involved in esterase control. The presence of several esterase isoenzymes in wheat is both evidence of their additivity with increasing ploidy level and biochemical support for the hypothesis that there is a higher possibility of adaptation of polyploids compared with diploid species.