Calcium Chloride Toxicology for Food Safety Assessment Using Zebrafish (Danio rerio) Embryos.

The salt calcium chloride (CaCl2) is widely used in industry as a food additive; levels for human consumption are regulated by international or governmental agencies. Generally, the food industry relies on toxicity studies conducted in mammals such as mice, rats, and rabbits for determining food safety. However, testing in mammals is time-consuming and expensive. Zebrafish have been used in a range of toxicological analyses and offer advantages with regard to sensitivity, time, and cost. However, information in not available with regard to whether the sensitivity of zebrafish to CaCl2 is comparable to the concentrations of CaCl2 used as food additives. The aim of this study was to compare the CaCl2 tolerance of zebrafish embryos and larvae with concentrations currently approved as food additives. Acute toxicity, embryotoxicity, cardiotoxicity, and neurotoxicity assays were used to determine the threshold toxic concentration of CaCl2 in zebrafish embryos and larvae. The data showed that doses above 0.4% had toxic effects on development and on the activity of the cardiac and neuronal systems. Furthermore, all embryos exposed to 0.8 and 1.6% of CaCl2 died after 24 hpf. These findings are consistent with the limits of CaCl2 concentrations approved by Codex Alimentarius. Therefore, zebrafish embryos could be suitable for screening food additives.

Comprehensive Assessment of Functional Effects of Commonly Used Sugar Substitute Sweeteners on Ex Vivo Human Gut Microbiome.

The composition and function of the human gut microbiome are often associated with health and disease status. Sugar substitute sweeteners are widely used food additives, although many studies using animal models have linked sweetener consumption to gut microbial changes and health issues. Whether sugar substitute sweeteners directly change the human gut microbiome functionality remains largely unknown. In this study, we systematically investigated the responses of five human gut microbiomes to 21 common sugar substitute sweeteners, using an approach combining high-throughput in vitro microbiome culturing and metaproteomic analyses to quantify functional changes in different taxa. Hierarchical clustering based on metaproteomic responses of individual microbiomes resulted in two clusters. The noncaloric artificial sweetener (NAS) cluster was composed of NASs and two sugar alcohols with shorter carbon backbones (4 or 5 carbon atoms), and the carbohydrate (CHO) cluster was composed of the remaining sugar alcohols. The metaproteomic functional responses of the CHO cluster were clustered with those of the prebiotics fructooligosaccharides and kestose. The sugar substitute sweeteners in the CHO cluster showed the ability to modulate the metabolism of Clostridia. This study provides a comprehensive evaluation of the direct effects of commonly used sugar substitute sweeteners on the functions of the human gut microbiome using a functional metaproteomic approach, improving our understanding of the roles of sugar substitute sweeteners on microbiome-associated human health and disease issues. IMPORTANCE The human gut microbiome is closely related to human health. Sugar substitute sweeteners as commonly used food additives are increasingly consumed and have potential impacts on microbiome functionality. Although many studies have evaluated the effects of a few sweeteners on gut microbiomes using animal models, the direct effect of sugar substitute sweeteners on the human gut microbiome remains largely unknown. Our results revealed that the sweetener-induced metaproteomic responses of individual microbiomes had two major patterns, which were associated with the chemical properties of the sweeteners. This study provided a comprehensive evaluation of the effects of commonly used sugar substitute sweeteners on the human gut microbiome.

A Pilot Safety Assessment for Recombinant Epinephelus lanceolatus Piscidin Yeast Powder as a Drug Food Additive after Subacute and Subchronic Administration to SD Rats.

Recombinant Epinephelus lanceolatus piscidin (RELP) was previously shown to improve growth performance and immune response when used as a feed additive for Gallus gallus domesticus. However, the long-term toxicity of RELP has not be thoroughly investigated. In the present study, we evaluated the subacute and subchronic oral toxicities of RELP in SD rats by hematological, biochemical, and histopathological analyses. To determine subacute and subchronic toxicities, male and female rats were fed with RELP 1000 mg/kg bodyweight/day for 28 and 90 days, respectively. Bodyweight and food intake were unchanged by RELP treatment over the course of the studies. After exposure, samples of blood, heart, lung, liver, and kidney were collected and analyzed. Results demonstrated that RELP exposure did not cause any observable hematological, biochemical, or histological abnormalities in SD rats. Thus, RELP may be a safe feed additive for use in agriculture and aquaculture.

Edible additive effects on zebrafish cardiovascular functionality with hydrodynamic assessment.

Food coloring is often used as a coloring agent in foods, medicines and cosmetics, and it was reported to have certain carcinogenic and mutagenic effects in living organisms. Investigation of physiological parameters using zebrafish is a promising methodology to understand disease biology and drug toxicity for various drug discovery on humans. Zebrafish (Danio rerio) is a well-acknowledged model organism with combining assets such as body transparency, small size, low cost of cultivation, and high genetic homology with humans and is used as a specimen tool for the in-vivo throughput screening approach. In addition, recent advances in microfluidics show a promising alternative for zebrafish manipulation in terms of drug administration and extensive imaging capability. This pilot work highlighted the design and development of a microfluidic detection platform for zebrafish larvae through investigating the effects of food coloring on cardiovascular functionality and pectoral fin swing ability. The zebrafish embryos were exposed to the Cochineal Red and Brilliant Blue FCF pigment solution in a concentration of (0.02‰, 0.2‰) cultured in the laboratory from the embryo stage to hatching and development until 9 days post fertilization (d.p.f.). In addition, zebrafish swimming behaviors in terms of pectoral fin beating towards the toxicity screening were further studied by visualizing the induced flow field. It was evidenced that Cochineal Red pigment at a concentration of 0.2‰ not only significantly affected the zebrafish pectoral fin swing behavior, but also significantly increased the heart rate of juvenile fish. The higher concentration of Brilliant Blue FCF pigment (0.2%) increased heart rate during early embryonic stages of zebrafish. However, zebrafish exposed to food coloring did not show any significant changes in cardiac output. The applications of this proposed platform can be further extended towards observing the neurobiological/hydrodynamic behaviors of zebrafish larvae for practical applications in drug tests.

Essential oil versus supercritical fluid extracts of winter savory (Satureja montana L.) - Assessment of the oxidative, microbiological and sensory quality of fresh pork sausages.

The effect of winter savory (Satureja montana L.) essential oil (SEO) and supercritical extracts (SE1; SE2) at concentrations of 0.075 and 0.150 µL/g on pH, lipid oxidation (TBARS), microbial growth and sensory quality of fresh pork sausages during storage were investigated. SEO, SE1 and SE2 improved oxidative and microbial stability of fresh pork sausages. The addition of SEO and SE1 at concentration of 0.150 µL/g affected the reduction of total Enterobacteriaeceae count to under 3 log cfu/g. Sausages produced with SE1 and SE2 had a higher scores for odor, flavor and overall acceptance compared to sausages treated with SEO and control sample. Therefore, the results of this paper revealed significant antioxidative and antimicrobial activity of SE1 and SE2, and consequently its high potential of utilization in processing of fresh pork sausages.

A comprehensive approach to formulation of seaweed-enriched meat products: From technological development to assessment of healthy properties.

Meat consumption is influenced by various kinds of factors, among them health implications. Different strategies can be effective in developing meat-based functional foods. These basically entail reducing the presence of compounds with negative health implications and enhancing the presence of beneficial compounds. This article reviews a comprehensive model for the development of meat-based functional foods based on a presentation of the research achieved in terms of the design and development of qualitatively and quantitatively modified meat products (frankfurters, patties and restructured steaks). These were reformulated to incorporate nutrients associated with three different seaweeds (wakame-Undaria pinnatifida; nori-Porphyra umbilicalis; and sea spaghetti-Himanthalia elongata) as sources of bioactive substances, while simultaneously reducing sodium and fat and improving fatty acid profiles. Those seaweeds were chosen, because in terms of composition and health implications, abundance on Spanish coasts, relatively widespread consumption, and suitability in terms of flavour and colour they are better suited than others for use as ingredients in new products. It also discusses the consequences of the use of this type of meat-based functional foods (combination of pork meat and 5% of each seaweed with or without hypercholesterolaemic agent included in the diets) on growing animals (Wistar male rats), and their effects on different aspects of lipoprotein metabolism, oxidative stress and liver structure. This article, then, reports a comprehensive approach to the production of seaweed-enriched meat products, considering aspects of technological development aimed at achieving the functional effect.

Assessment of the functional properties of protein extracted from the brown seaweed Himanthalia elongata (Linnaeus) S. F. Gray.

A protein extract from the brown seaweed Himanthalia elongata (Linnaeus) S. F. Gray was prepared and its functional properties, colour and amino acid composition were assessed for its potential future use by the food industry. The total content of amino acids was determined as 54.02±0.46gaminoacids/kg dry weight, with high levels of the essential amino acids lysine and methionine. SDS-PAGE showed 5 protein bands with molecular weights of 71.6, 53.7, 43.3, 36.4 and 27.1kDa. The water holding capacity and oil holding capacity were determined as 10.27±0.09gH2O/g and 8.1±0.07goil/g respectively. Foaming activity and stability were higher at alkaline pH values. The emulsifying capacity and stability of the extract varied depending on the pH and oil used. These results demonstrate the potential use of Himanthalia elongata protein extract in the food industry.

Assessment of the activity of directly acting antivirals and other products against different genotypes of hepatitis C virus prevalent in resource-poor countries.

Certain food additives and drugs used for other indications have been shown to inhibit in vitro replication of HCV and have been proposed as cheap options for the treatment of HCV infections in resource-poor countries. We here report that the in vitro anti-HCV (genotypes 1a, 1b, 2a and 4b) activity of nitazoxanide, silymarin, silibinin and the green tea extract EGCG is very weak when compared to directly acting antivirals. HCV-infected patients in resource-poor countries should receive the best possible treatment (if possible via expanded access programs); it is therefore advisable not to plan clinical studies with drugs/compounds with weak anti-HCV activity.

[Assessment of the using effectiveness of iodine containing additives in development of meat products for child nutrition].

The effectiveness of iodine containing additives on the basis of whey protein and milk protein casein compared to iodized salt in the composition of meat minced semi-finished products for child nutrition was examined in the experiment on laboratory animals. Four variants of the semi-finished products were investigated: 1 - control; 2 - enriched with iodine containing milk protein casein; 3 - enriched with iodine containing whey proteins; 4 - enriched with iodized salt. The semi-finished products were enriched at the level of 15% of the daily norm of iodine requirement for children at the age of 7-12 years. Iodine content in 100 g of product was 20 µkg. Rats (initial body weight 140±20 g, n=80) were divided into five groups (control, intact and three experimental groups). Groups 1 and 5 included the animals fed with a standard vivarium diet throughout the experiment. The rats from groups 2-4 were fed with the iodine enriched diet: group 2 received diet containing semi-finished products No. 2; group 3 sample No. 3 and group 4 - sample No. 4. The first stage of the experiment was aimed at accumulation of iodine in tissues and organs of animals consumed the tested iodine containing additives in the composition of semi-finished products. The second stage of the experiment consisted in simulation of the mercazolilum-induced (50 mg/kg b.w.) hypothyroidism (iodine deficiency) and detection of preventive effects of iodine containing meat semi-finished products in a model of experimental hypothyroidism in rats. The data obtained upon the end of the experiment suggest that the highest effect for correction of iodine deficiency was achieved when using the culinary products enriched with iodine containing whey proteins (sample No. 3): the level of thyroxine (T4) was restored by 98.7% in the animals from group 3 compared to the indices of the intact group, Т3 by 100%, TSH - by 89.3%. This effect was confirmed by the hematological and biochemical blood indexes, as well as the dynamics of their weight change: the level of white blood cells was significantly lower by 28%, granulocytes by 44%, monocytes by 42% compared to control rats; the weight gain of the animals of the 3 group was 20.3%, closer to that of intact animals - 26.4%, while in the control group it was 2.6 %.

Effect of olive powder and high hydrostatic pressure on the inactivation of Bacillus cereus spores in a reference medium.

The aim of this work was to study the effect of olive powder combined with high hydrostatic pressure (HHP) on the inactivation of Bacillus cereus spores, to use it as an additional control hurdle in beverages pasteurised by this technology. With this purpose, reference medium prepared at different concentrations of olive powder was inoculated with B. cereus spores and subjected to different pressure treatments. The outgrowth capacity of the treated spores was then determined at 20°C and 32°C. The addition of olive powder was found to slightly reduce the effectiveness of HHP, although in post-treatment storage there was an increased bacteriostatic effect in the samples with 2.5% of olive powder at both temperatures in the samples pressurised at 400 and 500 MPa, and only at 20°C in the samples pressurised at 200 MPa. The addition of olive powder therefore had an additive effect with storage temperature and HHP processing and could act as an additional control hurdle during the shelf-life of products pasteurised by HHP technologies or in the case of cold-chain breakage.

Safety assessment of sandalwood oil (Santalum album L.).

Sandalwood (Santalum album L.) is a fragrant wood from which oil is derived for use in food and cosmetics. Sandalwood oil is used in the food industry as a flavor ingredient with a daily consumption of 0.0074 mg/kg. Over 100 constituents have been identified in sandalwood oil with the major constituent being alpha-santalol. Sandalwood oil and its major constituent have low acute oral and dermal toxicity in laboratory animals. Sandalwood oil was not mutagenic in spore Rec assay and was found to have anticarcinogenic, antiviral and bactericidal activity. Occasional cases of irritation or sensitization reactions to sandalwood oil in humans are reported in the literature. Although the available information on toxicity of sandalwood oil is limited, it has a long history of oral use without any reported adverse effects and is considered safe at present use levels.

Determination of the presence of antioxidants deriving from sage and oregano extracts added to animal fat by means of assessment of the radical scavenging capacity by photochemiluminescence analysis.

Herbs and their extracts with antioxidant capacity could be used directly as stabilisers of fat and indirectly as feed additives, in order to improve quality and shelf-life of meat and fat-containing food. In this work a sensitive analytical method is proposed for determination of the antioxidant activity measured by photochemiluminescence (PCL) in lard stabilised with extracts of sage (Salvia officinalis L.) or oreganum (Origanum vulgare L.). A prior step of purification of fat samples is required, in order to separate and concentrate the phenolics from lipidic substances. The method was validated by determination of recovery rate and repeatability. In addition fat samples originating from pigs fed with feed additives of Salviae folium or Origani herba were analysed to investigate the supposed antioxidative effects, that could increase the shelf-life of meat products. In contrast with lard mixed with extracts of sage or oregano, back fat samples originating from pigs fed with feed additives of the same herbs didn't show a higher antioxidant activity than the control group. On the one hand it seems possible to keep perishable fat-containing food longer by an addition of an extract of sage or oregano due to their antioxidative properties, on the other hand administration of feed additives of dried herbs to pigs had no effect on quality and shelf-life of fat obtained from these animals.

Feed additives.

Feed additives used to supplement dairy rations can increase daily feed cost from 2 to 65 per animal. The economic response (benefit to cost ratio) varies from $2.00 to $12.00 for each dollar invested in a specific additive. Knowledgeable dairy veterinarians must understand the role of each additive, when it will be beneficial, inclusion level, strategies and limitations associated with an additive, research and field recommendations, and measurable responses to monitor. Dairy additives and various methods to evaluate their role and economic response objectively are discussed.

Improving food quality through new technology.

New technological developments make it possible to improve the quality of animal disease therapy, prophylaxis and diagnosis, and to improve animals' growth and fertility. The term 'quality' includes not only objective measurements, such as the fatness or leanness of meat, but also organoleptic factors such as flavour and others which are of increasing importance to consumers, such as animal welfare. Is new technology consistent with the improvement of quality? For example, beta-agonists and porcine somatotrophin reduce the fatness and increase the protein content of carcases, but there are also subtle positive relationships between fatness and eating quality. In contrast, bovine somatotrophin appears to have no effect on milk composition but there are indications that it may affect the perception of milk quality by some consumers. Improved vaccines can increase food quality by improving animal health and welfare and increasing the uniformity of the product; immunological techniques may also be used to improve meat quality. A gulf has developed between the benefits from new technology and consumer perceptions; indeed there is evidence of political resistance to some technological advances. Despite the stringent regulation of veterinary medicinal products in the United Kingdom and other countries, there is continued pressure for greater political control over their approval. The exchange of information between scientists, industry, the legal regulators and consumers must be improved so that advances in technology are acceptable to the majority and used to the advantage of all.

Assessment of immunobiological effects induced by chemicals, drugs or food additives. I. Tier testing and screening approach.

The indications of immunologic dysfunction following low level exposure of rodents or even man to certain chemicals and drugs have raised concern regarding methodology and approaches for routine assessment of immunobiological effects. The immunogiological effect observed may either indicate impaired immunopotentiation or hypersensitization. The assays selected for testing should be relevant to the human experience and adaptable to certain practical considerations such as cost, reproducibility of data, ease of performance and application to routine toxicology studies. Using these considerations, a tier approach was proposed consisting of assays for screening for immunologic effects (Tier I) and assays to help define the mechanisms responsible for the immunobiological effects observed (Tier II). A tier of assays was also proposed for measuring the sensitization potential of certain compounds. Certain assays from the screening tier were assembled into a hypothetical and practical test battery to screen for immunological effects of a chemical with potential immunosuppressive properties. Information provided by this test battery should provide a reasonable and sensitive data base from which a standard of evaluation could be made regarding the safety of the test (1) compound.