Nile red staining for rapid screening of plastic-suspect particles in edible seafood tissues.

Concerns regarding microplastic (MP) contamination in aquatic ecosystems and its impact on seafood require a better understanding of human dietary MP exposure including extensive monitoring. While conventional techniques for MP analysis like infrared or Raman microspectroscopy provide detailed particle information, they are limited by low sample throughput, particularly when dealing with high particle numbers in seafood due to matrix-related residues. Consequently, more rapid techniques need to be developed to meet the requirements of large-scale monitoring. This study focused on semi-automated fluorescence imaging analysis after Nile red staining for rapid MP screening in seafood. By implementing RGB-based fluorescence threshold values, the need for high operator expertise to prevent misclassification was addressed. Food-relevant MP was identified with over 95% probability and differentiated from natural polymers with a 1% error rate. Comparison with laser direct infrared imaging (LDIR), a state-of-the-art method for rapid MP analysis, showed similar particle counts, indicating plausible results. However, highly variable recovery rates attributed to inhomogeneous particle spiking experiments highlight the need for future development of certified reference material including sample preparation. The proposed method demonstrated suitability of high throughput analysis for seafood samples, requiring 0.02-0.06 h/cm2 filter surface compared to 4.5-14.7 h/cm with LDIR analysis. Overall, the method holds promise as a screening tool for more accurate yet resource-intensive MP analysis methods such as spectroscopic or thermoanalytical techniques.

Preparation of pure lower-order myo-Inositol phosphates on laboratory scale for physiological and enzymatic studies.

A simple method for the preparative production of lower-order myo-inositol phosphates was developed. Enzymatic phytate dephosphorylation was applied, because phytate-degrading enzymes generate usually predominantly one single myo-inositol phosphate isomer with five, four, three, two and one phosphate residue(s) bound to the myo-inositol ring in a regio- and stereoselective manner. The relative concentrations of the different lower-order myo-inositol phosphates in the reaction mixture were controlled by adjusting incubation time at 37 °C and a fixed phytate concentration and phytase activity. Purification of the individual lower-order myo-inositol phosphates was realized by anion-exchange chromatography on Q-Sepharose using a stepwise elution with ammonium formate:formic acid pH 2.5. Ethanol precipitation was successfully used to concentrate the pure lower-order myo-inositol phosphates. In a single approach 2-3 mg of pure myo-inositol tetrakis- or -trisphosphate isomers were obtained. About 60% of the initially applied phytate were converted into pure lower-order myo-inositol phosphates. The purified myo-inositol phosphate isomers were virtually free of other myo-inositol phosphate esters and could be used for enzymatic and physiological studies.

Limitations of an in vitro model of the poultry digestive tract on the evaluation of the catalytic performance of phytases.

BACKGROUND: The study aims to investigate the limitation of a poultry digestive tract model developed by Menezes-Blackburn et al. [J Agric Food Chem 63: 6142-6149 (2015)] on the evaluation of the bioefficacy of phytases. RESULTS: It was confirmed that the in vitro model does not mimic the in vivo situation in the birds sufficiently well to identify the best phytase product under real conditions, or to draw conclusion on the effect of phytate concentration, phytate source or feed composition on the bioefficacy of phytase. Addition of calcium ion (Ca2+ ) up to a concentration of 10 g kg-1 to the feed substrate, for example, did not affect enzymatic phytate dephosphorylation in the in vitro model in contrast to the observation in poultry. CONCLUSION: The in vitro approach was shown to be applicable as a complementary tool in the pre-selection of promising phytase candidates, resulting in a reduction in the number of feeding trials in the initial screening phase. © 2020 The Author. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Influence of salt concentration and iodized table salt on the microbiota of fermented cucumbers.

The fermentation of vegetables is a traditional preservation method, that experiences a renaissance even in domestic households. Table salt is added to the fermentation batches to favor the growth of lactic acid bacteria usually. On an industrial scale, the fermentation brine is typically prepared with non-iodized table salt. In our study, we investigated the microbiota of cucumber fermentations using culture-dependent and -independent methods. We could show that the fermentation process of cucumbers and the involved microbiota is influenced by the concentration of table salt and not by the use of iodized table salt. Therefore, we conclude that the use of iodized table salt does not negatively affect the fermentation process. We could verify that iodine permeates the cucumbers by diffusion, leading to satisfactory iodine concentrations in the final food product. The industrial use of iodized table salt in food fermentations could contribute to maintain a constant iodine supply to the general public.

Effect of phytase treatment of sorghum flour, an alternative for gluten free foods and bioaccessibility of essential minerals.

This study evaluated the effect of phytase treatment on the bioavailability of iron (Fe), calcium (Ca), zinc (Zn), and myo-inositol phosphate fractions in sorghum flour; and characterized its macronutrients and minerals. The proximate composition and mineral content indicated that, sorghum flour has a nutritional potential superior to wheat and maize. The results obtained in the solubility and dialysis assays indicated that, naturally occurring minerals (without phytase treatment) in sorghum flour, presented considerable bioaccessibility; reaching 32, 47 and 67% of dialyzable Fe, Zn, and Ca respectively. The use of phytase had a positive influence on the reduction of myo-inositol phosphates, mainly the IP6 fraction, present in sorghum flour samples, and an increase in the soluble percentage (Fe 52% for one sample, for Zn higher than 266%) and dialyzed minerals (Fe 7.8-150%; Zn 19.7 for one sample; and Ca 5-205%) for most samples. Therefore, the essential minerals naturally occurring in sorghum have an absorption potential; and the use of phytase reduced the IP6 fraction and improved the availability of the minerals evaluated.

Particle size of milled chokeberry pomace did not influence in vitro cellular absorption and transport efficiencies of anthocyanins, phenolic acids and flavonols.

Industrial chokeberry pomace is very rich in polyphenols. The main focus here lies on the possible relationship between the particle size of chokeberry milled pomace and an enhanced absorption and transport of polyphenols by Caco-2 cells. Wet milling was used to produce materials with particle size distributions in the micrometre and in the sub-micrometre to nanometre ranges starting from chokeberry pomace. Milled materials with about 50% of the particles with a mean size (x50,3) of 223 ± 13 µm (coarse milling) and about 90% of the particles with x50,3 of 160 ± 40 nm (fine milling, sonication) were obtained. None of the milled materials exhibited cytotoxic effects within the tested concentration-ranges. The polyphenol absorption and the transport efficiencies from the fine and the coarse milled materials were similar. Thus, no effect of the particle size upon cellular uptake and transport could be established, but agglomeration of particle during incubation cannot be excluded as the cause. Furthermore, based on polyphenol stability we postulate that direct milling may be applied to valorise the processing by-product from commercial fruit juice production.

Isolation and identification of temperature tolerant phosphate solubilizing bacteria as a potential microbial fertilizer.

Isolation and identification of temperature tolerant phosphate solubilizing bacteria (TTPSB) and their use as microbial fertilizers was the main goal of the study. In this study, TTPSB were isolated from soil samples treated for 16 h at 55 °C. Their phosphate solubilizing activity was either evaluated in solid media by forming a clear zone (halo) or in liquid media by quantification of the soluble phosphate in the growth medium. Five colonies (RPS4, RPS6, RPS7, RPS8 and RPS9) were identified to be able to form a halo and two of the isolates (RPS9 and RPS7) tolerated a temperature of 55 °C. With tricalcium phosphate (TCP) as the sole P-source, the phosphate solubilizing capacity of RPS9 and RPS7 was determined to be 563.8 and 324.1 mg P L-1 in liquid Sperber medium, respectively. Both bacterial isolates were identified as Pantoea agglomerans by molecular and biochemical characterization. To be used as a microbial fertilizer a carrier system for the temperature tolerant bacteria consisting of rock phosphate, sulfur and bagasse was used. It could be established that the bacterial cell counts of the microbial fertilizers were acceptable for application after storage for 4 months at 28 °C. In a greenhouse experiment using pot cultures, inoculation of maize (S.C.704) with the microbial fertilizers in an autoclaved soil resulted in a significant effect on total fresh and dry weight of the plant root and shoot as well as on the P content of the root and shoot. The effects observed with RPS9 as a component of the microbial fertilizer on plant growth and P nutrition was comparable with the addition of 50% of recommended triple superphosphate (TSP) dose. Using temperature tolerant bacteria in microbial fertilizers will overcome limitations in production and storage of the microbial fertilizers and contribute to a environmentally-friendly agriculture. The temperature tolerant P. agglomerans strain RPS9 was shown to be effective as part of a microbial fertilizer in supporting the growth and P uptake in maize.

The effects of food essential oils on cardiovascular diseases: A review.

Essential oils (EO) are complex secondary metabolites, which are produced by aromatic plants and identified by their powerful odors. Present studies on EO and their isolated ingredients have drawn the attention of researchers to screen these natural products and evaluate their effect on the cardiovascular system. Some EO, and their active ingredients, have been reported to improve the cardiovascular system significantly by affecting vaso-relaxation, and decreasing the heart rate and exert a hypotension activity. Several mechanisms have been proposed for the role of EO and their main active components in promoting the health of the cardiovascular system. The objective of this review is to highlight the current state of knowledge on the functional role of EO extracted from plants for reducing the risk of cardiovascular diseases and their mechanisms of action. Research on EO has the potential to identify new bioactive compounds and formulate new functional products for the treatment of cardiovascular diseases such as arterial hypertension, angina pectoris, heart failure, and myocardial infarction.

Screening and Characterization of Phytases from Bacteria Isolated from Chilean Hydrothermal Environments.

Phytases are enzymes involved in organic phosphorus cycling in nature and widely used as feed additives in animal diets. Thermal tolerance is a desired property of phytases. The objectives of this study were to screen and characterize bacterial phytases from Chilean hydrothermal environments. In this study, 60% (30 of 63) of screened thermophilic (60 °C) isolates showed phytase activity in crude protein extracts. The characterization of phytase from two selected isolates (9B and 15C) revealed that both isolates produce phytases with a pH optimum at 5.0. The temperature optimum for phytate dephosphorylation was determined to be 60 and 50 °C for the phytases from the isolates 9B and 15C, respectively. Interestingly, the phytase from the isolate 15C showed a residual activity of 46% after incubation at 90 °C for 20 min. The stepwise dephosphorylation of phytate by protein extracts of the isolates 9B and 15C was verified by HLPC analysis. Finally, the isolates 9B and 15C were identified by partial sequencing of the 16S rRNA gene as members of the genera Bacillus and Geobacillus, respectively.

Influence of iodized table salt on fermentation characteristics and bacterial diversity during sauerkraut fermentation.

The effect of iodine present in 1.0% table salt in combination with the use of starter cultures in sauerkraut fermentations were investigated in order to determine whether iodine interferes with lactic acid bacteria responsible for the fermentation. The effect of iodine was tested in fermentations performed using selected starter cultures or without starters (spontaneous fermentation). Lactobacillus plantarum and Leuconostoc mesenteroides used as starters at levels of ca. 1 × 107 cfu ml-1 led to a quick establishment of lactic acid bacteria (LAB) as predominant microorganisms, reaching 1 × 109 cfu ml-1 after 24 h decreasing the pH to below 4.0. In contrast, LAB counts in control fermentations without starters increased slower from 1 × 105 cfu ml-1 to 1 × 109 cfu ml-1 and a pH reduction below 4.0 was achieved only after 3 days fermentation. A metagenomic investigation showed a more diverse bacterial community in fermentations without starters, consisting of enterobacteria and pseudomonads in the first days of fermentation, and of LAB such as lactococci in the later stages. In fermentations with starters, lactobacilli predominated. Leuconostocs also occurred, but at much lower sequence abundance than lactobacilli, and thus were not able to predominate. Determination of iodine in the fermentation with starter bacteria and with iodized salt showed that the fermentation did not affect iodine concentration. The use of iodized salt did not statistically significantly influence microbial populations in the fermentation. Thus, there is no basis for the popular held belief that the use of iodized salt inhibits the growth of the bacteria important for the sauerkraut fermentation. A statistically near significant effect (p = 0.06), however, was noted for the effect of iodine on yeasts and mould populations in the fermentations performed without starter cultures. As sauerkraut is usually produced without starters, this should be further investigated.

Dephosphorylation of myo-inositol phosphates in the in vitro intestinal Caco-2 cell model.

Plant and microbial phytases present in raw materials can cause a dephosphorylation of phytate (myo-inositol hexakisphosphate) (InsP6)) during food processing resulting in a broad range of different myo-inositol phosphates such as pentakisphosphate (InsP5) and tetrakisphosphate (InsP4) in foods. Here, we investigated whether the human intestinal epithelium is able to dephosphorylate myo-inositol phosphates (InsP6, InsP5-, InsP4-, InsP3-isomers) using an in vitro model with differentiated human Caco-2 cells cultured on semipermeable inserts. Incubation of InsP6 and an InsP5-isomer with cells for 3 h showed no dephosphorylation of both InsPs. Treatment of cells with a mixture of different InsP4-isomers, however, caused a formation of about 3.5% of an InsP3-isomer (Ins(1,5,6)P3) and treatment with a mixture of different InsP3-isomers caused about 20% formation of InsP2-isomers, respectively. Thus, human intestinal cells can contribute to the dephosphorylation of myo-inositol phosphates of partly dephosphorylated forms such as InsP3 and InsP4.

A novel purple acid phytase from an earthworm cast bacterium.

BACKGROUND: Phytases are a diverse group of enzymes initiating the dephosphorylation of phytate. Phytate is considered as an anti-nutritional compound because of its capability to chelate nutrients such as Fe2+ , Zn2+ , Mg2+ , and Ca2+ . In this study, several bacterial isolates obtained from earthworm casts were evaluated for their phytate degrading capability. Enzymatic properties and the sequence of the corresponding phytase-encoding gene of the selected isolate were determined. RESULTS: The phytase exhibited its highest activity at pH 4.0 and was stable from pH 3 up to pH 9. The temperature optimum was determined to be 65 °C. The strongest inhibitors of enzymatic activity were identified as vanadate, Cu2+ , and Zn2+ . High-performance ion chromatography analysis of enzymatic phytate dephosphorylation revealed that the first dephosphorylation product was d/l-myo-inositol(1,2,3,4,5)pentakisphosphate. CONCLUSION: Owing to its enzymatic properties, such as tolerance to tartrate and the presence of the consensus motifs PDTVY, GNHE, DLG, VLFH, and GHDH, this phytase could be classified as a purple acid phytase. To the best of our knowledge, this is the first report describing a bacterial purple acid phytase. © 2017 Society of Chemical Industry.

Effects of natural blends of garlic and eucalypt essential oils on biogas production of four fibrous feeds at short-term of incubation in the ruminal anaerobic biosystem.

BACKGROUND: The present study explored the effect of garlic and/or eucalypt oils on biogas production during in vitro ruminal fermentation of four agro industry byproducts. For this, 0-180 mg oil L-1 incubation medium was added and gas volumes were recorded from 2 to 48 h of incubation. Dry matter substrate degradability and neutral as well as acid detergent fibre were determined after 72 h. RESULTS: Gas production and nutrient degradability was oil type dependent. The oils enhanced (P < 0.05) biogas and asymptotic biogas production for corn stalks and oat straw, although no effect was observed on asymptotic biogas production for sorghum straw and sugarcane bagasse. Addition of both oils decreased (P < 0.05) fermentation pH for corn stalks, sorghum straw and oat straw and also increased (P < 0.05) dry matter degradability for all four byproducts. Neutral detergent fibre degradability for all byproducts was higher (P < 0.05) with garlic oil. Eucalypt oil, however, decreased (P < 0.05) neutral detergent fibre degradability for sugarcane bagasse and corn stalks, although only weak effects were observed for sorghum and oat straws. CONCLUSION: With respect to ruminal biogas production, the addition of garlic oil showed better environmental effects than the addition of eucalypt oil and increasing oil concentrations resulted in enhanced fermentation characteristics. © 2018 Society of Chemical Industry.

The effect of exogenous phytase supplementation on nutrient digestibility, ruminal fermentation and phosphorous bioavailability in Rambouillet sheep.

BACKGROUND: The effect of phytase supplementation with respect to a high sorghum grain diet on sheep voluntary feed intake, apparent nutrient digestibility, ruminal fermentation, phosphorus (P) excretion and blood serum P concentration was evaluated. RESULTS: Phytase supplementation significantly decreased fecal P excretion (P = 0.003), resulting in a 26% decrease in relation to the phytase free diet. Dry matter intake, nutrient digestibility, ruminal butyrate and serum P were not (P > 0.10) affected by the phytase level. Neutral detergent digestibility showed a tendency to increase linearly (P = 0.10) with increasing phytase levels. Ruminal pH was lower for phytase supplemented sheep, with a significant decrease (P = 0.007) at 9 h post feeding, whereas ruminal ammonia-N at 3 h post feeding was lower (P = 0.004) for the phytase treatment groups, resulting in a decreasing linear response (P = 0.001) with an increasing phytase dose. Duodenal pH was significantly reduced at 6 h post feeding. Propionate tended (P = 0.051) to be increased linearly as the phytase supplementation level increased. CONCLUSION: Exogenous phytase supplementation of high sorghum grain diets significantly decreased fecal P excretion in Rambouillet rams. Phytase supplementation appears to affect neutral detergent fiber digestibility, duodenal and ruminal pH, ammonia and propionate. © 2018 Society of Chemical Industry.

New Trends in the Microencapsulation of Functional Fatty Acid-Rich Oils Using Transglutaminase Catalyzed Crosslinking.

Preparing stable protein-based microcapsules containing functional fatty acids and oils for food applications has been a big challenge. However, recent advances with transglutaminase (TGase) enzyme as an effective protein cross-linker could provide workable solutions for the encapsulation of omega-3 and omega-6 fatty acids without compromising their targeted release and their biological and physicochemical characteristics. The recent and available literature related to the microencapsulation techniques, physical and oxidative properties, and core retention and release mechanisms of TGase-crosslinked microcapsules entrapping edible oils were reviewed. The effects of factors involved in microencapsulation processes, on the efficiency and quality of the produced innovative microcapsules were also discussed and highlighted. A brief focus has been finally addressed to new insights and additional knowledge on micro- and nanoencapsulation of lipophilic food-grade ingredients by TGase-induced gelation. Two dominant microencapsulation methods for fish, vegetable, and essential oils by TGase-crosslinking are complex coacervation and emulsion-based spray drying. The developed spherical particles (<100 µm) with some wrinkles and smooth surfaces showed an excellent encapsulation efficiency and yield. A negligible release rate and a substantial retention level can result for different lipid-based cores covered by TGase-crosslinked proteins during the oral digestion and storage. A significant structural, thermal and oxidative stability for edible oils-loaded microcapsules in the presence of TGase can be also obtained.

Recent advances in γ-aminobutyric acid (GABA) properties in pulses: an overview.

Beans, peas, and lentils are all types of pulses that are extensively used as foods around the world due to their beneficial effects on human health including their low glycaemic index, cholesterol lowering effects, ability to decrease the risk of heart diseases and their protective effects against some cancers. These health benefits are a result of their components such as bioactive proteins, dietary fibre, slowly digested starches, minerals and vitamins, and bioactive compounds. Among these bioactive compounds, γ-aminobutyric acid (GABA), a non-proteinogenic amino acid with numerous reported health benefits (e.g. anti-diabetic and hypotensive effects, depression and anxiety reduction) is of particular interest. GABA is primarily synthesised in plant tissues by the decarboxylation of l-glutamic acid in the presence of glutamate decarboxylase (GAD). It is widely reported that during various processes including enzymatic treatment, gaseous treatment (e.g. with carbon dioxide), and fermentation (with lactic acid bacteria), GABA content increases in the plant matrix. The objective of this review paper is to highlight the current state of knowledge on the occurrence of GABA in pulses with special focus on mechanisms by which GABA levels are increased and the analytical extraction and estimation methods for this bioactive phytochemical. © 2017 Society of Chemical Industry.

Novel Glucose-1-Phosphatase with High Phytase Activity and Unusual Metal Ion Activation from Soil Bacterium Pantoea sp. Strain 3.5.1.

Phosphorus is an important macronutrient, but its availability in soil is limited. Many soil microorganisms improve the bioavailability of phosphate by releasing it from various organic compounds, including phytate. To investigate the diversity of phytate-hydrolyzing bacteria in soil, we sampled soils of various ecological habitats, including forest, private homesteads, large agricultural complexes, and urban landscapes. Bacterial isolate Pantoea sp. strain 3.5.1 with the highest level of phytase activity was isolated from forest soil and investigated further. The Pantoea sp. 3.5.1 agpP gene encoding a novel glucose-1-phosphatase with high phytase activity was identified, and the corresponding protein was purified to apparent homogeneity, sequenced by mass spectroscopy, and biochemically characterized. The AgpP enzyme exhibits maximum activity and stability at pH 4.5 and at 37°C. The enzyme belongs to a group of histidine acid phosphatases and has the lowest Km values toward phytate, glucose-6-phosphate, and glucose-1-phosphate. Unexpectedly, stimulation of enzymatic activity by several divalent metal ions was observed for the AgpP enzyme. High-performance liquid chromatography (HPLC) and high-performance ion chromatography (HPIC) analyses of phytate hydrolysis products identify dl-myo-inositol 1,2,4,5,6-pentakisphosphate as the final product of the reaction, indicating that the Pantoea sp. AgpP glucose-1-phosphatase can be classified as a 3-phytase. The identification of the Pantoea sp. AgpP phytase and its unusual regulation by metal ions highlight the remarkable diversity of phosphorus metabolism regulation in soil bacteria. Furthermore, our data indicate that natural forest soils harbor rich reservoirs of novel phytate-hydrolyzing enzymes with unique biochemical features.

Characteristics, antimicrobial capacity, and antioxidant potential of electrospun zein/polyvinyl alcohol nanofibers containing thymoquinone and electrosprayed resveratrol nanoparticles.

The aim of the present study was to fabricate, characterize, and evaluate the in vitro antimicrobial and antioxidant properties of zein/polyvinyl alcohol (ZN/PVA) nanofibers containing 2% and 4% of thymoquinone (TQ), either alone or in combination with electrosprayed ZN nanoparticles containing 1% and 2% of resveratrol (RS). According to scanning electron microscopy analysis, the diameter of nanofibers and nanoparticles increased with increasing TQ and RS concentrations, respectively. The molecular interaction between ZN or PVA polymers and TQ or RS was confirmed by Fourier transform infrared spectroscopy. Thermogravimetric analysis showed that the thermal stability of nanofibers did not change with the addition of TQ and RS. Moreover, incorporation of TQ in nanofibers along with RS nanoparticles increased their antibacterial and free radical scavenging activities based on broth dilution and DPPH methods, respectively (p ≤ .05). Escherichia coli O157:H7 (as a Gram-negative pathogenic bacteria) was more resistant to all treatments than Staphylococcus aureus (as a Gram-positive pathogenic bacteria). In addition, the combined use of TQ in nanofibers and RS nanoparticles had antagonistic antibacterial and synergistic antioxidant effects. The best results were obtained with ZN/PVA nanofiber containing 4% TQ and electrosprayed with 2% RS nanoparticles (p ≤ .05). According to the results of the present study, biodegradable ZN/PVA nanofiber containing TQ and electrosprayed with RS nanoparticles can be used as a novel active packaging material in the food industry.

Safety evaluation of an extension of use of the food enzyme thermolysin from the non-genetically modified Anoxybacillus caldiproteolyticus strain AE-TP.

The food enzyme thermolysin (EC. 3.4.24.27) is produced with the non-genetically modified Anoxybacillus caldiproteolyticus strain AE-TP by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in eight food manufacturing processes. Subsequently, the applicant has requested to extend its use to one additional process, to withdraw two processes and to revise the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme for use in a total of seven food manufacturing processes. The dietary exposure to the food enzyme-total organic solids (TOS) was calculated to be up to 0.989 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level reported in the previous opinion (700 mg TOS/kg bw per day, the mid-dose tested), the Panel derived a revised margin of exposure of at least 708. Based on the data provided for the previous evaluation and the revised margin of exposure in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.