Use of a commercial protease and yeasts to obtain CGRP-like molecules from saithe protein

Different bioactive molecules, such as CGRP-like peptides, can be found in fish protein hydrolysates. Calcitonin gene-related peptide (CGRP) is a neuropeptide known to act as a potent arterial and venous vasodilator in humans. This study focuses on the industrial obtaining of CGRP-like molecules from saithe (Pollachius virens) byproduct, derived from the filleting process. Protein from P. virens was primarily hydrolyzed with Alcalase and later treated with Saccharomyces cerevisiae live cells. Treatment with Saccharomyces doubled the quantity of bioactive molecules obtained. The CGRP-like molecules were partially purified by chromatography, and the immunoreactive material was further analyzed for its CGRP-like bioactivity, using a specific radioreceptor assay. The concentration of CGRP-like molecules increased over 100-fold after purification. The bioactive molecules were able to induce cyclic AMP stimulation in rat liver membranes. Finally, partial sequencing of the bioactive peptide was performed, showing some homology with alpha-actin and myosin of several fish species.     

Investigations into inhibitor type and mode, simulated gastrointestinal digestion, and cell transport of the angiotensin I-converting enzyme-inhibitory peptides in Pacific hake (Merluccius productus) fillet hydrolysate

Fish protein hydrolysate (FPH) produced by incubation of Pacific hake fillet with 3.00% Protamex at pH 6.5 and 40 degrees C for 125 min demonstrated in vitro ACE-inhibitory activity (IC50 = 165 microg/mL), which was enhanced by ultrafiltration through a 10 kDa molecular weight cutoff membrane (IC50 = 44 microg/mL). However, after simulated gastrointestinal digestion, FPH and ultrafiltrate had similar ACE-inhibitory activity (IC 50 = 90 microg/mL), indicating that FPH peptides act as "pro-drug type" inhibitors and that enrichment by ultrafiltration may be unnecessary. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry confirmed that the molecular weights of major peaks were <1 kDa regardless of ultrafiltration. ACE-inhibitory activities of digested hydrolysates were not significantly affected by preincubation with ACE ( P > 0.05) and exhibited a competitive inhibitory mode. A permeability assay using fully differentiated colorectal adenocarcinoma (Caco-2) cells showed an apical to basolateral transport of peptides that ranged from approximately 2 to 20% after 2 h at 37 degrees C. Pacific hake fillet hydrolysates are a potentially bioavailable source of ACE-inhibitory peptides awaiting further in vivo study.     

Angiotensin I-converting enzyme inhibitory peptide derived from glycinin, the 11S globulin of soybean (Glycine max)

Angiotensin I-converting enzyme (ACE), a dipeptidyl carboxypeptidase, catalyzes the conversion of Angiotensin I to the potent vasoconstrictor Angiotensin II and plays an important physiological role in regulating blood pressure. Inhibitors of angiotensin 1-converting enzyme derived from food proteins are utilized for pharmaceuticals and physiologically functional foods. ACE inhibitory properties of different enzymatic hydrolysates of glycinin, the major storage protein of soybean, have been demonstrated. The IC50 value for the different enzyme digests ranges from 4.5 to 35 microg of N2. The Protease P hydrolysate contained the most potent suite of ACE inhibitory peptides. The ACE inhibitory activity of the Protease P hydrolysate after fractionation by RP-HPLC and ion-pair chromatography was ascribed to a single peptide. The peptide was homogeneous as evidenced by MALDI-TOF and identified to be a pentapeptide. The sequence was Val-Leu-Ile-Val-Pro. This peptide was synthesized using solid-phase FMOC chemistry. The IC50 for ACE inhibition was 1.69 +/- 0.17 microM. The synthetic peptide was a potent competitive inhibitor of ACE with a Ki of 4.5 +/- 0.25 x 10(-6) M. This peptide was resistant to digestion by proteases of the gastrointestinal tract. The antihypertensive property of this peptide derived from glycinin might find importance in the development of therapeutic functional foods.     

Liquid chromatographic determination of barbaloin (aloin) in foods

A simple and rapid liquid chromatographic method is described for the determination of barbaloin (aloin, 10-D-glucopyranosyl-1,8-dihydroxy-3-(hydroxymethyl)-9(10H)-anthraceno ne) in foods. Barbaloin is extracted with water from foods containing aloe and the extract is cleaned up on a disposable cartridge by using methanol-water (55 + 45) as eluant. The eluted barbaloin is separated by liquid chromatography on a YMC A-302 column with methanol-water (50 + 50) mobile phase, and detected at 293 nm. Recoveries of barbaloin added to foods at the levels of 0.05 and 0.50 mg/g were 94.4-100%. Assay results for commercial food samples indicated that the present method is applicable to a variety of foods supplemented with aloe.     

Determination of polysorbates in foods by colorimetry with confirmation by infrared spectrophotometry, thin-layer chromatography, and gas chromatography

A method is presented for the detection of polysorbates (PSs) in 8 kinds of processed foods by colorimetric and thin-layer chromatographic (TLC) techniques. The PSs are extracted from processed foods with a mixture of methylene chloride and ethanol by using an Extrelut column. The extract is further purified by using a silica gel column. The PS extract is complexed with cobalt-thiocyanate (Cothiocyanate) reagent and is determined spectrophotometrically at 620 nm. The recoveries and coefficients of variation for 8 kinds of processed foods fortified with 0.1% PS 80 were 67.9-94.6% and 4.0-11.3%, respectively. The detection limit of TLC corresponded to 50 mg PS 80/kg. PS identity was confirmed by infrared spectrophotometry of PS extract, and gas chromatography of fatty acids and thin layer chromatography of POE-sorbitan residues after saponification.     

Complete amino acid analysis in hydrolysates of foods and feces by liquid chromatography of precolumn phenylisothiocyanate derivatives

The amino acid analysis method using precolumn phenylisothiocyanate (PITC) derivatization and liquid chromatography was modified for accurate determination of methionine (as methionine sulfone), cysteine/cystine (as cysteic acid), and all other amino acids, except tryptophan, in hydrolyzed samples of foods and feces. A simple liquid chromatographic method (requiring no derivatization) for the determination of tryptophan in alkaline hydrolysates of foods and feces was also developed. Separation of all amino acids by liquid chromatography was completed in 12 min compared with 60-90 min by ion-exchange chromatography. Variation expressed as coefficients of variation (CV) for the determination of most amino acids in the food and feces samples was not more than 4%, which compared favorably with the reproducibility of ion-exchange methods. Data for amino acids and recoveries of amino acid nitrogen obtained by liquid chromatographic methods were also similar to those obtained by conventional ion-exchange procedures.     

Determination of the diglycidyl ether of bisphenol A and its derivatives in canned foods.

Migration of the diglycidyl ether of bisphenol A (DGEBA) to food from can enamels and can pull-top seals is reported. Derivatives of DGEBA are also determined in some foods. Levels of DGEBA in the foods surveyed in this study range from nondetected (<0.3 ppb) to 50 mg/kg as determined by liquid-liquid extraction or solid-phase extraction coupled with high-pressure liquid chromatography using fluorescence detection. Confirmation of the analytes is by gas and/or liquid chromatography with mass spectral analysis. Fourier transform infrared spectroscopy with 30 degrees specular reflectance/transmittance is used to characterize the coated food contact surfaces. Stability studies with DGEBA in water, acid, and saline solutions show conversion to the hydrolysis products and chloro adducts occurs readily. The presence of DGEBA derivatives in food demonstrates that analysis for DGEBA migration alone is not a good indicator of total migration from can coatings to foods.     

荔枝低分子量多糖的分离纯化及抗氧化吸湿保湿性能分析

为进一步开发和利用荔枝中的多糖成分,该文对荔枝低分子量多糖组分进行分离纯化,并对其理化性质、抗氧化和吸湿保湿性进行研究。采用超声波辅助提取、分级醇沉、DEAE-cellulose 52和Sephadex G-100柱分离纯化荔枝多糖;紫外-可见光谱扫描法、比旋光度法、渗透凝胶色谱法3种方法验证纯度;高效凝胶渗透色谱法测定相对分子量,高效阴离子交换色谱测定单糖组成;清除DPPH自由基和羟基自由基评价体外抗氧化活性;体外法测定吸湿保湿性。结果表明:荔枝多糖经分离纯化后获得组分荔枝多糖PLC-1,经3种方法验证PLC-1为精多糖,相对分子量为2.35×104 Da,是由半乳糖、鼠李糖、葡萄糖组成,摩尔比为:1.00∶3.52∶5.89。抗氧化活性研究结果表明PLC-1对DPPH和羟基自由基呈良好的量效关系,半数清除浓度IC50分别为0.41和0.31 mg/m L。吸湿保湿性的结果表明,PLC-1具有良好的吸湿和保湿性,在32 h时的吸湿率为58.3%,32 h时的失水率为45.3%,荔枝多糖PLC-1为具抗氧化和吸湿保湿活性的多糖,研究结果可为荔枝的深加工和进一步研究开发提供一定的理论基础。     

Chemical and cellular antioxidant activity of phytochemicals purified from olive mill waste waters

The isolation and identification of a phytocomplex from olive mill waste waters (OMWW) was achieved. The isolated phytocomplex is made up of the following three phenolic compounds: hydroxytyrosol (3,4-DHPEA), tyrosol (p-HPEA) and the dialdehydic form of decarboxymethyl elenolic acid, linked with (3,4-dihydroxyphenyl)ethanol (3,4-DHPEA-EDA). The purification of this phytocomplex was reached by partial dehydration of the OMWW, followed by liquid-liquid extraction with ethyl acetate and middle pressure liquid chromatography (MPLC) on a Sephadex LH-20 column. The phytocomplex accounted for 6% of the total phenolic content of the OMWW. The phytocomplex and individual compounds were tested for antioxidant capacity by the oxygen radical absorbance capacity (ORAC) method. The ORAC phytocomplex produced 10,000 ORAC units/g dry weight, whereas the cellular antioxidant activity, measured by the cellular antioxidant activity in red blood cell (CAA-RBC) method, demonstrated that the phytocomplex and all of the components are able to permeate the cell membrane thus exhibiting antioxidant activity inside the red blood cells. Our phytocomplex could be employed in the formulation of fortified foods and nutraceuticals, with the goal to obtain substantial health protective effects due to the suitable combination of the component molecules.     

ACE inhibitory activity in enzymatic hydrolysates of insect protein

In this paper, ACE inhibitory activity in insect protein hydrolyzed by various enzymes (gastrointestinal proteases, alcalase, and thermolysin) is reported for the first time. Four insects of different insect orders were tested: Spodoptera littoralis (Lepidoptera), Bombyx mori (Lepidoptera), Schistocerca gregaria (Orthoptera), and Bombus terrestris (Hymenoptera). ACE inhibitory activity was measured by two different methods: a spectrophotometric method using FAPGG (2-furanacryloyl-phenylalanyl-glycyl-glycine) as substrate, and an HPLC method using dansyltriglycine (DTG) as substrate. Hydrolysis of the insect protein resulted in an increased ACE inhibitory activity. Overall, the highest ACE inhibitory activity was obtained after gastrointestinal digestion. These results suggest a role for insect protein as antihypertensive component in functional foods and nutraceuticals. Furthermore, the ACE inhibitory activity differed according to the method used. As a consequence, there is a need to standardize methodologies to evaluate ACE inhibitory activity.     

Simple and sensitive method for pyrroloquinoline quinone (PQQ) analysis in various foods using liquid chromatography/electrospray-ionization tandem mass spectrometry

Pyrroloquinoline quinone (PQQ) is believed to be an important factor for mammalian growth and development and has, therefore, been declared a vitamin by some researchers. However, this issue remains controversial, and from a nutritional viewpoint, accurate determination of PQQ levels in a variety of foods is very important. Here, we describe a simple, highly sensitive, and highly selective method for quantitative analysis of PQQ. Liquid foods or aqueous extracts of solid foods were analyzed using high-performance liquid chromatography (HPLC) combined with electrospray-ionization (ESI) tandem mass spectrometry (MS/MS). (15)N-labeled PQQ was added to the samples as an internal standard. Quantitative analyses of PQQ were performed by multiple reaction monitoring (MRM) with LC/MS/MS. Free PQQ was detected in almost all food samples in the range 0.19-7.02 ng per g fresh weight (for solid foods) or per mL (liquid foods). This method will enable the rapid and simple determination of PQQ levels in many samples.     

Bioguided Fraction of Antioxidant Activity of Ethanol Extract from Tartary Buckwheat Bran

This study describes the activity‐guided isolation of antioxidant agents from tartary buckwheat bran (TBB). The ethanol crude extract of oil‐free TBB was partitioned sequentially into ethyl acetate, n‐butanol, and residual aqueous fractions. The ethyl acetate fraction (EAF) had the highest phenolic and flavonoid contents and showed the strongest antioxidant activity. EAF was superior to rutin and inferior to vitamin C (Vc) in DPPH radical scavenging ability, had an advantage over rutin and Vc in ABTS radical scavenging ability, and again surpassed rutin and quercetin in reducing power. Then EAF was subjected to column chromatography, and isolated compounds were identified using nuclear magnetic resonance data by comparing with those reported in the literature. Finally, the bioassay‐guided fractionation of the crude ethanol extract of TBB afforded three known compounds (quercetin, p‐hydroxybenzoic acid, and daucosterol) responsible for antioxidant activity. p‐Hydroxybenzoic acid and daucosterol were isolated from buckwheat grain for the first time. Taken together, establishing of the three pure compounds is of paramount importance to the understanding of antioxidant activity of TBB, and there is an immense potential to process TBB or its EAF into value‐added functional foods and beverages.     

Validation of thermally assisted hydrolysis and methylation-gas chromatography using a vertical microfurnace pyrolyzer for the compositional analysis of Fatty Acid components in microalgae

Thermally assisted hydrolysis and methylation-gas chromatography (THM-GC) in the presence of trimethylsulfonium hydroxide, using a vertical microfurnace pyrolyzer, was validated for the compositional analysis of fatty acid components in microalgae. The chromatograms of a microalga, Pavlova lutheri , obtained under optimized THM conditions clearly showed a series of fatty acid methyl esters including thermally labile polyunsaturated fatty acid components. On the basis of these peak areas, their chemical compositions were rapidly determined without using any tedious sample pretreatment with a precision of <8% relative standard deviation. Moreover, the compositions thus obtained were in good agreement with those obtained by the conventional technique involving solvent extraction. Finally, the THM-GC technique was applied for the compositional analysis of fatty acid components in a newly found microalga, Coccomyxa gloeobotrydiformis . The obtained data showed a high abundance (24 mol %) of α-linolenic acid components, suggesting its potential usefulness as feed sources and/or functional foods.     

Polyphenol content and antioxidative activity in some species of freshly consumed salads

Ten genotypes belonging to Lactuca sativa, Cicorium intybus, Plantago coronopus, Eruca sativa, and Diplotaxis tenuifolia and used in fresh mixed salads were investigated for their polyphenol contents. Flavonoids and hydroxycinnamic acids were characterized by high-performance liquid chromatography (HPLC)/diode array detection/mass spectrometry. Quercetin, kaempferol, luteolin, apigenin, and crysoeriol derivatives were identified; hydroxycinnamic acids were all caffeoyl derivatives. The total polyphenol content was obtained through the Folin-Ciocalteu test and from the HPLC data. The amounts ranged between 0.9 and 4.7 mg/g fresh weight. The antiradical activity was determined by the reaction with the stable DPPH* radical. The Fe2+ chelating activity was determined with a spectrophotometric test. From the complex of data, a quite complete picture of the characteristics of the vegetables emerges. A cultivated C. intybus cultivar exhibited the highest polyphenol content, while a wild C. intybus genotype exhibited the highest antiradical activity. In every case, the characteristics of the different salads as functional foods have been pointed out.     

Screening of commercial hydrolases for the degradation of ochratoxin A

Ochratoxin A (OTA) is a nephrotoxic and carcinogenic mycotoxin. The toxin is a common contaminant of various foods and feeds and poses a serious threat to the health of both humans and animals. A number of commercial hydrolases were screened for the ability to degrade OTA to nontoxic compounds. A crude lipase from Aspergillus niger (Amano A) proved to substantially hydrolyze OTA to the nontoxic OTalpha and phenylalanine, as confirmed by HPLC with fluorescence detection. The enzyme was purified by anion exchange chromatography to homogeneity. Activity staining of the purified enzyme with alpha-naphthyl acetate/Fast Red revealed only one band exhibiting hydrolytic activity. The specific activity of the purified enzyme toward OTA was 2.32 units/mg.     

Profiles of carotenoids, anthocyanins, phenolics, and antioxidant activity of selected color waxy corn grains during maturation

Waxy corns are becoming increasingly consumed as fresh foods or as raw materials for whole grain foods facilitating human consumption in China, so they are usually harvested before complete maturity. Unfortunately, information on functional properties of immature waxy corns is very limited. Therefore, we investigated the profiles of carotenoids, anthocyanins, phenolics, and the antioxidant activity in three types of waxy corn with different colors (white, yellow, and black) during maturation, as well as a normal corn (yellow) used as control. The results showed that black waxy corn had the highest quantity of anthocyanins, phenolics and the best antioxidant activity, yellow corn contained a relatively large amount of carotenoids, while white corn had the lowest amounts of carotenoids, anthocyanins, phenolics, and antioxidant capacity. For each type of waxy corn, the higher carotenoids were found at the M2 stage (no major difference between the M1 and M2 stages for yellow corn). The levels of anthocyanin and phenolics decreased for white and yellow corns, contrary to those for black corn during maturation. The antioxidant activity determined by scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH), the ferric reducing antioxidant power (FRAP), and the Trolox equivalent antioxidant capacity (TEAC) assays increased with ripening, but no difference was found between the M2 and maturity stages for yellow and black corns. For white corn, the DPPH radical scavenging activity first increased and then decreased, while the antioxidant activity determined by TEAC and FRAP assay decreased during maturation. Differences in these parameters indicate that types and harvesting time have significant influences on functional properties of waxy corns.     

A gamma-glutamyl peptide isolated from onion (Allium cepa L.) by bioassay-guided fractionation inhibits resorption activity of osteoclasts

One gram of onion added to the food of rats inhibits significantly (p < 0.05) bone resorption as assessed by the urinary excretion of tritium released from bone of 9-week-old rats prelabeled with tritiated tetracycline from weeks 1 to 6. To isolate and identify the bone resorption inhibiting compound from onion, onion powder was extracted and the extract fractionated by column chromatography and medium-pressure liquid chromatography. A single active peak was finally obtained by semipreparative high-performance liquid chromatography. The biological activity of the various fractions was tested in vitro on the activity of osteoclasts to form resorption pits on a mineralized substrate. Medium, containing the various fractions or the pure compound, was added to osteoclasts of new-born rats settled on ivory slices. After 24 h of incubation, the tartrate-resistant acid phosphatase positive multinucleated cells, that is, osteoclasts, were counted. Subsequently, the number of resorption pits was determined. Activity was calculated as the ratio of resorption pits/osteoclasts and was compared to a negative control, that is, medium containing 10% fetal bovine serum only and to calcitonin (10(-12) M) as a positive control. Finally, a single peak inhibited osteoclast activity significantly (p < 0.05). The structure of this compound was elucidated with high-performance liquid chromatography-electrospray ionization-mass spectrometry, time-of-flight electrospray ionization mass spectrometry, and nuclear magnetic resonance spectroscopy. The single peak was identified as gamma-L-glutamyl-trans-S-1-propenyl-L-cysteine sulfoxide (GPCS). It has a molecular mass of 306 Da and inhibits dose-dependently the resorption activity of osteoclasts, the minimal effective dose being approximately 2 mM. As no other peak displayed inhibitory activity, it likely is responsible for the effect of onion on bone resorption.     

Determination of carotenoids, total phenolic content, and antioxidant activity of Arazá (Eugenia stipitata McVaugh), an Amazonian fruit

The fruit of Arazá (Eugenia stipitata McVaugh) native to the Colombian Amazon is considered a potentially economically valuable fruit for the Andean economy due to its novel and unique taste. The fruit has an intense yellow color, but its chemical composition and properties have not been well studied. Here we report the identification and quantitation of carotenoids in the ripe fruit using high performance liquid chromatography (HPLC) with photodiode array detector (PDA) and atmospheric pressure chemical ionization (APcI) mass spectrometry (MS/MS). The qualitative carotenoid profile of the fruit according to maturity stage was also observed. Furthermore, antioxidant activity of the peel and pulp were assessed using the ferric reducing ability of plasma (FRAP), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) methods, in addition to chemical indexes and total phenolic content. Multiple carotenoids were identified in the peel and pulp including four xanthophylls (free and esterified as their mono and diesters) and two carotenes. One of the xanthophylls was tentatively identified as zeinoxanthin, while the others were identified as lutein, zeaxanthin, and β-cryptoxanthin. Carotenes included α-carotene and β-carotene. The total carotenoid content was significantly higher in the peel (2484 ± 421 μg/100 g FW) than in the pulp (806 ± 348 μg/100 g FW) with lutein, β-cryptoxanthin, and zeinoxanthin as the major carotenoid components. The unique carotenoid composition of this fruit can differentiate it from other carotenoid-rich fruits and perhaps be useful in authentication procedures. Overall, results from this study suggest that Colombian Arazá may be a good edible source of carotenoids important in retinal health as well as carotenoids with provitamin A activity. Therefore, Arazá fruit can be used as a nutraceutical ingredient and in production of functional foods in the Colombian diet.     

Stimulation of benzyladenine-induced in vitro shoot organogenesis and endogenous proline in melon (Cucumis melo L.) by fish protein hydrolysates in combination with proline analogues

A previous study demonstrated that proline is beneficial for improving melon in vitro shoot organogenesis. A natural source of proline and proline precursors can be obtained from fish protein hydrolysates (FPH), a byproduct of the fishery industry. Proline analogues azetidine-2-carboxylate and hydroxyproline in combination with standardized FPH were used to stimulate proline synthesis and benzyladenine-induced shoot organogenesis by exploiting the proposed proline-linked pentose phosphate pathway (PPP). In the presence of elevated levels of endogenous proline, potential stimulation of cytokinins and auxin may occur via the PPP and shikimate pathways, respectively. Treatments with FPH singly and in combination with the above proline analogues significantly increased the endogenous proline content and the extent of differentiation, suggesting that in vitro organogenesis is closely linked to proline synthesis, strengthening the hypothesis that purine metabolism via the proline-linked PPP may be important for organogenesis. Thioproline addition resulted in increased proline levels but without corresponding stimulation of organogenesis. This study also provides potential use of fishery waste for value-added application in plant micropropagation industry.     

Carotenoid content of pandanus fruit cultivars and other foods of the Republic of Kiribati

BACKGROUND: Kiribati, a remote atoll island country of the Pacific, has serious problems of vitamin A deficiency (VAD). Thus, it is important to identify locally grown acceptable foods that might be promoted to alleviate this problem. Pandanus fruit (Pandanus tectorius) is a well-liked indigenous Kiribati food with many cultivars that have orange/yellow flesh, indicative of carotenoid content. Few have been previously analysed. AIM: This study was conducted to identify cultivars of pandanus and other foods that could be promoted to alleviate VAD in Kiribati. METHOD: Ethnography was used to select foods and assess acceptability factors. Pandanus and other foods were analysed for beta- and alpha-carotene, beta-cryptoxanthin, lutein, zeaxanthin, lycopene and total carotenoids using high-performance liquid chromatography. RESULTS: Of the nine pandanus cultivars investigated there was a great range of provitamin A carotenoid levels (from 62 to 19,086 microg beta-carotene/100 g), generally with higher levels in those more deeply coloured. Seven pandanus cultivars, one giant swamp taro (Cyrtosperma chamissonis) cultivar and native fig (Ficus tinctoria) had significant provitamin A carotenoid content, meeting all or half of estimated daily vitamin A requirements within normal consumption patterns. Analyses in different laboratories confirmed high carotenoid levels in pandanus but showed that there are still questions as to how high the levels might be, owing to variation arising from different handling/preparation/analytical techniques. CONCLUSIONS: These carotenoid-rich acceptable foods should be promoted for alleviating VAD in Kiribati and possibly other Pacific contexts where these foods are important. Further research in the Pacific is needed to identify additional indigenous foods with potential health benefits.