In vitro and in vivo protein hydrolysis of beans (Phaseolus vulgaris) genetically modified to express different phaseolin types

Experiments were conducted to study whether phaseolin type could influence proteolysis susceptibility and nutritional value of total bean protein. The DOR-390 bean cultivar was genetically modified to express different phaseolin types (S, T or I). Beans were soaked and autoclaved. A sequential hydrolysis was carried out in vitro with pepsin and pancreatin. Differences in the degree of protein hydrolysis among bean lines started at 30 min and remained until 240 min, with the S bean proteins presenting lower values (P < 0.05). Subsequently, rats were fed with diets containing beans expressing different phaseolin types as the only source of protein for N digestibility and nutritional value determination. No differences (P > 0.05) in ileal protein digestibility and rat growth were observed. In conclusion, the differences in in vitro hydrolysis between bean lines expressing different phaseolin types had no consequences on growth and N retention in rats.     

Phenolic profiles of in vivo and in vitro grown Coriandrum sativum L.

Coriandrum sativum L. is a source of a variety of polyphenols and other phytochemicals, related to its high antioxidant activity and to its use for indigestion, rheumatism, and prevention of lipid peroxidation damage. Plant cell cultures are a means to study or to produce some active metabolites, such as polyphenols. This technique was applied to the investigation of coriander, and a detailed analysis of individual polyphenols in vivo and in vitro grown samples was performed. The in vivo vegetative parts showed quercetin derivatives as the main flavonoids and quercetin-3-O-rutinoside (3296 mg/kg dw) was the main polyphenol found in this part of coriander. The fruits revealed only phenolic acids and derivatives, caffeoyl N-tryptophan hexoside (45.33 mg/kg dw) being the most abundant phenolic derivative. In vitro samples also gave a high diversity of polyphenols, being C-glycosylated apigenin (2983 mg/kg dw) the main compound. Anthocyanins were only found in clone A, which was certainly related to its purple pigmentation, and peonidin-3-O-feruloylglucoside-5-O-glucoside was the major anthocyanin found (1.70 μg/kg dw). In vitro culture can be used to explore new industrial, pharmaceutical, and medicinal potentialities, such as the production of secondary metabolites like flavonoids.     

In vitro starch digestibility,expected glycemic index,and thermal and pasting properties of flours from pea,lentil and chickpea cultivars

In vitro starch digestibility, expected glycemic index (eGI), and thermal and pasting properties of flours from pea, lentil and chickpea grown in Canada under identical environmental conditions were investigated. The protein content and gelatinization transition temperatures of lentil flour were higher than those of pea and chickpea flours. Chickpea flour showed a lower amylose content (10.8–13.5%) but higher free lipid content (6.5–7.1%) and amylose–lipid complex melting enthalpy (0.7–0.8 J/g). Significant differences among cultivars within the same species were observed with respect to swelling power, gelatinization properties, pasting properties and in vitro starch digestibility, especially chickpea flour from desi (Myles) and kabuli type (FLIP 97-101C and 97-Indian2-11). Lentil flour was hydrolyzed more slowly and to a lesser extent than pea and chickpea flours. The amount of slowly digestible starch (SDS) in chickpea flour was the highest among the pulse flours, but the resistant starch (RS) content was the lowest. The eGI of lentil flour was the lowest among the pulse flours.     

The in vitro and in vivo antioxidant properties of seabuckthorn (Hippophae rhamnoides L.) seed oil

The antioxidant capacity of seabuckthorn (Hippophae rhamnoides L.) seed oil was investigated with a number of established in vitro assays and in an in vivo study of carbon tetrachloride (CCl₄)-induced oxidative stress in mice. The results showed that DPPH radical scavenging activity, ferrous ion chelating activity, reducing power and inhibition of lipid peroxidation activity all increased with increasing concentrations of seabuckthorn seed oil. Moreover, the EC₅₀ values of seabuckthorn seed oil from the hydrogen peroxide, superoxide radical, hydroxyl radical scavenging assays were 2.63, 2.16 and 0.77 mg/ml, respectively. In the in vivo study, seabuckthorn seed oil inhibited the toxicity of CCl₄, as seen from the significantly increased activities of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase. The GSH content in the liver was also increased, whereas hepatic malondialdehyde was reduced. Taken together, these results clearly indicate that seabuckthorn seed oil has significant potential as a natural antioxidant agent.     

Sequential extraction of polysaccharides from enzymatically hydrolyzed okara byproduct: Physicochemical properties and in vitro fermentability

Okara, a byproduct of soymilk production, has been upgraded through the use of an enzymatic treatment with Ultraflo L® to give a product (okara^ET) which has a higher content of soluble dietary fibre and an enhanced ratio of soluble: insoluble fibre than is found in okara without treatment. Polysaccharides were isolated from okara^ET by sequential extraction to yield soluble fractions in water (22%), CDTA (8.7%), alkali (37.7%) without and NaClO₂ (9.1%) and the cellulosic residue represents a (22.5%). The physicochemical properties of okara^ET were improved due to the enzymatic treatment: oil retention capacity (6.94 g/g), water retention capacity (10.76 g/g) and swelling capacity (13.85 g/g) were higher than in okara that had not undergone enzymatic treatment. The gelation capacity (8%) and the cation exchange capacity (8.96 mEq/kg) of okara^ET were lower than that of other byproducts. Short chain fatty acid production during in vitro fermentation of okara^ET by a pure culture of Bifidobacterium bifidus was mainly represented by acetic acid, followed by propionic and butyric acids. In addition, the decreases in pH and substrate consumption demonstrated the bifidogenic capacity of okara^ET.     

Phenolic profiles of cultivated, in vitro cultured and commercial samples of Melissa officinalis L. infusions

Melissa officinalis L. (lemon balm) is normally consumed as an infusion and presents therapeutic properties, such as sedative, carminative and antispasmodic, also being included in some pharmaceutical preparations. The phenolic profiles of different samples of lemon balm, prepared as infusions, were evaluated by HPLC-DAD-ESI/MS. The profiles were compared in order to understand the differences between cultivated, in vitro cultured and commercial (bags and granulated) samples. All the samples showed a similar phenolic profile, presenting differences only in the quantities found of each compound. Rosmarinic acid was the most abundant compound, being higher in commercial samples, especially in tea bag sample (55.68 mg/g of infusion) and lower in in vitro cultured sample (15.46 mg/g). Moreover, dimers, trimers and tetramers of caffeic acid were identified and quantified for the first time in lemon balm. Only one flavonoid, luteolin-3′-O-glucuronide was found in all the samples, ranging from 8.43 mg/g in commercial granulate sample to 1.22 mg/g in in vitro cultured sample. Overall, cultivated and in vitro cultured samples presented the lowest amounts of phenolic compounds (59.59 and 30.21 mg/g, respectively); otherwise, commercial samples showed the highest contents (109.24 mg/g for tea bag and 101.03 mg/g for granulate sample). The present study shows that infusion of lemon balm can be a source of phenolic compounds, known for their bioactive effects.     

Thermal aggregation,amino acid composition and in vitro digestibility of vicilin-rich protein isolates from three Phaseolus legumes: A comparative study

The heat-induced aggregation and the in vitro digestibility of vicilin-rich protein isolates from three Phaseolus legumes, including kidney bean, red bean and mung bean were investigated and compared, and their amino acid composition and free sulfhydryl (SH) group contents also evaluated. The results showed that the extent in the heat-induced aggregation varied with the type of the protein isolates, and the formation of new disulphide bonds (at the expense of free SH contents) was involved in the formation of the aggregates. The protein isolates with higher levels of hydrophobic and uncharged polar amino acids, and lower basic amino acid contents exhibited lower extent of their heat-induced aggregation. The in vitro pepsin plus trypsin digestibility was different for various native protein isolates. The digestibility was to a varying extent affected by the heat treatment. The influences of heating on the digestibility of these proteins were closely related to the extent of their heat-induced aggregation. The results suggest that the improvement of nutritional property of those vicilin-rich protein isolates by heat treatment is largely dependent upon their amino acid composition as well as the extent of heat-induced aggregation.     

Antiacetylcholinesterase and antioxidant activities of Plectranthus barbatus tea,after in vitro gastrointestinal metabolism

This study describes the in vitro metabolism, by the gastrointestinal tract, together with the biological activity of the herbal tea obtained from Plectranthus barbatus leaves. The results showed that there was no appreciable degradation and that the activity was kept constant after gastric juice digestion. In the presence of pancreatic juice, degradations varied from negligible, in the case of flavonoid glucuronides, to almost complete, in the case of the abietane diterpenoid. The digested decoction contained only 50% of its initial biological activity, after pancreatic digestion. The action of Caco-2 cells on the extract revealed that neither rosmarinic acid, the main compound of the extract, nor the other components present in minute quantities were metabolised by the intact cells. Rosmarinic acid could be found inside Caco-2 cells although in trace amount. Glucoronidase from Escherichia coli, a gut bacterium, was able to hydrolyse the flavonoid derivatives, thus the aglycones were formed and permeate the Caco-2 cells.     

Effect of soaking and hydrothermal processing methods on the levels of antinutrients and in vitro protein digestibility of Bauhinia purpurea L. seeds

The effects of various domestic processing methods such as soaking, cooking and autoclaving on the levels of certain antinutritional factors and in vitro protein digestibility of seeds of Bauhinia purpurea L., an underutilised legume collected from South India, were investigated. The raw seeds were found to contain antinutritional factors like total free phenolics (2.75 g/100 g), tannins (2.35 g/100 g), phytic acid (692 mg/100 g) and flatulence factors, raffinose (0.54 g/100 g), stachyose (1.17 g/100 g) and verbascose (0.95 g/100 g). Soaking the seeds in distilled water caused maximum reduction in the phytic acid content (37%), whereas soaking in NaHCO₃ solution reduced significant levels of phenolics and tannins (72% and 78%, respectively). A reduction in the levels of oligosaccharides (raffinose by 63%, stachyose by 42% and verbascose by 79%) was observed during cooking. Of the attempted treatments, autoclaving appeared to be most effective in reducing levels of all the investigated antinutrients, except phytic acid, and also improved the in vitro protein digestibility of B. purpurea seeds.     

Preparation and in vitro evaluation of calcium-induced soy protein isolate nanoparticles and their formation mechanism study

Soy protein isolate (SPI) nanoparticles (28–179 nm) were prepared by employing a cold gelation method with a slight modification. The obtained nanoparticles exhibited uniform size distribution and spherical shape with a unique honeycomb-like core structure. Nanoparticle characteristics including size, surface charge and hydrophobicity could be adjustable by changing calcium concentration and environmental pH. Generally, higher calcium concentration and lower pH led to formation of nanoparticles with larger size, lower surface charge and hydrophobicity. Both protein conformation and nanoparticle dissociation studies indicated that calcium likely shielded negative charges on the SPI polypeptide chains, and functioned as a salt-bridge to permit polypeptide chains to approach one another. In this process, calcium favoured the development of β-sheet structures to form SPI aggregates stabilised by hydrogen bonding. These aggregates were then associated to build SPI nano-networks through hydrophobic interactions. In vitro study indicated that the SPI nanoparticles were non-toxic and mainly located in the cytoplasm when uptaken into Caco-2 cells.     

Predicting relative concentrations of bioavailable iron in foods using in vitro digestion: New developments

In vitro methods have been developed for the prediction of iron bioavailability from foods and supplements. The dialyzability method measures dialyzable iron, released during a simulated gastrointestinal digestion, as an index of iron bioavailability. A new setup, that involves six-well plates and a ring insert that holds the dialysis membrane, is proposed for the application of the dialyzability method with the objective to increase efficiency and to allow testing small-volume samples. A series of solutions (water, ascorbic acid, and phytate), liquid foods (fresh milk and condensed milk), and solid foods (bread + meat meal, corn flakes), were tested in the presence or absence of added iron and digested with the new setup and the setup previously described for the dialyzability method. In both cases, percent dialyzable iron in each treatment remained similar (P > 0.05). These results suggest that the new setup can be employed in future applications with similar food matrices of the dialyzability method.     

Effect of ultrasound treatments of tomato pulp on microstructure and lycopene in vitro bioaccessibility

The influence of ultrasound treatments of tomato pulp on microstructure and lycopene in vitro bioaccessibility was investigated. To this purpose, samples were subjected to ultrasound at a frequency and amplitude of 24 kHz and 100 μm, respectively, for increasing lengths of time. Results showed that ultrasound was responsible for loss of tomato cell integrity, as well as a decrease in the degree of pectin esterification. In contrast, rheological measurements showed that ultrasonically treated tomato pulp had greater gel-like properties than an untreated sample. It was inferred that ultrasound promoted the formation of a new network due to hydrogen bonding and hydrophobic interactions among the de-esterified pectin molecules. Such a reinforcement of the tomato pulp structure resulted in a decrease in lycopene in vitro bioaccessibility of the ultrasonically treated tomato pulp, probably due to the fact that the presence of a stronger network may make lycopene less available to the digestion process.     

Impact of salt and lipid type on in vitro digestion of emulsified lipids

This study examined the effects of oil type and NaCl addition on the micro-structural changes that occur to emulsified lipids as they pass through a model gastrointestinal tract (GIT). Oil-in-water emulsions stabilized by a non-ionic surfactant (Tween 20) were prepared using different kinds of lipids (3% soybean oil, corn oil, olive oil or lard). The emulsified lipids were passed through an in vitro digestion model that simulated the composition (pH, minerals, surface active components, and enzymes) of the human GIT. Prior to digestion, emulsified lipid droplets appear to be bridging flocculation in 1% NaCl added emulsified lipids, moreover lipid droplets of 1% NaCl added emulsified lipids seems to be more disrupted than no NaCl added emulsified lipids. Mean particle size prepared with lard was smaller than those of other emulsified lipids. Free fatty acid contents increased after in vitro digestion in all emulsified lipids. Especially, free fatty acid content of emulsified lipid made from lard and olive oil were significantly higher than those of other emulsions after in vitro digestion.     

Characterization,amino acid composition and in vitro digestibility of hemp (Cannabis sativa L.) proteins

The protein constituents and thermal properties of hemp (Cannabis sativa L.) protein isolate (HPI) as well as 11S- and 7S-rich HPIs (HPI-11S and HPI-7S) were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and different scanning calorimetry (DSC), and their amino acid composition and in vitro digestibility were also evaluated, as compared to soy protein isolate (SPI). SDS-PAGE analysis showed that the edestin (consisting of acidic and basic subunits, AS and BS) was the main protein component for HPI and HPI-11S, while HPI-7S was composed of the BS of edestin and a subunit of about 4.8 kDa. DSC analysis characterized thermal transition of the edestin component and the possible present form of different subunits. Except lysine and sulfur-containing amino acids, the essential amino acids of various HPIs met the suggested requirements of FAO/WHO for 2–5 year old infants. The proportion of essential amino acids to the total amino acids (E/T) for HPI (as well as HPI-11S) was significantly higher than that of SPI. In an in vitro digestion model, various protein constituents of various HPIs were much easily digested by pepsin plus trypsin, to release oligo-peptides with molecular weight less than 10.0 kDa (under reduced condition). Only after pepsin digestion, in vitro digestibility of HPIs was comparable to that of SPI, however after pepsin plus trypsin digestion, the digestibility (88–91%) was significantly higher than that (71%) of SPI (P < 0.05). These results suggest that the protein isolates from hempseed are much more nutritional in amino acid nutrition and easily digestible than SPI, and can be utilized as a good source of protein nutrition for human consumption.     

Physicochemical, in vitro digestibility and functional properties of carboxymethyl rice starch cross-linked with epichlorohydrin

Cross-linked carboxymethyl rice starches (CL-CMRSs) were prepared from reactions between native rice starch and varied concentrations (0.1–15% w/w, M-0.1 to M-15) of epichlorohydrin (ECH) in a simultaneous carboxymethylation-crosslinking reaction setup using methanol as the solvent. The degree of carboxymethyl substitution was between 0.24 and 0.28, while apparent amylose contents were lowered due to modification. SEM images showed minor change on the granule surface, while XRD profiles indicated slight loss of crystallinity. DSC thermograms revealed no transition peak in all treated samples. The water uptake (WU), swelling volume (SV) and free swelling capacity (FSC) of CL-CMRSs increased significantly as a result of the modification, while swelling of CMRSs cross-linked with 2% (M-2) and 3% (M-3) ECH yielded FSC values and WU values that were much greater than those of native starches and were comparable to that of Explotab®. All modified starch samples showed increased amount of rapidly digestible starch (RDS), while cross-linking with 5–7.5% ECH raised the resistant starch (RS) content, compared to native starch. M-2 also showed promising results in tablet disintegration test. ECH–CL–CMRSs could potentially be used as an excipient in pharmaceutical and food/food supplement products.     

Effect of heat,rutin and disulfide bond reduction on in vitro pepsin digestibility of Chinese tartary buckwheat protein fractions

Protein fractions (albumin, globulin, prolamin and glutelin) were extracted from defatted tartary buckwheat flour. The in vitro pepsin digestibilities of the four protein fractions were different, and albumin was more susceptible to pepsin hydrolysis. The native structure of the four protein fractions may be destroyed by heat treatment, and the digestibilities were all improved significantly (P < 0.05). Adding rutin to the digestion mixture of the four fractions did not cause a decrease in pepsin digestibility, although it did cause a significant increase in certain instances (P < 0.05). Treatment with β-mercaptoethanol (2-ME) only caused a higher initial proteolysis rate and did not increase the final digestibility distinctly except for prolamin. After pepsin digestion, the remaining proteins of unhydrolyzed albumin, globulin, prolamin and glutelin (untreated) shared some similarities. They also exhibited a minor band at 20,000 Da and a broad band at 10,000–14,000 Da.     

Lutein-enriched frankfurter-type products: Physicochemical characteristics and lutein in vitro bioaccessibility

This paper reports a study of the physicochemical characteristics of lutein-enriched frankfurter-type products with two fat levels, and the lutein in vitro bioaccessibility in these products. In general, the high-fat products possessed better emulsion stability and greater hardness and chewiness than low-fat samples. For each fat level, the addition of lutein extract in olive oil reduced emulsion stability in meat products. The presence of lutein reduced the lightness and increased the redness of samples. Lutein was highly stable upon in vitro digestion, with overall recovery of over 84% at the end of the duodenal phase and very low isomerisation. Micellarisation was high but depended on the fat content, ranging from 29–34% (for the low-fat sample) up to 73–81% of the amount initially present (for the high-fat sample). Storage (22 days at 4 °C) did not significantly affect lutein content or bioaccessibility. Our results support the suitability of meat products as lutein carriers and as a means to increase the systematic intake of lutein.