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.     

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.     

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.     

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.     

Effect of alkaline pH-shift processing on in vitro gastrointestinal digestion of herring (Clupea harengus) fillets

The effect of alkaline pH-shift processing on herring (Clupea harengus) protein oxidation, salt solubility and digestibility, has been evaluated. For the latter, herring mince and pH-shift produced herring protein isolate, both raw and heat-treated, were digested using a static gastrointestinal in vitro model. The pH-shift process resulted in drastically lowered protein salt solubility and increased lipid oxidation while protein carbonyl formation was unaffected. Yet, no significant differences in the degree of hydrolysis (DH) were observed between mince and isolates after completed gastrointestinal digestion, something which was confirmed by a similar release of proteinaceous material <3 kDa and similar free amino acid profiles. The polypeptide profiles of digested samples however revealed that two peptides (33 and 36 kDa) were present in larger amounts in the digested protein isolate compared to the digested herring mince. The results indicate that alkaline pH-shift processing had limited quantitative influence on the gastrointestinal digestibility of herring proteins despite its negative effects on protein salt solubility and lipid oxidation.     

Extrusion of a hard-to-cook bean (Phaseolus vulgaris L.) and quality protein maize (Zea mays L.) flour blend

Heated extrusion was tested as an alternative process for incorporating “hard-to-cook” beans into food products. A 3² factorial design was used to evaluate extrusion conditions for a 40/60 (w/w) blend of “hard-to-cook” beans and quality protein maize. Tested extrusion variables were temperature (155, 170 and 185 °C) and moisture content (15.5, 17.5 and 19.5 g/100 g). Screw speed was fixed at 130 rpm. The extrudates obtained at 155 and 170 °C with 15.5% moisture had the best physical characteristics and were chosen for comparative analysis of nutritional changes between the unprocessed “hard-to-cook” bean/quality protein maize flour blend and the resulting extrudates. In vitro protein digestibility was higher in the extrudates (80%) than in the flour blend (76%). In vitro starch digestibility was higher at 155 °C (89%) and 170 °C (92%) than in the flour blend (12%). Processing conditions decreased dietary fibre content by 38% at 155 °C and 44% at 170 °C.     

Isolation, solubility and in vitro hydrolysis of chickpea vicilin-like protein

The chickpea vicilin-like globulin was isolated and chromatographed on Sepharose CL-6B and Sephacryl S-300. The native globulin with a molecular weight of 140 kDa was resolved in Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in seven polypeptide bands in the range of 12.4-67 kDa. The solubility profile of the protein in water and NaCl solutions was typical of a legume globulin. The purified vicilin-like globulin, native and heated, was hydrolyzed by pepsin, trypsin and chymotrypsin. The hydrolysis patterns indicated that the native vicilin-like protein was only partially degraded by the enzymes in comparison with casein. Heating increased its susceptibility to hydrolysis relative to the native form, for all the enzymes. However, the results obtained by the pH-drop method revealed that the in vitro digestibility of the vicilin-like protein was not altered by heating, while 11S-like and total globulins suffered a small increase, indicating that the structural characteristics of storage globulins may be important factors limiting the protein digestion.     

In vitro starch hydrolysis and estimated glycaemic index of bread substituted with different percentage of chempedak (Artocarpus integer) seed flour

Chempedak (Artocarpus integer) seed flour (CSF) was substituted for wheat flour at different levels (0%, 10%, 20%, 30% w/w) in bread. Assessment on the in vitro starch hydrolysis was carried out to evaluate the hydrolysis index (HI) and estimated glycaemic index (EGI) of bread substituted with different levels of CSF. Kinetics of in vitro starch hydrolysis in all bread samples (with the exception for white bread) indicated a gradual increase with respect to time intervals. Bread of 30% CSF exhibited significantly lower (p < 0.05) in vitro starch hydrolysis, as compared with the other samples. Results showed that HI value decreased significantly (p < 0.05) as the levels of CSF substitution increased. Resistant starch (RS) content in bread samples was inversely related with HI value as CSF substitution levels increased, thus lowering the EGI value.     

In vitro human digestion models for food applications

In vitro digestion models are widely used to study the structural changes, digestibility, and release of food components under simulated gastrointestinal conditions. However, the results of in vitro digestion models are often different to those found using in vivo models because of the difficulties in accurately simulating the highly complex physicochemical and physiological events occurring in animal and human digestive tracts. This paper provides an overview of current trends in the development and utilisation of in vitro digestion models for foods, as well as information that can be used to develop improved digestion models. Our survey of in vitro digestion models found that the most predominant food samples tested were plants, meats, fish, dairy, and emulsion-based foods. The most frequently used biological molecules included in the digestion models were digestive enzymes (pancreatin, pepsin, trypsin, chymotrypsin, peptidase, α-amylase, and lipase), bile salts, and mucin. In all the in vitro digestion models surveyed, the digestion temperature was 37 °C although varying types and concentrations of enzymes were utilised. With regard to digestion times, 2 h (the stomach, small intestine, and large intestine each) was predominantly employed. This survey enhances the understanding of in vitro digestion models and provides indications for the development of improved in vitro digestion models for foods or pharmaceuticals.     

Physicochemical changes of myofibrillar proteins during processing of Cantonese sausage in relation to their aggregation behaviour and in vitro digestibility

The physicochemical changes of myofibrillar proteins, especially oxidation behaviour, were measured to determine their mechanism of action on in vitro protein digestibility during Cantonese sausage processing. The results indicated that the carbonyl level significantly increased (p < 0.05) during the process. The SH group level decreased, while S–S group level increased gradually. Protein aggregation was induced by oxidation and heat treatment. Result from Fourier transform infrared (FTIR) spectroscopy confirmed protein aggregation occurred. The analysis of in vitro digestibility showed a highly significant (p < 0.05) correlation between pepsin activity and carbonyl group formation, S–S group level, protein surface hydrophobicity, D_4,3. A negative and highly significant correlation between trypsin, α-chymotrypsin activity and carbonyl group formation was measured, while no significant correlation with S–S groups, protein surface hydrophobicity, D_4,3 was observed. It indicated that not only protein oxidation and aggregation but also degradation by pepsin would influence proteolysis with trypsin and α-chymotrypsin.     

Amaranthus hypochondriacus and Amaranthus caudatus germplasm: Characteristics of plants,grain and flours

In the present study, the plant, grain and flour characteristics of 48 Amaranthus hypochondriacus and 11 Amaranthus caudatus lines are reported. A. hypochondriacus grains had a higher thousand kernel weight (0.62–0.88 g), a creamish yellow colour (L∗ = 61.38–68.29, b∗ = 5.26–6.8 and a∗ = 19.71–23.84), whereas A. caudatus grains had a lower thousand kernel weight (0.46–0.70 g) and a reddish-brown colour (L∗ = 47.1–51.2, b∗ = 11.82–14.02 and a∗ = 7.72–13.29). The A. caudatus lines had a higher protein content, fat content and tendency for retrogradation, and lower α-amylase activity as compared to A. hypochondriacus lines. The Amaranthus lines with higher a∗-values and lower thousand kernel weight, L∗- and b∗-values, had a higher fat, crude fibre and protein content. The correlation of fat and crude fibre content with the thousand kernel weight, L∗-, a∗- and b∗-values, was significant. The A. hypochondriacus lines showed higher pasting temperature, and lower peak viscosity, breakdown and setback, as compared to the A. caudatus lines. The result reflected that the lines with higher α-amylase activity showed a lower peak viscosity, breakdown, final viscosity and gel hardness, and a higher pasting temperature.     

A study of the in vitro protein digestibility of indica and japonica cultivars

Protein digestibility was determined in 18 indica and japonica raw milled rices using an in vitro pH-drop method with three- or four-enzyme system. Similar protein digestibility was found between indica and japonica rices, which is in agreement with the in vivo digestibilities in human. Cooking improved protein digestibility in the four-enzyme assay, while reducing agents exhibited apparent inhibition in multienzyme digestibility of indica and japonica rices. A significant correlation was detected between protein content and the estimates of digestibility, whereas no significant correlation was found between amylose content and digestibility estimates. SDS–PAGE analysis showed a significant difference in the degradation extent of prolamin between multienzyme and pepsin digestion, which might contribute to the inconsistence between results of this study and previous findings that in vitro protein digestibility of japonica rice was higher than that of indica rice. In addition, our results supported the previous report that waxy gene product level is not a major determinant of protein digestibility in milled rice.     

Effects of baking on protein digestibility of organic spelt products determined by two in vitro digestion methods

Consumption of organic foods is steadily increasing because it is believed to be healthier than conventional foods. This study was designed to investigate protein digestibility of organic spelt bread, biscuit, cookie and muffin in comparison to their corresponding normal wheat products. Three types of fermented bread products namely, yeast leavened, sour and yeast/sour dough were evaluated. Protein digestibility was assessed based on two methods, three-enzyme one-step and two-enzyme two-step digestion in vitro. The one-step digestion method produced results that were comparable with in vivo (rat) methods whereas the two-step digestion method was more reliable in determining differences among the examined wheat products. Organic spelt used in the present study was comparable to common wheat in protein content averaging 15.4 g/100 g dry matter. Slight differences were observed between organic spelt and common wheat products in protein digestibility determined by the two digestion methods. However, significant differences were found among each wheat products. In general, after baking protein digestion was significantly increased. Spelt and common wheat bread products had similar protein digestibility within each type of bread with sour dough breads had the highest protein digestibility. Biscuit, cookie and muffin products possessed lower protein digestibility than breads. In general, variations in protein digestibility due to baking were more noticeable than that found between the two wheats.     

Emulsifying and foaming properties of Phaseolus vulgaris and coccineus proteins

Protein isolates from two Phaseolus cultivars, common bean (Phaseolus vulgaris L.) and scarlet runner bean (Phaseolus coccineus L.), were prepared by wet extraction methods (isoelectric precipitation – 4000 rpm, ultrafiltration, extraction with NaCl 2%, and isoelectric precipitation – 9900 rpm). The protein isolates were characterized by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then evaluated for their solubility. The emulsion stability of emulsions produced at pH 7.0 and 5.5 with 1% or 2% or 3% w/v protein isolate was evaluated by average droplet size diameter, viscosity and creaming measurements. Emulsions with 1% protein content were unstable through storage. Emulsions with 3% w/v protein isolate concentration, extracted by ultrafiltration at pH 5.5 from both cultivars, were flocculated; this was more pronounced for coccineus isolates. The foaming properties, for the respective foams, were investigated. Foams with 1% w/v protein showed little foaming ability Ultrafiltration isolates produced more foam, which was especially stable at pH 5.5.     

Common bean (Phaseolus vulgaris L.) hydrolysates inhibit inflammation in LPS-induced macrophages through suppression of NF-κB pathways

The objectives of this study were to evaluate the antioxidant capacity of protein hydrolysates of the common bean (Phaseolus vulgaris L.) varieties Negro 8025 and Pinto Durango and determine their effect on the markers of inflammation in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Cell viability was determined and the percentage of viable cells was calculated and concentrations that allowed >80% cell viability were used to determine the markers of inflammation. Alcalase hydrolysates and pepsin–pancreatin hydrolysates showed the highest antioxidant capacity after 80 and 120 min of hydrolysis, respectively. Alcalase hydrolysates of the common bean Pinto Durango at 120 min inhibited inflammation, with IC₅₀ values of 34.9 ± 0.3, 13.9 ± 0.3, 5.0 ± 0.1 and 3.7 ± 0.2 μM, while var. Negro needed 43.6 ± 0.2, 61.3 ± 0.3, 14.2 ± 0.3 and 48.2 ± 0.1 μM for the inhibition of cyclooxygenase-2 expression, prostaglandin E₂ production, inducible nitric oxide synthase expression and nitric oxide production, respectively. Also, hydrolysates significantly inhibited the transactivation of NF-κB and the nuclear translocation of the NF-κB p65 subunit. In conclusion, hydrolysates from the common bean can be used to combat inflammatory and oxidative-associated diseases.     

Influence of naturally acid-soluble proteins from beans (Phaseolus vulgaris L.) on in vitro digestibility determination

The in vitro digestibility of bean (Phaseolus vulgaris) protein fractions was studied using a pepsin-pancreatin system. Enzymatic hydrolysis was stopped by adding a strong acid and the extent of proteolysis determined by measurement of free amino groups in the soluble fraction. The in vitro digestibility of bean protein fractions was low when in the native state and was differently affected by denaturation. For phaseolin, the main reserve protein, heating caused a significant increase of susceptibility to hydrolysis, whereas heat had no apparent effect on digestibility of glutelins and albumins (II). For the PIL (protease inhibitor-lectin rich) fraction, which was shown to have a composition similar to total albumins, there was a decrease of digestibility, probably associated to disulfide bond formation upon heating. Results of in vitro digestibility were shown to be strongly dependent on the utilization of a sample blank to account for proteins naturally soluble in the acid used to interrupt hydrolysis, which would otherwise be estimated as digested protein. These proteins are characterized by a high carbohydrate content, probably responsible for their high solubility and low digestibility.     

Thermal inactivation of Salmonella spp. during conching

Although chocolate is a microbiologically stable product it has been described as a vehicle for Salmonella spp. Because of the low water activity (a_w) and the high fat content of chocolate Salmonella spp. shows an increased heat resistance, even during the thermal process of chocolate making. The aim of this study was to evaluate the thermal inactivation of Salmonella spp. during conching in various masses of chocolate and cocoa butter at different temperatures (50-90 °C). The effect of thermal treatment on Salmonella spp. was determined with the MPN (Most-Probable-Number) method. Results of thermal treatment showed approximate D-values for cocoa butter from D_50°C = 245 min to D_60°C = 306 min, for cocoa liquor from D_50°C = 999 min to D_90°C = 26 min and for dark chocolate of D_50°C = 1574 min. z-values were found to be z = 20 °C in cocoa liquor and z = 14 °C in dark chocolate. This study demonstrates that the conching process alone does not ensure the inactivation of Salmonella spp. in different chocolate masses and that an additional decontamination step at the beginning of the process as well as an HACCP concept is necessary during chocolate production to guarantee the absence of Salmonella spp. in chocolates and related products.     

Hypocholesterolaemic effect of Bifidobacterium animalis subsp. lactis (Bb12) and trypsin casein hydrolysate

Hypercholesterolaemia is one of the most important risk factors in the development of cardiovascular disease. In this study, we report the in vitro potential of Bifidobacterium animalis subsp. lactis (Bb12) cultures and bovine casein hydrolysates formed by trypsin and Bb12 culture to reduce cholesterols levels. Cholesterol levels in vitro were reduced by up to 48% after incubation with Bb12 and up to 87% after incubation with trypsin hydrolysates, whereas unhydrolysed bovine casein did not affect cholesterol levels. Individual peptide fractions, obtained from size-exclusion chromatography, from casein hydrolysates formed by trypsin after a 48 h hydrolysis, reduced cholesterol levels by 2.7–50%. The molecular masses of these fractions, containing hypocholesterolaemic peptides, were below 1200 Da, as determined by LC-MS.     

Phytochemical composition,anti-inflammatory and antitumour activities of four Teucrium essential oils from Greece

The essential oils of four Teucrium species were studied and 150 components, in all, were identified. All oils were rich in sesquiterpenes (50.1–55.8%). Spathulenol and δ-cadinene were the main compounds of Teucrium brevifolium oil; caryophyllene and 4-vinyl guaiacol predominated in Teucrium flavum. Carvacrol and caryophyllene oxide predominated in Teucrium montbretii ssp. heliotropiifolium, while carvacrol and caryophyllene were the most abundant components in Teucrium polium ssp. capitatum. The oil which most effectively inhibited LPS-induced NO production in macrophage cell line RAW 264.7 was that from T. brevifolium (IC₅₀ = 7.1 μg/ml), followed by T. montbretii ssp. heliotropiifolium and T. polium ssp. capitatum (IC₅₀ = 16.5 and 29.4 μg/ml, respectively). The in vitro cytotoxic assay on three human cancer cell lines showed that the most antiproliferative oils were those from T. polium ssp. capitatum and T. montbretii ssp. heliotropiifolium on CACO-2 cell lines (IC₅₀ = 52.7 and 92.2 μg/ml, respectively). The T. brevifolium oil showed a selective cytotoxicity on COR-L23 while significant activity was exerted by T. polium oil on C32.     

Purification and identification of novel antioxidant peptides from enzymatic hydrolysates of sardinelle (Sardinellaaurita) by-products proteins

In order to utilise sardinelle (Sardinellaaurita) protein by-products, which is normally discarded as industrial waste in the process of fish manufacturing, heads and viscera proteins were hydrolysed by different proteases to obtain antioxidative peptides. All hydrolysates showed different degrees of hydrolysis and varying degrees of antioxidant activities. Hydrolysate generated with crude enzyme extract from sardine (Sardinapilchardus) displayed high antioxidant activity, and the higher DPPH radical-scavenging activity (87 ± 2.1% at 2 mg/ml) was obtained with a degree of hydrolysis of 6%. This hydrolysate was fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P₁–P₈). Fraction P₄, which exhibited the highest DPPH scavenging activity, was then fractionated by reversed-phase high performance liquid chromatography (RP-HPLC). Seven antioxidant peptides were isolated. The molecular masses and amino acids sequences of the purified peptides were determined using ESI-MS and ESI-MS/MS, respectively. Their structures were identified as Leu-His-Tyr, Leu-Ala-Arg-Leu, Gly-Gly-Glu, Gly-Ala-His, Gly-Ala-Trp-Ala, Pro-His-Tyr-Leu and Gly-Ala-Leu-Ala-Ala-His. The first peptide displayed the highest DPPH radical-scavenging activity (63 ± 1.57%; at 150 μg/ml) among these peptides.