Antioxidant and angiotensin converting enzyme (ACE) inhibitory activities of cocoa (Theobroma cacao L.) autolysates

The present study investigated antioxidant and angiotensin converting enzyme (ACE) inhibitory activities of cocoa autolysates. After removal of cocoa fat, alkaloids and polyphenols, the remaining proteinous powder was autolyzed at pH 3.5 and 5.2. At similar concentrations, autolysates produced at pH 3.5 indicated the highest reducing power and ACE inhibition activity. However, those generated at pH 5.2 showed the highest antioxidant activity based on ??-carotene bleaching assay. The results displayed a dose-dependent trend. Based on amino acids composition, slight differences were detected between autolysates, and as it was found, they were rich in hydrophobic amino acids. Qualitative and quantitative tests were applied to assure that the results from the assays were not due to the polyphenols of cocoa autolysates. Based on the results no polyphenols could be detected from cocoa autolysates. It can be indicated that among other useful substances of cocoa, its peptides and amino acids could contribute to its health-promoting properties. Furthermore, these bioactive substances can be exploited into functional foods or used as a source of nutraceuticals.     

Dietary fibre composition,antioxidant capacity and physico-chemical properties of a fibre-rich product from cocoa (Theobroma cacao L.)

The proximate composition and dietary fibre (DF) content of a fibre-rich product obtained from cocoa were studied. This product contained 60.54% (dry matter basis) of DF, made of mainly insoluble fibre although with appreciable amounts of soluble dietary fibre (10.09% d.m.). The presence of associated polyphenolic compounds (1.32% and 4.46% of soluble polyphenols and condensed tannins, respectively) provides this fibre material with intrinsic antioxidant capacity as determined by the FRAP and TEAC methods. Hydration properties (swelling and water holding capacity) and the glucose retardation index of cocoa fibre were similar to other natural commercial insoluble fibres. The antioxidant capacity of this fibre-rich cocoa powder and its physico-chemical properties render it a suitable product to be used in the preparation of low-calorie, high-fibre foods.     

Dynamics of volatile and non-volatile compounds in cocoa (Theobroma cacao L.) during fermentation and drying processes using principal components analysis

Different volatile and non-volatile compounds produced during the fermentation-drying process are considered as indicatives of cocoa beans quality. We found thirty-nine different compounds identified by SPME-HS/GC-MS and related to the desirable notes and off-flavor that have been reported. Volatile and non-volatile compounds were associated with acidity and changes of pH, such as acetic and lactic acid. Using the principal component analysis (PCA), relations between compounds and fermentation and drying day were associated with dynamics of these compounds. The identification of principal compound produced during the fermentation and drying processes can be helpful in searching for off-flavor indicator and as a fermentation index, such as isobutyric, isovaleric and propionic acids. Oxidation of 3-methyl-1-butanol-to-3-methyl-1-butanol acetate can be of use in evaluating the degree of fermentation. At drying, the compounds with the highest levels were acetic and isobutyric acid, ethyl and 3-methyl-1-butanol acetate, pentanal and 2,3-pentanedione, and 1,3-butanediol and 2,3-butanediol. Therefore, acetic acid and isobutyric acid, due to their high levels and their low threshold value could play an important role in the aromatic quality of cacao drying.     

The content of protein and non-protein (free and protein-bound) tryptophan in Theobroma cacao beans

The contents of protein and non-protein (free and protein-bound) tryptophan and of proteins in cocoa beans of various origin were determined. Protein concentrations varied from 11.8 g/100 g in beans from the Dominican Republic to 15.7 g/100 g in roasted beans from the Ivory Coast. The highest protein tryptophan content was found in cocoa beans from Ecuador. Madagascar beans had the highest value of free tryptophan and Echeandia the lowest (17.26 and 6.39 mg/100 g, respectively). Tryptophan was bound to water-soluble proteins as well as to proteins soluble in buffer solution (pH 8.9) and in 70% ethanol. In particular, Dominican Republic cocoa contained the highest amount of tryptophan bound to water-soluble proteins. Very little tryptophan was linked to proteins soluble in alkaline or ethanol solutions, and values ranged from 0.96 to 3.04 and from 0.24 to 1.21 mg/100 g of dry defatted cocoa sample, respectively.     

Highly acetylated pectin from cacao pod husks (Theobroma cacao L.) forms gel

A pectin (OP) obtained from cacao pod husk with a high acetyl content, which is a structural feature that could disturb the pectins' gel formation, was able to form gels at low pH and a high sucrose content. Pectin gels (1.32% GalA equivalent, w/w) were prepared at pH 2.5–3.3 in the presence of 60% sucrose (w/w). Rheological analyses were performed to determine the optimal pH for further studies. Next, the OP samples were prepared at pH 2.7 in concentrations ranging from 0.33 to 1.98% GalA (w/w) with 60% sucrose (w/w) and subjected to rheological analysis. Dynamic oscillatory experiments at 25 °C indicated the presence of gels for all of the analysed concentrations. Measurements of the elastic (G′) and viscous (G″) moduli at 25 °C also indicated that increasing the pectin concentration resulted in stronger gels. Rotational experiments revealed a shear-thinning behaviour in which the apparent viscosities of the samples increased as the concentration increased. Although the OP had a high degree of acetylation, this pectin was able to form gels, which suggests its potential for use as a gelling and thickening additive.     

Development and characterisation of functional cocoa (Theobroma cacao L.)-based edible films

The aim of this study was to develop functional edible films containing cocoa (Theobroma cacao L.) powder extract using alginate, pectin and chitosan in combination with proteins (whey protein isolate, soya and hemp protein). The films were examined for their physico-chemical (dry matter content, colour, thickness), mechanical (elongation at break (EAB)), bioactive (the content of total polyphenols (TPC), flavan-3-ols (F3olC), antioxidant capacity (AC)) and sensory properties. The plain alginate film exhibited the highest EAB (29.1%). The highest TPC and F3olC were determined in plain alginate (29 mg GAE/g and 2.75 mg (+)-catechin/g) and pectin (29 mg GAE/g and 2.25 mg (+)-catechin/g) films. The addition of proteins resulted in prolonged release of polyphenols and enhanced functional properties; however, the formation of protein–polyphenol complexes caused slight alterations in the bioactive composition of the films. The obtained results indicate a high potential of the developed films as functional, biodegradable form of active food packaging.     

Extraction of cocoa butter alternative from kokum (Garcinia indica) kernel by three phase partitioning

Kokum kernel is a byproduct of agro-processing industry in India containing about 40–50% fat which has the potential as a worthy cocoa butter alternative (CBA). However, inefficient extraction techniques that are practiced at cottage level restrict its industrial applications. This work reports on the optimization of the technique of three phase partitioning (TPP) for efficient extraction of kokum kernel fat. The parameters of TPP were optimized with respect to ammonium sulphate concentration, ratio of slurry to t-butanol and pH of slurry. The optimized protocol resulted in maximum recovery of 95% (w/w) fat recovery within 2 h. The technique is economical and eco-friendly, and is promising for utilization of agro-processing waste in India to a product of commercial significance.     

Effect of different treatments on the quality of cuttlefish (Sepia officinalis L.) viscera

The effects of different preservation times (cuttlefish auction), transformation factory treatments, different times of frozen storage at −20 °C and various defrosting temperatures were investigated with respect to the chemical changes of the viscera of cuttlefish (Sepia officinalis). During auction storage, pH and TCA (trichloroacetic acid)-soluble protein concentration decreased, whereas intracellular (cathepsins, total acid proteases and acid phosphatases) and extracellular (amylase, chymotrypsin, trypsin and total alkaline proteases) enzymatic activities increased. In cuttlefish transformation factories, pH value and TCA-soluble protein concentration increased. In transformation factory conditions, lipase and amylase levels varied, while other assayed enzymes were stable. Moreover, during transformation factory treatment, the molecular weight of proteins/peptides decreased and the oil composition evolution reflected hydrolysis of cuttlefish viscera. Thus, cuttlefish viscera fatty acid composition differed between fresh viscera and factory viscera. Despite changes in the fatty acid chain composition during factory transformation, the percentage of poly-unsaturated fatty acids remained high. Frozen storage implied major changes in viscera quality such as an increase in pH and a decrease in the quantity of high molecular weight protein. Moreover, the higher the defrosting temperature, the higher was the pH. During storage at −20 °C the quantities of proteins, carbohydrates and lipids decreased slightly.     

Characterization of the constituents and antioxidative activity of cocoa tea (Camellia ptilophylla)

Three main types of cocoa tea derived from Camellia ptilophylla, white cocoa tea (WCT), green cocoa tea (GCT) and black cocoa tea (BCT) were prepared. A comparison between cocoa tea and commonly consumed tea Biluochun cha (BLC), Longjing cha (Lj), Qimenhong cha (Qmh) and Yinghong 9^# (Yh9) was studied. Levels of catechins and purine alkaloids were determined by HPLC. Total polyphenol contents were determined by ferrous tartrate assay. Antioxidative activity was evaluated using ABTS decolorization assay, DPPH radical assay and ferric thiocyanate (FTC) method. The results showed that WCT contained the highest polyphenol content and showed the highest antioxidative activity. The radical scavenging ability of the tea extracts followed this order: WCT > GCT > Blc > Lj > BCT > Yh9 > Qmh. Tea extracts measured by the linoleic acid system also showed the same patterns of activity as the ABTS and DPPH method. A high correlation between polyphenol content and antioxidative activity of tea extracts was observed.     

Behavior of Salmonella during fermentation,drying and storage of cocoa beans

Due to cocoa being considered a possible source of Salmonella contamination in chocolate, the behavior of Salmonella during some cocoa pre-processing stages (fermentation, drying and storage) was investigated. The fermentation process was carried out on a pilot scale (2 kg beans/box) for 7 days. Every day a fermentation box was inoculated with a Salmonella pool (ca. 4 log MPN/g). The results showed that Salmonella did not affect (P > 0.05) the growth of the main microorganism groups involved in cocoa fermentation. On the other hand, the pathogen was influenced (P < 0.05) by yeast, acetic acid bacteria and pH. In spite of Salmonella showing counts ≤ 1 log MPN/g in the first days, at the end of fermentation it grew in all samples, reaching counts as high as 7.49 log MPN/g. For drying and storage, cocoa beans were inoculated during the fermentation (experiment A) or during the drying (experiment B). In these stages the decline of the water activity affected the pathogen behavior. In experiment A during the drying, Salmonella count increased in most of the samples. In experiment B either a slight growth or no growth in the samples inoculated up to 48 h was observed, whereas the other samples showed reductions from the initial count. After 30 days of storage at room temperature, the water activity decreased to 0.68, and reductions of Salmonella ranged from 0.93 to 2.52 log MPN/g. Despite the reductions observed during the storage, the pathogen was detected even after 120 days. Therefore, the results showed that Salmonella growth or survival depends on when the contamination occurs.     

Yeast derivatives (extracts and autolysates) in winemaking: Release of volatile compounds and effects on wine aroma volatility

A qualitative study of volatile compounds in three commercial yeast extracts and autolysates was performed by solid-phase microextraction-gas chromatography with mass spectrometric and olfactometric detection; their addition to white wines and their effect on wine aroma composition were investigated by analytical, olfactometric and sensory evaluations. More than 160 volatile compounds were detected in the headspace of the commercial powders (some not previously reported in literature), and their olfactory characters were described. Yeast derivatives strongly modified wine aroma composition, either affecting the volatility of wine aroma compounds or by releasing exogenous volatiles. Dosage appeared to be fundamental: low amounts increased the volatility of some esters, giving more flowery and fruity notes to the wine; higher amounts increased fatty acid content in the wine headspace, producing yeasty, herbaceous and cheese-like smells. Sensory tests demonstrated that yeast derivatives would not be suitable for wines with a typical varietal aroma.     

Antioxidative effects of daikon sprout (Raphanus sativus L.) and ginger (Zingiber officinale Roscoe) in rats

The antioxidative effects of vegetables are expected to prevent carcinogenesis. The intake of daikon sprout (Japanese name “kaiware-daikon”, Raphanus sativus L.) or ginger (Zingiber officinale Roscoe) significantly decreased the concentration of urinary thiobarbituric acid-reactive substances (TBARS) in rats as compared with those before the intake. Moreover, the intake of these vegetables reduced urinary 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels in lipopolysaccharide (LPS)-treated rats as compared with those fed a basal diet only. These results show that these vegetables suppress lipid peroxidation and the formation of malonaldehyde, and protect DNA from LPS-induced oxidative damage in rats. The suppression of lipid peroxidation and oxidative DNA damage in rats by the intake of daikon sprout or ginger indicates that these vegetables have an antioxidative effect in vivo which could be related to the prevention of carcinogenesis.     

Effects of milking interval and cisternal udder evaluation in Tunisian Maghrebi dairy dromedaries (Camelus dromedarius L.)

Effects of 4 different milking intervals (8, 12, 16, and 24 h) on milk yield and milk composition were studied in Tunisian Maghrebi dairy dromedaries (n = 6) at late lactation [240 ± 14 days in milk (DIM), 5.84 ± 1.62 L/d]. Camel-cows suckled their calves for 2 mo, were hand milked while suckling until mo 4 of lactation (calf weaning) and machine milked thereafter. Intravenous injection of oxytocin was administered before machine milking at each experimental milking to induce complete milk ejection and to avoid carryover effects of milking intervals. Cisternal and alveolar milk were measured at 380 ± 16 DIM for a 24-h milking interval. Milk accumulated logarithmically (R² = 0.95) in the udder from 8- to 24-h milking interval without reaching a plateau. Consequently, milk secretion rate decreased exponentially (R² = 0.93) according to milking interval. Compared with 12-h milking interval (6.1 L/d), estimated daily milk yield was 113, 87, and 70% for 8-, 16-, and 24-h intervals, respectively. Total milk solids, milk fat content, and milk pH decreased with increasing milking interval, showing the greatest value at 8-h intervals (14.1 ± 0.4%, 4.6 ± 0.5%, and 6.66 ± 0.05, respectively) and the lowest at 24-h intervals (12.3 ± 0.9%, 2.9 ± 0.6%, and 6.54 ± 0.02, respectively). Milk protein (3.9 ± 0.1%), lactose (4.5 ± 0.2%), ash (0.84 ± 0.01%) and density (1.028 ± 0.01) remained constant for all milking intervals. Milk K, Ca, and Mg contents increased as milking interval increased, but Na content did not change (0.06 ± 0.01%, on average). Milk Na:K ratio tended to decrease from 0.35 (1:2.9) to 0.22 (1:4.5) for the extreme milking intervals. Plasma lactose concentration steadied from 8- to 16-h (67 ± 32 μmol) but increased dramatically at 24-h intervals (338 ± 118 μmol), indicating that mammary tight junctions became permeable after 24 h of milk accumulation. Camel udders showed small cisterns (19.3% of total milk in the udder at 24 h) when compared with other dairy animals; we recommend the use of prestimulation for machine milking and selection for larger udder cisterns. Alveolar milk contained more fat (5.16 vs. 1.75%; SEM, 0.39%) and protein (3.23 vs. 2.73%; SEM, 0.15%) than cisternal milk. Despite the increase of plasma lactose during tight junction leakiness, the tendency for the Na:K ratio to decrease may be indicative of a camel's specific regulatory mechanism for controlling Na and K concentrations in milk and delaying the inhibitory effect of milk stasis on milk secretion rate. In conclusion, this short-term study proved the low storage capacity of the Tunisian Maghrebi camel udder but also showed their moderate ability to adapt to extended milking intervals at late lactation.     

知母-三七粉对糖尿病大鼠的降血糖和降血脂作用研究

目的：研究知母-三七粉、知母提取物及三七提取物对四氧嘧啶造模糖尿病大鼠的降血糖和降血脂作用。方法：通过正常动物降糖试验和高血糖模型降糖试验来研究知母-三七粉对的降血糖和降血脂作用,并采用最大给药量试验法评价其急性毒性。结果：正常大鼠降血糖试验表明,知母-三七粉、知母提取物、三七提取物均对正常大鼠的体重及空腹血糖无明显影响(P<0.05)。四氧嘧啶造模的高血糖模型大鼠降血糖试验表明,各剂量组对糖尿病大鼠的体重无明显影响,且均能显著降低糖尿病大鼠的空腹血糖值及餐后血糖值(P<0.01)。与模型组相比,知母-三七粉、知母提取物高剂量组及三七提取物高剂量组的糖化血清蛋白(glycated serum protein, GSP)水平均显著降低(P<0.05),血清胰岛素(serum insulin, INS)含量均显著上升(P<0.05);此外,各剂量组均能降低糖尿病大鼠的总胆固醇(total cholesterol, TC)、甘油三酯(triglyceride, TG)及低密度脂蛋白胆固醇(low-density lipoprotein cholesterol, LDL-C)水平,同时升高其高密度脂蛋白胆固醇(high density lipoprotein ch, HD)水平,其中知母-三七粉的效果更加显著。肾脏的病理切片也表明,知母和三七提取物有改善肾脏病变、缓解肾小管肿大的作用。最后,急性毒性试验表明,在观察的14 d内各组小鼠无明显中毒症状、无死亡现象,体重增长正常,解剖未见明显异常。结论：通过实验可以看出知母-三七粉、知母提取物、三七提取物对正常大鼠的体重与空腹血糖无明显影响,对高血糖大鼠表现出良好的降血糖和降血脂作用,且知母-三七粉安全性良好,属于实际无毒级别,为知母-三七粉的开发应用提供一定的理论依据。     

Phenolic compounds content and antioxidant activity in pomace from selected red grapes (Vitis vinifera L. and Vitis labrusca L.) widely produced in Brazil

The phenolic compounds content and antioxidant activity of pomace from the vinification of grape varieties widely produced in Brazil (Cabernet Sauvignon, Merlot, Bordeaux and Isabel) were investigated with a view to their exploitation as a potential source of natural antioxidants. Cabernet Sauvignon grape pomace was found to have the highest content of total phenolic compounds (74.75 mg gallic acid equivalent (GAE)/g), the highest antioxidant activity (determined using the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging methods; 485.42 and 505.52 μMol Trolox equivalent antioxidant capacity (TEAC)/g, respectively), and the highest reducing power (determined using the FRAP method; 249.46 μMol TEAC/g). The Bordeaux variety showed the highest oxidation inhibition power (41.13%), determined using the β-carotene/linoleic acid method and the highest content of total anthocyanins (HPLC; 29.17 mg/g). Catechin was the most abundant non-anthocyanic compound identified in the grape pomace (150.16 mg/100 g) for all varieties. In this study, pomaces of the red wine vinification of Cabernet Sauvignon and Bordeaux varieties showed the highest potential as a source of antioxidant compounds and natural colourants, respectively.     

The effects of carbon monoxide on Atlantic salmon (Salmo salar L.)

Atlantic salmon were exposed to carbon monoxide (CO) before the fish were percussively killed and gill cut. The fish were compared against a control group treated identically, without CO. Salmon exposed to CO expressed no adverse reactions and were easily stunned by percussion. CO-treated salmon had an earlier onset of rigor mortis and a faster decrease in muscle pH than the control group. No significant difference in drip loss was found between salmon treated with CO and the control. A significantly deeper red colour of both gills and fillets of CO-treated salmon was observed 10 days post mortem. Significantly higher levels of plasma lactate and potassium were found in CO-treated salmon compared to control, as well as a lower level of pCO₂. Exposure to CO did not increase plasma cortisol, sodium, haematocrit or glucose; however, lactate was high. Exposure of salmon or other fish to CO could improve quality and welfare when slaughtered.     

Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus predominate during well-performed Malaysian cocoa bean box fermentations,underlining the importance of these microbial species for a successful cocoa bean fermentation process

Two spontaneous Malaysian cocoa bean box fermentations (one farm, two plantation plots) were investigated. Physical parameters, microbial community dynamics, yeast and bacterial species diversity [mainly lactic acid bacteria (LAB) and acetic acid bacteria (AAB)], and metabolite kinetics were monitored, and chocolates were produced from the respective fermented dry cocoa beans. Similar microbial growth and metabolite profiles were obtained for the two fermentations. Low concentrations of citric acid were found in the fresh pulp, revealing low acidity of the raw material. The main end-products of the catabolism of the pulp substrates glucose, fructose, and citric acid by yeasts, LAB, and AAB were ethanol, lactic acid, acetic acid, and/or mannitol. Hanseniaspora opuntiae, Lactobacillus fermentum, and Acetobacter pasteurianus were the prevalent species of the two fermentations. Saccharomyces cerevisiae, Lactobacillus plantarum, Lactobacillus pentosus, and Acetobacter ghanensis were also found during the mid-phase of the fermentation processes. Leuconostoc pseudomesenteroides and Acetobacter senegalensis were among the prevailing species during the initial phase of the fermentations. Tatumella saanichensis and Enterobacter sp. were present in the beginning of the fermentations and they could be responsible for the degradation of citric acid and/or the production of gluconic acid and lactic acid, respectively. The presence of facultative heterofermentative LAB during the fermentations caused a high production of lactic acid. Finally, as these fermentations were carried out with high-quality raw material and were characterised by a restricted microbial species diversity, resulting in successfully fermented dry cocoa beans and good chocolates produced thereof, it is likely that the prevailing species H. opuntiae, S. cerevisiae, Lb. fermentum, and A. pasteurianus were responsible for it.     

Volatile compounds of copoazú (Theobroma grandiflorum Schumann) fruit

Volatile compounds were isolated from copoazú (Theobroma grandiflorum Schumann) fruit by liquid–liquid extraction at two different pH values of 3.3 (natural) and 7. The volatile extracts were analyzed by capillary GC–FID and GC–MS. Among the 56 compounds identified at two pH values, many of them were only present in the lower pH extract, confirming the presence of bound compounds in copoazú. Ethyl butanoate, ethyl hexanoate and linalool were the major constituents found in copoazú fruit.     

Saffron (Crocus sativus L.) increases glucose uptake and insulin sensitivity in muscle cells via multipathway mechanisms

Saffron (Crocus sativus Linn.) has been an important subject of research in the past two decades because of its various biological properties, including anti-cancer, anti-inflammatory, and anti-atherosclerotic activities. On the other hand, the molecular bases of its actions have been scarcely understood. Here, we elucidated the mechanism of the hypoglycemic actions of saffron through investigating its signaling pathways associated with glucose metabolism in C₂C₁₂ skeletal muscle cells. Saffron strongly enhanced glucose uptake and the phosphorylation of AMPK (AMP-activated protein kinase)/ACC (acetyl-CoA carboxylase) and MAPKs (mitogen-activated protein kinases), but not PI 3-kinase (Phosphatidylinositol 3-kinase)/Akt. Interestingly, the co-treatment of saffron and insulin further improved the insulin sensitivity via both insulin-independent (AMPK/ACC and MAPKs) and insulin-dependent (PI 3-kinase/Akt and mTOR) pathways. It also suggested that there is a crosstalk between the two signaling pathways of glucose metabolism in skeletal muscle cells. These results could be confirmed from the findings of GLUT4 translocation. Taken together, AMPK plays a major role in the effects of saffron on glucose uptake and insulin sensitivity in skeletal muscle cells. Our study provides important insights for the possible mechanism of action of saffron and its potential as a therapeutic agent in diabetic patients.