Fractionation of lipids and purification of γ-linolenic acid (GLA) from Spirulinaplatensis

The microalgae, Spirulinaplatensis, is an excellent source of γ-linolenic acid (GLA), an essential polyunsaturated fatty acid and a potent nutraceutical. The fatty acid composition of S.platensis ARM 740 was determined after transmethylation by gas chromatography (GC). Lipid fractionation was achieved on silica gel column chromatography and preparative TLC. Neutral lipids, glycolipids and phospholipids accounted for 77.0%, 15.6% and 7.4%, respectively, of the total lipid fraction. S.platensis ARM 740 was found to contain 94% of the total GLA in the glycolipid fraction. Attempts were made to purify GLA methyl ester by using urea to form inclusion complexes with the saturated and the less unsaturated FAMEs (fatty acid methyl esters), which enhanced the purity of GLA methyl ester to 84%. A further approach to isolate GLA methyl ester with higher purity involved the use of argentated silica gel chromatography. An initial PUFA concentration step frequently adopted by most researchers to increase GLA purity was not necessary in the isolation of GLA from S.platensis. A GLA methyl ester with a purity of >96% and a recovery of 66% was obtained. Purity of the isolated GLA methyl ester was confirmed by GC and IR analysis with respect to authentic standard.     

In situ production of γ-aminobutyric acid in breakfast cereals

Breakfast cereals are an important part of an equilibrated diet in the Western world, making them extremely suited for carrying health benefits. Intake of γ-aminobutyric acid (GABA), a major inhibitory neurotransmitter of the nervous system, has been related to blood pressure lowering in hypertensive individuals. In vivo, GABA is formed from glutamic acid (GA) by glutamic acid decarboxylase (GAD), a widely distributed enzyme in prokaryotic and eukaryotic species. We here enriched breakfast cereals with GABA by recipe and process optimisation. The dynamics of GA and GABA were monitored throughout the production process. Addition of exogenous recombinantly produced GAD of Yersinia intermedia increased GABA levels by 2- to 5-fold. As only trace levels of GABA (<15 ppm) and relatively low levels of its precursor (GA, <100 ppm) are present in the wheat and rice flour used, a well-thought ingredient choice (inclusion of quinoa flour (ca. 90 ppm GABA and 700 ppm GA) or bran enrichment (ca. 66 ppm GABA and 500 ppm GA)) also significantly increases the GABA content in the final flakes. Finally, a strict control of the heating steps during the production process reduces GA and GABA losses. Consumption of one portion (30 g) of the here produced enriched breakfast cereals can even meet up to 55% of the daily intake earlier reported to lower blood pressure (ca. 10 mg).     

Antioxidant activity and γ-aminobutyric acid (GABA) content in sea tangle fermented by Lactobacillus brevis BJ20 isolated from traditional fermented foods

GABA-producing lactic acid bacteria were isolated from kimchi and salt-fermented Jot-gal, which are traditional Korean fermented foods. The strain, BJ-20, isolated from salt-fermented Jot-gal (cod gut), possessed the highest GABA-producing ability in MRS broth with 1% monosodium glutamate (MSG), as determined by thin layer chromatography. The BJ-20 strain was identified as Lactobacillus brevis and designated as L. brevis BJ20. A sea tangle solution was fermented over 5 days to produce GABA using L. brevis BJ20. During fermentation, the GABA concentration dramatically increased, while the glutamic acid concentration decreased. This result indicates that the glutamic acid was converted to GABA by L. brevis BJ20 in the fermented sea tangle solution. Furthermore, the fermented solution exhibited strong antioxidant activities, such as DPPH scavenging, superoxide scavenging, and xanthine oxidase inhibition, which were higher than those of BHA as a positive control.     

Effects of components in culture medium on glutamate decarboxylase activity and γ-aminobutyric acid accumulation in foxtail millet (Setaria italica L.) during germination

The effects of glutamic acid (Glu), pyridoxal-5-phosphate (PLP) and calcium chloride (CaCl₂) in culture medium on glutamate decarboxylase (GAD) activity and γ-aminobutyric acid (GABA) accumulation in foxtail millet (Setaria italica L.) during germination were investigated in this study. The components in culture medium for GABA accumulation were optimised using response surface methodology (RSM). Results showed that GAD activity and GABA yield were dependent on the addition of Glu, PLP and CaCl₂ into the culture medium. Box–Behnken design indicated that the optimal culture components for GABA accumulation were: Glu at a concentration of 1.2 mg/ml, PLP at a concentration of 50 μM and CaCl₂ at a concentration of 2.5 mM. Under the optimal conditions, the maximal production of GABA (42.9 mg/100 g FW) was obtained. Analysis of variance for the regression model suggested that the model can quite exactly predict GABA accumulation in millet during germination.     

Study of nonenzymic browning in α-amino acid and γ-aminobutyric acid/sugar model systems

The reactivities, in nonenzymic browning, of γ-aminobutyric acid (GABA), a non-protein amino acid with wide natural occurrence and potential health benefits as it occurs in foods, and of the α-l-amino acids arginine, glutamic acid, glutamine, leucine, lysine, and phenylalanine were investigated by heating equimolar mixtures of glucose and the cited amino acids at 110 °C at pH 6.0 for different times (0–4 h). Linear regression analysis indicated that the colour development in a GABA/glucose mixture was slower than that of a lysine/glucose mixture and comparable to that of a phenylalanine/glucose mixture. High-performance anion-exchange chromatography (HPAEC) with integrated pulsed amperometric detection (IPAD) showed that the decrease in GABA levels (ca. 10% after heating for 4 h) as a function of heating time was smaller than that of glucose (ca. 30% after heating for 4 h). At the same time, glucose to fructose isomerisation took place. After 20 min of heating at pH 6.0, all mixtures showed a fructose peak, the area of which increased with heating time. However, after correcting for fructose isomerisation, glucose losses were still higher than amino acid losses. In contrast to its precursor glutamic acid, GABA was stable during heating of a solution containing it alone. Heating of GABA-containing d-sugar solutions (xylose, fructose, glucose, maltose and sucrose) showed that the relative order of colour yield was pentose > hexose > disaccharides. As well as glucose to fructose isomerisation, HPAEC–IPAD allowed monitoring of the different isomerisation reactions occurring, and also disaccharide hydrolysis in the different GABA/sugar mixtures.     

Isolation and characterisation of κ-casein/whey protein particles from heated milk protein concentrate and role of κ-casein in whey protein aggregation

Milk protein concentrate (79% protein) reconstituted at 13.5% (w/v) protein was heated (90 °C, 25 min, pH 7.2) with or without added calcium chloride. After fractionation of the casein and whey protein aggregates by fast protein liquid chromatography, the heat stability (90 °C, up to 1 h) of the fractions (0.25%, w/v, protein) was assessed. The heat-induced aggregates were composed of whey protein and casein, in whey protein:casein ratios ranging from 1:0.5 to 1:9. The heat stability was positively correlated with the casein concentration in the samples. The samples containing the highest proportion of caseins were the most heat-stable, and close to 100% (w/w) of the aggregates were recovered post-heat treatment in the supernatant of such samples (centrifugation for 30 min at 10,000 × g). κ-Casein appeared to act as a chaperone controlling the aggregation of whey proteins, and this effect was stronger in the presence of α_S- and β-casein.     

Effect of oral γ-aminobutyric acid (GABA) administration on sleep and its absorption in humans

Food Science and Biotechnology - The effects of γ-aminobutyric acid (GABA) on sleep and its levels in blood after oral administration were investigated in humans. A randomized, single-blind,...     

Quantitative analysis of triterpenoids in different parts of Ilex hainanensis, Ilex stewardii and Ilex pubescens using HPLC–ELSD and HPLC–MS and antibacterial activity

An HPLC–ELSD method was first developed for the quantitative analysis of principle triterpenoids in Ilex hainanensis, Ilex stewardii and Ilex pubescens. The established method, with excellent precision, repeatability and recovery, was successfully applied to determine four triterpenoids in 24 samples from different species and medicinal parts of Ilex, and the changing trend was discussed. HPLC–ESI–MSⁿ was used for the identification of constituents in samples. The proposed method was simple, effective and suitable for investigations of these plants. Furthermore, the ethanol extracts from leaves of Ilex species, as well as their main components, were assessed for their antibacterial activities. The results indicated that the extracts of I. hainanensis, I. stewardii and I. pubescens could inhibit the growth of the tested oral pathogens, Streptococcus mutans and Actinomyces viscosu. Particularly, ilexgenin A was the most effective with MIC values of 7.8 and ?3.9 μg/ml, respectively.     

Effects of different ATP contents on phosphorylation level of glycogen phosphorylase and its activity in lamb during incubation at 4 ℃ in vitro

The aim of this study was to investigate the effects of ATP on glycogen phosphorylase activity in lamb during incubation at 4℃ in vitro. Sarcoplasmic proteins from lamb were extracted and treated with different contents of ATP to get three groups of glycogen phosphorylase with low, middle and high ATP content groups, the amount of ATP were 0.5, 2.0 and 2.5 μM per 100 μg protein, respectively. The control group was without ATP adding. The results showed that ATP promoted the phosphorylation of glycogen phosphorylase, and phosphorylation modification promoted its activity. But ATP inhibited the activity of glycogen phosphorylase and ATP preferentially participated in phosphorylation. When ATP concentration was 0.5 μM per 100 μg protein, the effect of phosphorylation modification on the activity of glycogen phosphorylase was equal to the inhibition of ATP. The effect of glycogen phosphorylase phosphorylation on its activity gradually became dominant as incubation time prolonged.     

Effect of γ-irradiation on antioxidant activity of black pepper (Piper nigrum L.)

Antioxidant activity and EPR investigations of irradiated ground black pepper (Piper nigrum L.) were evaluated. The black pepper was exposed to γ-irradiation at doses from 5 to 30 kGy. The effect of irradiation on antioxidant properties of black pepper extracts was investigated by radical-scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, by determination of reducing power and content of thiobarbituric acid-reactive substances. Some significant changes were observed in creation of thiobarbituric acid-reactive substances (TBARS). Difference between non-irradiated and irradiated samples at the legal European limit dose of 10 kGy reached, on average, 23% and, at the Food and Drug Administration (FDA) 30 kGy limit, 33%. Irradiation affected significantly the DPPH radical-scavenging activity and reducing power of ground black pepper extracts. The γ-radiation treatment of ground black pepper samples observed by EPR, resulted in the production of three paramagnetic species (GI–GIII) characterized by different origin, thermal behaviour and stability. The axially symmetric EPR resonances, GI and GII, were assigned to the carbohydrate radical structures. The spin Hamiltonian parameters of GIII possessed the characteristic features of “cellulosic” radical species. The EPR measurements, performed 20 weeks after the radiation process, confirmed that temperature increase from 298 to 353 K, caused significant decrease of integral EPR signal intensity for γ-irradiated samples (∼40%), compared to the reference (non-irradiated) ground black pepper, where only 13% drop was found. Significant correlation between EPR and thiobarbituric acid methods was assessed by study of antioxidant activity changes in relation to irradiation doses and also in the case of spice storage, between EPR and reducing power methods.     

Development and validation of a UPLC–ELSD method for fast simultaneous determination of five bile acid derivatives in Calculus Bovis and its medicinal preparations

A reverse phase ultra-performance liquid chromatography coupled with evaporative light scattering detection (UPLC–ELSD) method was successfully established for simultaneous determination of five bile acid derivatives in Calculus Bovis and its medicinal preparations using a Waters Acquity UPLC T3 C₁₈ column (2.1 × 50 mm, 1.8 μm) with gradient elution of 0.2% aqueous formic acid and acetonitrile. The influence of liquid flow-rate and column temperature, gas flow-rate and drift tube temperature on resolution was investigated to get the optimized parameters and conditions. This method was validated according to the regulatory guidelines with respect to precision, accuracy and linearity. Under optimum conditions, the five analytes were baseline separated in 11 min with detection and quantification limits ranged in 3.0–6.0 ng and 6.0–18.0 ng, respectively, which provided about a twofold reduction in analysis time and good efficiency and sensitivity by comparing a conventional high performance liquid chromatography (HPLC) method.     

Identification of an acidic α-amylase from Alicyclobacillus sp. A4 and assessment of its application in the starch industry

An acidic α-amylase was purified from thermoacidophilic Alicyclobacillus sp. A4 by ion exchange chromatography with 22% recovery, and showed a molecular mass of 64 kDa by SDS-PAGE. Its amino acid sequence was determined by sequencing three internal peptides and the complete genome of strain A4, and shared highest identity (64%) with Alicyclobacillusacidocaldarius α-amylase. Compared with other reported α-amylases, the purified enzyme had some distinct characteristics. The optimal activity was found to occur at 75 °C and pH 4.2, similar to the glucamylase widely used in the starch industry. The enzyme was Ca²⁺ independent, and had strong ability to digest raw starch (96.71%) with commercial glucamylase in one step. These properties of the purified enzyme make up the deficiency of the commercial α-amylases currently used and avoid repeated adjustment of pH and temperature in double-enzymatic sugar-making process. The purified enzyme will be commercially valuable in the starch industry.     

6-shogaol attenuates H2O2-induced oxidative stress via upregulation of Nrf2-mediated γ-glutamylcysteine synthetase and heme oxygenase expression in HepG2 cells

Food Science and Biotechnology - The signaling pathway by which 6-shogaol protects HepG2 cells against H2O2-induced oxidative stress was investigated. Cellular anti-oxidant activities, the GSH...     

Enhancement of γ-aminobutyric acid (GABA) in Nham (Thai fermented pork sausage) using starter cultures of Lactobacillus namurensis NH2 and Pediococcus pentosaceus HN8

The aim was to produce Nham that was enriched with γ-aminobutyric acid (GABA); therefore two GABA producing lactic acid bacteria (Pediococcus pentosaceus HN8 and Lactobacillus namurensis NH2) were used as starter cultures. By using the central composite design (CCD) we showed that addition of 0.5% monosodium glutamate (MSG) together with an inoculum size of roughly 6 log CFU/g of each of the two strains produced a maximal amounts of GABA (4051 mg/kg) in the ‘GABA Nham’ product. This was higher than any current popular commercial Nham product by roughly 8 times. ‘GABA Nham’ with the additions of both starters and MSG (T_SM) supported maximum populations of lactic acid bacteria (LAB) with a minimum of yeasts and no staphylococci or molds when compared to the controls that had no addition of any starters or MSG (T_NN), or only the addition of MSG (T_NM), or with only the starter (T_SN). Based on proximate analysis among the Nham sets, ‘GABA Nham’ was low in fat, carbohydrate and energy although its texture and color were slightly different from the control (T_NN). However, sensory evaluations of ‘GABA Nham’ were more acceptable than the controls and commercial Nham products for all tested parameters. Hence, a unique novel ‘GABA Nham’ fermented pork sausage was successfully developed.