Improved synthesis of isomaltooligosaccharides using immobilized α-glucosidase in organic–aqueous media

Food Science and Biotechnology - α-Glucosidase was immobilized onto an epoxy-activated resin (Eupergit C) to catalyze maltose into isomaltooligosaccharides (IMO), and then the effects of...     

Enzymatic properties and transglycosylation of α-galactosidase from Penicillium oxalicum SO

Penicillium oxalicum SO α-galactosidase demonstrated weak hydrolysing activity but a high rate of transglycosylation in the reaction with melibiose, where the major product was 6-α-galactosyl melibiose. The transfer ratio was 83.6% and was maintained over a long reaction time of 80 h. The molecular weight was estimated to be 124,000 by SDS–PAGE. The optimal pH was ∼3 and a stable pH, with a range of 2.4–9.5, was found. The optimal temperature was ∼60 °C and the activity was stable below 60 °C. With respect to acceptor specificity, mono-alcohols, sugar alcohols and sugars were poor acceptors, but the di-alcohol ethylene glycol and the tri-alcohol glycerin were good acceptors. The percentage of transglycosylation to glycerin increased up to 41.7%, as that to melibiose decreased, with the initial glycerin concentration of 40%. The production of α-d-galactosylglycerol was 293 mg for each gram of melibiose used by the enzymatic reaction.     

Optimization of conditions for galactooligosaccharide synthesis during lactose hydrolysis by β-galactosidase from Kluyveromyces lactis (Lactozym 3000 L HP G)

A study on optimisation of the conditions for galactooligosaccharide (GOS) formation during lactose hydrolysis, produced by Lactozym 3000 L HP G, was carried out. The synthesis was performed during times up to 300 min at 40, 50 and 60 °C, pH 5.5, 6.5 and 7.5, lactose concentration 150, 250 and 350 mg/mL and enzyme concentration 3, 6 and 9 U/mL. The product mixtures were analysed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). During the hydrolysis of lactose, besides glucose and galactose, galactobiose, allolactose and 6′ galactosyl lactose were also formed as a result of transgalactosylation catalysed by the enzyme. The effect of the reaction conditions was different in the formation of di- and the trisaccharide. Thus, the optimal conditions for galactobiose and allolactose synthesis were 50 °C, pH 6.5, 250 mg/mL of lactose, 3 U/mL of enzyme and 300 min, whereas the best reaction conditions for 6′ galactosyl lactose production were 40 °C, pH 7.5, 250 mg/mL of lactose, 3 U/mL of enzyme and 120 min. These results show the possibility to obtain reaction mixtures with Lactozym 3000 L HP G, with different composition, depending on the assayed conditions.     

Enzymatic synthesis and identification of oligosaccharides obtained by transgalactosylation of lactose in the presence of fructose using β-galactosidase from Kluyveromyces lactis

The enzymatic transgalactosylation of lactose in the presence of fructose using β-galactosidase from Kluyveromyces lactis (KlβGal) leading to the formation of oligosaccharides was investigated in detail. The reaction mixture was analyzed by high performance liquid chromatography with differential refraction detector (HPLC-RI) and two main transgalactosylation products were discovered. To elucidate their overall structures, the products were isolated and purified using preparative liquid chromatography and analyzed by LC/MS, one-dimensional (1D) and two-dimensional (2D) NMR studies. Allo-lactulose(β-d-galactopyranosyl-(1→1)-d-fructose) with two main isomers in D(2)O was identified to be the major transgalactosylation product while lactulose(β-d-galactopyranosyl-(1→4)-d-fructose) turned out to be the minor one, indicating that KlβGal was regioselective with respect to the primary C-1 hydroxyl group of fructose. The maximum yields of allo-lactulose and lactulose were 47.5 and 15.4g/l, respectively, at 66.5% lactose conversion (200g/l initial lactose concentration).     

Characterization of Aspergillus oryzae glycoside hydrolase family 43 β-xylosidase expressed in Escherichia coli

This is the first report of glycoside hydrolase family 43 β-xylosidase from Aspergillus oryzae. To characterize this enzyme, the recombinant enzyme was expressed in Escherichia coli. Unlike known β-xylosidases from fungal origins, the enzyme did not show substrate ambiguity and was stable at alkaline pH.     

Characterisation of phenolic antioxidants in aqueous extract of Orthosiphon grandiflorus tea by LC–ESI-MS/MS coupled to DPPH assay

Aqueous extract from Orthosiphon grandiflorus tea was on-line screened for its antioxidant components based on their capacity to scavenge free DPPH (1,1-diphenyl-2-picrylhydrazyl) radical after separation by a LC gradient. The structural elucidation of the active compounds was achieved by negative ionisation LC–ESI-MS/MS. Based on their mass spectra and fragmentation patterns related to antioxidant activity trace, seven compounds showing strong DPPH scavenging were identified to be danshensu, caffeic acid, caftaric acid, rosmarinic acid, caffeic acid derivative, sagerinic acid and salvianolic acid B. It is noted that danshensu, caftaric acid, sagerinic acid and salvianolic acid B have been reported in O. grandiflorus for the first time. In addition, the quantification of antioxidant compounds was performed using LC–MS/MS in multiple reaction monitoring (MRM) mode. Rosmarinic acid was found as a major component responsible for the antioxidant activity of this plant extract.     

Rapid screening and identification of antioxidants in aqueous extracts of Houttuynia cordata using LC–ESI–MS coupled with DPPH assay

Houttuynia cordata Thunb. has been used traditionally as immune stimulant and anticancer agent. An aqueous extract of H. cordata tea showed high radical scavenging activity determined by off-line DPPH assays. Then, it was screened for its antioxidant components via an on-line DPPH radical scavenging technique coupled with a liquid chromatography–electrospray ionization mass spectrometer (LC–ESI–MS). Based on their mass spectra and fragmentation patterns; the antioxidant compounds were identified as quinic acid derivative, caffeic acid derivatives, procyanidin B, neo-chlorogenic acid, catechin, chlorogenic acid, crypto-chlorogenic acid and quercetin hexoside. LC–MS/MS in multiple reactions monitoring (MRM) mode was used to quantify these antioxidant compounds. Chlorogenic acid was found to be a major component in H. cordata tea.     

Identification and quantification of leaf surface flavonoids in wild-growing populations of Dracocephalum kotschyi by LC–DAD–ESI-MS

Dracocephalum kotschyi Boiss. (Lamiaceae) is an aromatic and perennial herb endemic to Iran with interesting pharmacological and biological properties. The flavonoids luteolin-7-O-glucoside, apigenin-7-O-glucoside (cosmosiin), luteolin 3′-O-β-d-glucuronide, luteolin, apigenin, cirsimaritin, isokaempferide, penduletin, xanthomicrol, calycopterin and the polyphenol rosmarinic acid were identified among 13 natural populations of the plant by ESI–MS, LC–DAD and LC–DAD–ESI-MS. The plant extracts containing the identified compounds showed significant antioxidant activity, which was correlated with the flavonoid content. Additionally, leaf and stem size and geographical variability among the studied populations were correlated with flavonoid accumulation. Canonical correlation analysis was used to find a relationship between plant dimensions and phytochemical composition, and the plants with the lowest growth indices were found to have the highest levels of methoxylated flavonoids.     

Volatile profiling of Ficus carica varieties by HS-SPME and GC–IT-MS

Aroma is one of the essential parameters for the evaluation of fruit quality, with volatile components being determinant for this characteristic. During this work, the volatile profile of fresh fruits (pulp and peel) and leaves of Portuguese Ficus carica L. white (“Pingo de Mel” and “Branca Tradicional”) and dark (“Borrasota Tradicional”, “Verbera Preta” and “Preta Tradicional”) varieties were characterised by HS-SPME/GC–IT-MS.     

Characterization of the components present in the active fractions of health gingers (Curcuma xanthorrhiza and Zingiber zerumbet) by HPLC–DAD–ESIMS

Curcuma xanthorrhiza and Zingiber zerumbet are two of the most commonly used ingredients in Indo-Malaysian traditional medicines, health supplements and tonics. Recently, a number of products derived from the aqueous extracts of these species have appeared in the market in the form of spray-dried powder packed in sachet or bottle. On-line high performance liquid chromatography, coupled with diode array detection and electrospray ion trap tandem mass spectroscopy (HPLC–DAD–ESI–MSⁿ), was used to analyze the components in the antioxidant-active fractions from the rhizomes of these species. Three components were identified from C. xanthorrhiza, including bisdemethoxycurcumin (1), demethoxycurcumin (2) and curcumin (3). The active fraction from Z. zerumbet consisted of five components, including kaempferol 3-O-rhamnoside (4), compound 5 [kaempferol 3-O-(2″-O-acetyl)rhamnoside (5a) or kaempferol 3-O-(3″-O-acetyl)rhamnoside (5b)], kaempferol 3-O-(4″-O-acetyl)rhamnoside (6), kaempferol 3-O-(3″,4″-O-diacetyl)rhamnoside (7) and kaempferol 3-O-(2″,4″-O-diacetyl)rhamnoside (8). To confirm their identities, the components from Z. zerumbet were isolated conventionally and were analyzed by spectroscopic techniques as well as by comparison with literature data.     

Assessment of GH1, CAPN1 and CAST polymorphisms as markers of carcass and meat traits in Bos indicus and Bos taurus–Bos indicus cross beef cattle

The objective of this study was to investigate the effects of bovine GH1, CAPN1 and CAST gene polymorphisms on carcass and meat traits in Nellore and Nellore x Bos taurus beef cattle. Three hundred animals were genotyped for GH1/MspI (TC/G in intron 3), CAPN316 (AF_252504.2:g.5709C > G) and CAST/RsaI (AY_008267.1:g282C > G) and phenotyped for rib eye area, backfat thickness, intramuscular fat, shear force (SF), and myofibrillar fragmentation index (MFI). No significant associations were observed between the GH1/MspI and CAST/RsaI polymorphisms and phenotypes, although the relation between the CAST/RsaI genotypes and meat tenderness evaluated by MFI approached significant. The fact that the CAPN316 polymorphism did not show adequate segregation in Nellore cattle confirms the difficulty of using this marker in breeding programs of different Bos indicus breeds. However, the positive results of the association analysis obtained for Nellore x B. taurus crosses contributed to the validation of previous findings.     

Antioxidant and antiacetylcholinesterase activities of essential oils from Cymbopogon schoenanthus L. Spreng. Determination of chemical composition by GC–mass spectrometry and C NMR

Cymbopogon schoenanthus L. Spreng, is an aromatic herb consumed in salads and used to prepare traditional meat recipes in Tunisia. The chemical composition, antioxidant activities and acetylcholinesterase inhibitory properties of the essential oils from fresh leaves, dried leaves and roots collected from three different locations in southern Tunisia, were evaluated. Essential oils were analysed by GC–mass spectrometry and ¹³C NMR. The major components were limonene (10.5–27.3%), β-phellandrene (8.2–16.3%), δ-terpinene (4.3–21.2%) and α-terpineol (6.8–11.0%). Antioxidant activity was measured by DPPH assay. The results ranged from 36.0% to 73.8% (2 μl of essential oil per mL of test solution).     

Identification and quantification of constituents of Gardenia jasminoides Ellis (Zhizi) by HPLC-DAD–ESI–MS

A simple, rapid and specific HPLC method was carried out for the analysis of characteristic constituents in Gardenia jasminoides Ellis (Zhizi), namely iridoids, caffeoyl quinic acid derivatives and crocins. The separation was successfully obtained using a C₁₈ column by gradient elution with mixtures of methanol and water as mobile phases; detection wavelength was set at 240 nm for iridoid glycosides, 315 nm for quinic acid derivatives and 438 nm for crocins.     

Effect of production system on physical–chemical,antioxidant and fatty acids composition of Longissimus dorsi and Serratus ventralis muscles from Iberian pig

The effect of three production systems of Iberian pigs namely Montanera (free-range system and feeding based on acorns and grass), Recebo (free-range system and nutrition based in combination of acorns, grass and mixed feeds) and Intensive (confinement with mixed feeds) on some quality traits of Longissimus dorsi (LD) and Serratus ventralis (SV) muscles were studied. Muscles from pigs raised in the Montanera system showed significantly higher CIE L^∗, a^∗ and b^∗ values and higher haem pigment content than those from Intensive system. Similarly, muscles from pigs raised in the Montanera system had significantly higher contents of α and γ-tocopherol and phenolic compounds contents and higher lipophilic and hydrophilic activity antioxidant than those from pigs raised in the Intensive system. Fatty acids profiles from Montanera pigs had significantly higher monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids and lower saturated fatty acids (SFA) than those from pigs raised in the Intensive system. In relation to muscle effect, LD showed lower intramuscular fat (IMF), α-tocopherol, phenolic compounds, lipid oxidation and PUFA, but higher MUFA than SV.     

Batch and continuous synthesis of lactulose from whey lactose by immobilized β-galactosidase

In this study, lactulose synthesis from whey lactose was investigated in batch and continuous systems using immobilized β-galactosidase. In the batch system, the optimal concentration of fructose for lactulose synthesis was 20%, and the effect of galactose, glucose and fructose on β-galactosidase activity was determined for hydrolysis of whey lactose and the transgalactosylation reaction for lactulose synthesis. Galactose and fructose were competitive inhibitors, and glucose acted as a noncompetitive inhibitor. The inhibitory effects of galactose and glucose were stronger in the transgalactosylation reaction than they were in the hydrolysis reaction. In addition, when immobilized β-galactosidase was reused for lactulose synthesis, its catalytic activity was retained to the extent of 52.9% after 10 reuses. Lactulose was synthesized continuously in a packed-bed reactor. We synthesized 19.1 g/l lactulose during the continuous flow reaction at a flow rate of 0.5 ml/min.     

Flavonol glucosides in Allium species: A comparative study by means of HPLC–DAD–ESI-MS–MS

Cultivars and consumption typologies of some Allium species can significantly vary from a chemical point of view and even small differences can be important for their characterization and differentiation. Bulbs of three varieties and four consumption typologies of onion (Allium cepa L.) and two varieties of shallot (Allium ascalonicum Hort.) were subjected to HPLC–DAD–ESI-MS–MS analysis. Seven flavonol glucosides were identified in all the samples, two of which, quercetin 3,4′-diglucoside and quercetin 4′-glucoside, represent about the 90% of the overall contents. Cultivars and consumption typologies of the Allium species under study show significant differences in flavonol contents, from the very low quantity of antioxidant compounds in white onion, about 7 mg/kg against 600–700 mg/kg that were found in red and gold varieties, to the enormous content of flavonols that are present in onions of prompt consumption, where quercetin 4′-glucoside exceeds 1 g/kg and quercetin 3-glucoside is present in a ratio higher then 10:1 with respect to its value in the other onion typologies. Shallots are very rich in the two major flavonols.     

Characterisation of aroma active compounds in black truffles (Tuber melanosporum) and summer truffles (Tuber aestivum) by gas chromatography–olfactometry

The aromatic composition of two different species of truffles (black and summer) was evaluated by gas chromatography–olfactometry (GC–O). Volatiles released by the truffles at 25 °C for 7.5 h were collected in a trapping system consisting of 400 mg of LiChrolut EN kept at 0 °C and further eluted with dichloromethane/methanol (95:5). The extract was analysed by two different GC–O strategies: (1) a semiquantitative GC–O study using a panel composed of nine individuals, (three of them truffle experts) and (2) an AEDA (aroma extract dilution analysis) experiment with a small panel of two judges. The results show that the aroma emitted by a typical black truffle is due to at least 17 different aroma molecules, six of which are reported for the first time: 1-hexen-3-one, 2-methyl-3-furanthiol, furaneol, 3-ethylphenol, 3-propylphenol and 5-methyl-2-propylphenol. The most important aroma compounds of black truffle aroma are 2,3-butanedione, dimethyl disulphide (DMDS), ethyl butyrate, dimethyl sulphide (DMS), 3-methyl-1-butanol and 3-ethyl-5-methylphenol. Quantitatively, black truffle emits mostly 3-ethyl-5-methylphenol (more than 50% of the total aroma molecules emitted), 5-methyl-2-propylphenol, β-phenylethanol and 3-ethylphenol. In the case of summer truffle, the most important aroma molecules are DMS, DMDS, methional, 3-methyl-1-butanol, 1-hexen-3-one and 3-ethylphenol. From the quantitative point of view, summer truffle emits mainly β-phenylethanol, DMS and 3-ethylphenol, but the emission is up to 100 times less than that of black truffles.     

Survey of T-2 and HT-2 toxins by LC–MS/MS in oats and oat products from European oat mills in 2005–2009

T-2 and HT-2 toxins were analysed in oats (n?=?243), oat flakes (n?=?529), oat meal (n?=?105) and oat by-products (n?=?209) from 11 European mills during 2005–2009 by high-performance liquid chromatography with a triple quadrupole mass spectrometer. Limits of quantification were 5?µg?kg^?1 for both T-2 and HT-2 toxins in oats. The incidence of T-2?+?HT-2 (>5?µg?kg^?1) in oats, oat flakes, oat meal and oat by-products was 93, 77, 34 and 99%, respectively. The mean values of T-2?+?HT-2 were 94, 17, 11 and 293?µg?kg^?1 for oats, oat flakes, oat meal and oat by-products, respectively. T-2 and HT-2 occurred together and the T-2 level was 52% of HT-2 in oats. Maximal T-2 and HT-2 concentration in oat flakes and oat meal were 197 and 118?µg?kg^?1. The toxins were reduced by 82–88% during processing, but increased 3.1 times in oat by-products.