Primary structure of 2S albumin from seeds of Lupinus albus

 The 2S albumin from the seeds of white lupin (Lupinus albus L.) was purified by ammonium sulfate precipitation and ultrafiltration followed by anion-exchange and reversed-phase HPLC. The complete amino acid sequences of the large and the dominating small subunit were determined by automated Edman degradation of the reduced and S-pyridylethylated polypeptides and of their enzymatic fragments. The small subunit of the 2S albumin (a) consists of 37 amino acid residues resulting in a molecular mass M _r of 4407. The large subunit (b) contains 75 amino acid residues (M _r=8827). The two polypeptide chains are linked by two interchain disulfide bonds (Cys^a8-Cys^b24 and Cys^a20-Cys^b13 or, more probably, Cys^a20-Cys^b12). In addition, the large polypeptide contains two intrachain disulfide bridges (Cys^b26-Cys^b68 and Cys^b12-Cys^b60 or, more probably, Cys^b13-Cys^b60) and one free sulfhydryl group (Cys^b40). A high degree of homology (88%) exists between the primary structure of the 2S albumin from L. albus and that of a comparable 2S protein from L. angustifolius, designated conglutin δ. Received: 4 April 1996     

Primary structure of 2S albumin from seeds of Lupinus cosentinii

 The 2S albumin from seeds of Lupinus cosentinii Guss. was purified, and the complete amino acid sequences of the dominating small and large subunit were determined by automated Edman degradation of the reduced and S-pyridylethylated polypeptides and of their enzymatic fragments. The small subunit of the 2S albumin consists of 35 amino acid residues resulting in a molecular mass (M _r) of 4233. The large subunit contains 73 amino acid residues (M _r = 8627). The two polypeptide chains are linked by two interchain disulphide bonds. In addition, the large polypeptide contains two intrachain disulphide bridges and one free sulphydryl group. A high degree of homology (88–89%) exists between the primary structure of the 2S albumin from L. cosentinii and those from other Lupinus species. The positions of the cysteines and of some other amino acids are conserved not only in most of the Dicotyledoneae 2S albumins sequenced so far but also in other storage proteins. Received: 26 May 1997     

Primary structure of 2S albumin from seeds of Lupinus cosentinii

 The 2S albumin from seeds of Lupinus cosentinii Guss. was purified, and the complete amino acid sequences of the dominating small and large subunit were determined by automated Edman degradation of the reduced and S-pyridylethylated polypeptides and of their enzymatic fragments. The small subunit of the 2S albumin consists of 35 amino acid residues resulting in a molecular mass (M _r) of 4233. The large subunit contains 73 amino acid residues (M _r = 8627). The two polypeptide chains are linked by two interchain disulphide bonds. In addition, the large polypeptide contains two intrachain disulphide bridges and one free sulphydryl group. A high degree of homology (88–89%) exists between the primary structure of the 2S albumin from L. cosentinii and those from other Lupinus species. The positions of the cysteines and of some other amino acids are conserved not only in most of the Dicotyledoneae 2S albumins sequenced so far but also in other storage proteins. Received: 26 May 1997     

Primary structure and polymorphism of 2S albumins from seeds of Andean lupin (Lupinus mutabilis Sweet)

 2S albumins were isolated from seeds of Andean lupin (Lupinus mutabilis Sweet) by buffer extraction, ammonium sulphate precipitation and ultrafiltration followed ion-exchange and reversed-phase HPLC. The 2S albumin preparation (LM2S) contained eight albumins. The complete amino acid sequences of small and large subunits of three major albumins (LM2S-4, -5 and -6) were determined by automated Edman degradation of S-pyridylethylated polypeptides and peptides obtained from them by enzymatic digestions. The small subunit of the dominant 2S albumin (LM2S-4) contains 39 amino acid residues and has a molecular mass of 4731 Da. The large subunit of LM2S-4 contains 74 amino acid residues (molecular mass=8708 Da). Two 2S albumin isoforms (LM2S-4 and -6) are due to the expression of two distinct genes; LM2S-6 isoform has eight amino acid replacements when its sequence is compared with the sequence of LM2S-4. The LM2S-5 isoform contains an identical small subunit to LM2S-4, and has in comparison with LM2S-4 two additional amino acid residues at the N-terminus of the large subunit. The amino acid sequences of 2S isoforms from L. mutabilis showed high homology (78–83% identity) with 2S albumins from different Old World Lupinus species. Received: 15 December 1998 / Revised version: 9 February 1999     

The fatty acid composition of the oil from Lupinus albus cv. Luxe as affected by environmental and agricultural factors

There is a growing interest in white lupin (Lupinus albus L.) seed for food or feed, favoured by the availability of well-performing varieties with low content of alkaloids. The objective of this study was to assess the influence of the environmental and agricultural factors on the content and fatty acid composition of lupin oil. The investigation was performed on the sweet variety Luxe grown in three Italian locations (one continental and two Mediterranean) and 13 environments in total. Statistical analyses (analysis of variance and principal component analysis) indicated that oil content and composition of fatty acids were affected largely by the growing location. Mediterranean sites tended to lower crop yield, but to increase oil content and absolute α-linolenic acid content compared to the continental location; large variation occurred also between the Mediterranean sites. The α-linolenic acid content ranged from 1.41 to 3.24 mg/g flour, highlighting the possible value of white lupin in order to reach the recommended daily intake of this fatty acid. The observed ω-3/ω-6 ratio, ranging from 0.45 to 0.63, was much higher than that of most vegetable oils.     

The subunit structure and stability of conglutin δ, a sulphurrich protein from the seeds of Lupinus angustifolius L.

The oligomeric structure and stability of conglutin δ, the 2S sulphur-rich lupin seed protein, has been studied. Molecular weights were determined by sedimentation equilibrium methods in the analytical ultracentrifuge. Conglutin δ₂ (M_r 14 000, 2S), the most abundant form, was composed of one large (?9600) and one small polypeptide chain linked by disulphide bonds. The minor oligomeric form, conglutin δ₁ (M_r 28 000, 2.8S), was a disulphide-linked dimer of conglutin δ₂. Each form was capable of reversible association and at low ionic strength (neutral pH), the conglutin δ₁ momomer associated to a homogeneous dimer (M_r 56 000, 4.1S). Calculated frictional ratios (f/f₀ ? 1.28–1.49) suggested that the three different forms were asymmetric. Spectral studies (ORD/CD) showed that conglutins δ₁ and δ₂ were rich in alpha-helix (?38%) unlike the major 7S and 11S legume seed storage proteins. The helical structure was unusually stable both to heat and chemical denaturants; below 60°C (at neutral pH) the helix remained unaffected and only partial denaturation occurred at higher temperatures. Nevertheless, complete denaturation was achieved (at 20°C) in high concentrations of guanidine hydrochloride (greater than 6.5 M). The stability was due to the presence of disulphide cross-links; with the addition of reducing agent, as little as 1 M guanidine hydrochloride (GuHCl) was sufficient for denaturation of the helical structure. Titration experiments showed a single buried tyrosine (pK 11.4) which could be exposed (pK 10.2) in 6 M GuHCl.     

Stereodifferentiation of 3-mercapto-2-methylpropanol in wine

 3-Mercapto-2-methylpropanol, an odorous compound recently identified in red wines, was extracted from a red wine variety, using a low-temperature vacuum distillation coupled with a specific reversible thiol capture. The analysis of the diacetylated wine extract by enantioselective multidimensional gas chromatography with mass spectrometry detection revealed the presence of only one enantiomer, (R)-3-mercapto-2-methylpropanol. Although both of the optical isomers are characterized by the same broth and sweat odour, they have very different odour thresholds. The amounts of (R)-3-mercapto-2-methylpropanol in young Cabernet-Sauvignon and Merlot wines can exceed its perception threshold, thus suggesting its contribution to the aroma of these wines. Received: 8 June 1999     

Determination of site-specific D/H isotope ratios of glycerol from different sources by 2H-NMR spectroscopy

 A method was developed for the preparation of glycerol from wines and from naturally occuring lipids. In addition, the D/H isotopic ratios of glycerol from these sources was determined using site specific natural isotopic fractionation. Glycerol from four different sources was examined: glycerol isolated from vegetable oils and animal fats, glycerol obtained by chemical synthesis and glycerol which is a byproduct of the alcoholic fermentation of grape musts. Depending on the origin of the glycerol, different isotopic ratios were found; these can be used for the detection of adulterated food or as proof of the authenticity of a given product, such as wine. Received: 13 May 1998     

Quantitative detection of the 35S promoter and the NOS terminator using quantitative competitive PCR

 DNA-based analytical methods are often used to verify the presence of genetically modified organisms (GMOs) in food. In Switzerland, a preliminary study, organized by a subcommission of the Swiss Food Manual, of different polymerase chain reaction (PCR) systems for the detection of GMOs showed that the application of qualitative PCR systems can lead to interlaboratory differences of at least a factor of 10. These differences can be diminished using internal standards (competitors). The quantitative competitive (QC) PCR for the detection of the 35S promoter or the NOS terminator in food samples is presented. The GMO content of food samples can be determined using QC-PCR. Received: 2 July 1998 / Revised version: 15 October 1998     

Isolation and characterization of mutatoxanthin-epimers from red paprika (Capsicum annuum)

 (3S,5R,8R,3′R)- and (3S,5R,8S,3′R)-mutatoxanthin were isolated from red spice paprika (Capsicum annuum) and characterized by their UV-VIS, CD, ¹H-NMR, ¹³C-NMR and mass spectra. Received: 22 November 1999 / Revised version: 11 February 2000     

Elucidation of the formation of CO2 in culinary dry products

 The formation of CO₂ in tomato powder, chosen as an example of a dry culinary product, was investigated at room temperature and at low values of water activity (a _w). CO₂ formation correlated well with parameters that represent the beginning and progression of the Maillard reaction. In the absence of O₂, CO₂ formation decreased. Pectin and depolymerized pectin did not influence CO₂ formation while galacturonic acid (GalA) had a large effect. Determination of ¹³CO₂ in low-moisture model systems revealed that CO₂ was not formed by decarboxylation of GalA alone. Only a small proportion of [1-¹³C]glycine and GalA was degraded by the Strecker pathway; however, glucose reacted with the labelled amino acid forming ¹³CO₂ which amounted to over 90% of the total CO₂ formed. Therefore, CO₂ could be used as an early indicator for the beginning of the Maillard reaction in dry culinary products. Received: 28 October 1996     

Elucidation of the formation of CO2 in culinary dry products

 The formation of CO₂ in tomato powder, chosen as an example of a dry culinary product, was investigated at room temperature and at low values of water activity (a _w). CO₂ formation correlated well with parameters that represent the beginning and progression of the Maillard reaction. In the absence of O₂, CO₂ formation decreased. Pectin and depolymerized pectin did not influence CO₂ formation while galacturonic acid (GalA) had a large effect. Determination of ¹³CO₂ in low-moisture model systems revealed that CO₂ was not formed by decarboxylation of GalA alone. Only a small proportion of [1-¹³C]glycine and GalA was degraded by the Strecker pathway; however, glucose reacted with the labelled amino acid forming ¹³CO₂ which amounted to over 90% of the total CO₂ formed. Therefore, CO₂ could be used as an early indicator for the beginning of the Maillard reaction in dry culinary products. Received: 28 October 1996     

大豆过敏原11S球蛋白G2中A2链结合表位的预测

采用生物信息学软件NCBI GenBank数据库、PDB数据库、SWISS-MODEL、Deep View 4.1等预测11S球蛋白G2中A2链的二级结构和三级结构模型,使用DiscoTope 2.0 Server软件预测B细胞构象表位,发现构象表位多位于无规则卷曲处。定位B细胞线性结合表位,结果表明11S球蛋白G2中A2链的可能线性表位有：37KPDNRIE43、79PSYTNGP85、114SQQRGRSQRP123、52WNPNNK57、196QQGGSQSQKGKQQE209、241QGENEEED248、267RKPQQEEDDDDEEEQ281、287TDKGC291。此表位预测为后续制肽、免疫等操作找出更准确的肽序,以便建立适合我国大豆过敏人群的食品大豆过敏原的检测技术。     

Distribution of microbes in supercritical CO2 extraction of sea buckthorn (Hippopha? rhamnoides) oils

 The distribution of vegetative microbial cells and their spores in a supercritical CO₂ extraction process was studied. The seed and flesh/skin fractions of the press residue of sea buckthorn berries (Hippopha? rhamnoides) from a juice factory were used as raw materials. A pilot-scale extraction plant was operated at 30 MPa at temperatures of 40 and 60°C. The number of yeasts, moulds and bacteria in the pulp/skin fraction, in the extraction residues, in the extracted oils as well as in the water phases separated from the extracted oils was estimated by the spread plate technique. The microbial content of the flesh/skin material was increased in some extractions by the addition of bacterial spores. In general, the extraction process led to a decrease in the bacterial count of the extracted material, whereas no microbial growth was detected in the oils extracted or in the water phases separated from them. Neither yeasts nor moulds were found in any samples after the extraction process. The microbial status of seed oil and flesh/skin oil obtained by industrial-scale CO₂ extraction at 40°C and at 30 MPa before and after gelatine encapsulation remained unchanged. This proves that supercritical CO₂ can be used to manufacture edible oil products free of living micro-organisms and their spores. Received: 14 May 1996     

Distribution of microbes in supercritical CO2 extraction of sea buckthorn (Hippophaë rhamnoides) oils

The distribution of vegetative microbial cells and their spores in a supercritical CO₂ extraction process was studied. The seed and flesh/skin fractions of the press residue of sea buckthorn berries (Hippophaë rhamnoides) from a juice factory were used as raw materials. A pilot-scale extraction plant was operated at 30?MPa at temperatures of 40 and 60°C. The number of yeasts, moulds and bacteria in the pulp/skin fraction, in the extraction residues, in the extracted oils as well as in the water phases separated from the extracted oils was estimated by the spread plate technique. The microbial content of the flesh/skin material was increased in some extractions by the addition of bacterial spores. In general, the extraction process led to a decrease in the bacterial count of the extracted material, whereas no microbial growth was detected in the oils extracted or in the water phases separated from them. Neither yeasts nor moulds were found in any samples after the extraction process. The microbial status of seed oil and flesh/skin oil obtained by industrial-scale CO₂ extraction at 40°C and at 30?MPa before and after gelatine encapsulation remained unchanged. This proves that supercritical CO₂ can be used to manufacture edible oil products free of living micro-organisms and their spores.     

Purification and characterisation of the 7S globulin storage protein from sesame (Sesamum indicum L.)

The 7S globulin from sesame seeds was purified by means of selective precipitation and anion-exchange chromatography on Q Sepharose Fast Flow. The 7S globulin migrated as a single band on native PAGE, which suggested homogeneity of the sample. The isolated protein was composed of at least eight polypeptide chains, ranging from 12.4 to 65.5 kDa, judged by SDS–PAGE analysis, and did not contain disulphide bonds. Furthermore, comparison of the polypeptide bands of the 7S and 11S globulins by SDS–PAGE indicated that the purified 7S globulin was free of legumin-like contaminant polypeptides and of 2S albumin. The identity of the purified polypeptides was verified by comparing the N-terminal amino acid sequences of the main polypeptide bands with the amino acid sequence deduced from a cDNA clone, which encoded the sesame 7S globulin precursor. Purification of the 7S globulin from sesame has not previously been reported.     

Stability of naturally occurring 2,5-dimethyl-4-hydroxy-3 [2H]-furanone derivatives

The stability, in aqueous buffer solutions at different pH values (pH 2.0–8.0, interval: 1.5 pH units), of 2,5-dimethyl-4-hydroxy-3[2H]-furanone (Furaneol, DMHF, 1), its methoxy derivative 2,5-dimethyl-4methoxy-3[2H]-furanone (methoxyfuraneol, mesifurane, DMMF, 2 and the glycosidically bound forms DMHF β-D-glucopyranoside 3 and DMHF 6′-O-malonyl-β-Dglucopyranoside 4 was investigated over a period of 32 days at 23 °C. Only slight decomposition of 2 and 3 was observed, whereas 1 and 4 were found to be unstable at all pH values. In addition, 3 and 4 were subjected to enzymatic hydrolysis. In contrast to the rapid hydrolysis of 3, the malonylated glycoside, 4, remained unaffected by enzymatic treatment with β-glucosidase (Emulsin). Using a pectinolytic enzyme preparation (Rohapect D5L; R?hm, Darmstadt, Germany) with esterase activities, hydrolysis of 4 was achieved. Received: 25 September 1996     

Stability of naturally occurring 2,5-dimethyl-4-hydroxy-3 [2H]-furanone derivatives

The stability, in aqueous buffer solutions at different pH values (pH?2.0–8.0, interval: 1.5?pH units), of 2,5-dimethyl-4-hydroxy-3[2H]-furanone (Furaneol, DMHF, 1), its methoxy derivative 2,5-dimethyl-4methoxy-3[2H]-furanone (methoxyfuraneol, mesifurane, DMMF, 2 and the glycosidically bound forms DMHF β-D-glucopyranoside 3 and DMHF 6′-O-malonyl-β-Dglucopyranoside 4 was investigated over a period of 32 days at 23?°C. Only slight decomposition of 2 and 3 was observed, whereas 1 and 4 were found to be unstable at all pH values. In addition, 3 and 4 were subjected to enzymatic hydrolysis. In contrast to the rapid hydrolysis of 3, the malonylated glycoside, 4, remained unaffected by enzymatic treatment with β-glucosidase (Emulsin). Using a pectinolytic enzyme preparation (Rohapect D5L; Röhm, Darmstadt, Germany) with esterase activities, hydrolysis of 4 was achieved.     

L-Rhamnose: progenitor of 2,5-dimethyl-4-hydroxy-3 [2H]-furanone formation by Pichia capsulata?

 2,5-Dimethyl-4-hydroxy-3[2H]-furanone (Furaneol, 1), an important aroma constituent, was detected at concentrations of up to 2 mg/l after 4 days of growth of Pichia capsulata on casein peptone culture medium containing L-(+)-rhamnose (2). Blank samples without yeast contained no 1 after the incubation period. In parallel experiments five samples of 2 exhibiting different [¹³C] abundance were given to P. capsulata. The volatile compounds formed were isolated and analysed for their [¹³C]/[¹²C] ratios by on-line gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS). A positive correlation between the isotopes in 1 and 2 was observed; thus, 2 served as the carbon source for 1. However, 1 was formed from a postulated intermediate of 2 generated during thermal sterilization, as 1 was neither detected after sterile filtration of the culture medium nor after separate thermal sterilization of the casein peptone and 2. This observation was confirmed by an experiment investigating the time course of the formation of 1. Received: 4 June 1996     

Microbiological oxidation of 2-methylbutanol of differing enantiomeric ratios by Gluconobacter species

 2-Methylbutanoic acid, an aroma compound of high economic interest, was synthesised using Gluconobacter species for bioconversion. The main focus was on enantioselective effects during metabolism. A preference for the (S)-enantiomer was observed when the microorganisms were fed with 2-methylbutanol of known enantiomeric ratios. Received: 15 December 1997