高效液相色谱串联质谱法检测马铃薯及马铃薯粉中α-茄碱和α-卡茄碱含量

建立了高效液相色谱-串联质谱同时测定马铃薯及马铃薯粉中α-茄碱与α-卡茄碱含量的方法。样品采用10%乙酸水溶液匀浆提取3 min,马铃薯粉提取液经Bond Elut C18 SPE柱净化后,采用C18色谱柱分离,液相色谱串联质谱仪检测。结果表明,α-茄碱和α-卡茄碱在0.01~1.00 μg/mL范围内线性关系良好,相关系数分别为0.9998和0.9997。马铃薯和马铃薯粉在低、中、高三个添加浓度范围内,回收率在90.0%~110%之间,相对标准偏差在1.6%~5.0%之间。马铃薯中α-茄碱和α-卡茄碱的定量限分别为0.80和0.42 μg/kg,马铃薯粉中α-茄碱和α-卡茄碱的定量限分别为25和14 μg/kg(S/N=10)。本方法简单快速,灵敏度高,重复性好,适用于马铃薯及马铃薯粉中的α-茄碱和α-卡茄碱检测。     

The incorporation of sweet potato application in the preparation of a rice beverage

Summary To increase the industrial applications of sweet potato, a rice beverage was prepared by adding barley sprouts, sweet potato, or a mixture of barley sprouts and sweet potato (1:1). Amylases from barley sprouts and sweet potatoes had a similar hydrolysis pattern to β‐amylase. Heat stability of this enzyme in sweet potato was higher than that in barley. Reducing sugar content in the mixture of barley sprouts and sweet potato was higher than in either barley sprouts or sweet potato alone. After the preparation of the rice beverage, the maltose content of the mixture with barley sprouts, either barley sprouts and sweet potato, or sweet potato was 37.2, 44.1 and 40.3 mg mL^?1 after 6 h, respectively. The amylase activity in the mixture with barley sprouts and sweet potato decreased more than that of the mixture with only sweet potato. The use of sweet potato resulted in an increase of sweetness, flavour and improved preference in rice beverage.     

浆水中微生物的分离与鉴定

以浆水为实验材料,在多种培养基平板上分离纯化其中的微生物,共得到24 株菌,其中细菌20 株,丝状真菌3 株,酵母菌1 株。对纯化后的各菌株进行菌落形态观察、革兰氏染色、生化特性研究以及分子生物学鉴定,结果表明：浆水中存在多种微生物,其细菌以乳杆菌属或双歧杆菌属等益生菌为主,酵母菌为酿酒酵母,丝状真菌为变灰青霉和橘青霉。结果说明浆水微生物组成优良,具有开发利用潜力,但其中还存在杂菌和有害菌,应在生产中引起进一步重视。     

Contents and its change during storage of alpha-solanine and alpha-chaconine in potatoes

Contents of alpha-solanine and alpha-chaconine in native species of potato (May Queen, Danshaku and Waseshiro), and in species (Jagakids Red '90 (Red) and Jagakids Purple '90 (Purple)) on the market, and their change during storage at room temparature were investigated. alpha-Solanine and alpha-chaconine were extracted from potatoes with methanol, cleaned up by using a Sep-Pak Plus C18 cartridge, and then subjected to HPLC. The recoveries of alpha-solanine and alpha-chaconine from potatoes were both more than 96%, and the quantitation limits were both 2 microg/g. alpha-Solanine and alpha-chaconine were detected in periderm in all samples at the levels of 260-320 microg/g in May Queen,190-240 microg/g in Danshaku, 43-63 microg/g in Waseshiro, 140-200 microg/g in Red and 84-130 microg/g in Purple, respectively. alpha-Solanine and alpha-chaconine were detected in the cortex in all samples of May Queen and Danshaku at the levels of 2.7-12 microg/g and 5.8-31 microg/g, respectively. Contents of alpha-solanine and alpha-chaconine in the cortex of May Queen and Danshaku were less than 10% of those in the periderm. When potatoes were stored for 90 days at room temparature in a dark place, no marked change in the contents of alpha-solanine and alpha-chaconine was observed in any of the potato samples.     

Purification and properties of an extracellular inulinase from Rhizopus sp. strain TN-96

A filamentous fungus, Rhizopus sp. strain TN-96, was isolated from rhizosphere soil samples. An extracellular inulinase was purified from the culture filtrate of strain TN-96 grown on inulin by DEAE-Cellulofine A-500 and Sephacryl S-200 HP chromatographies. The enzyme was homogeneous as judged by SDS-polyacrylamide gel electrophoresis, with an apparent M(r) of 83 kDa. The purified enzyme had specific activities of 17 U/mg toward inulin (I) and 0.32 U/mg toward sucrose (S) (I/S ratio, 53). Inulinase activity was optimal at pH 5.5 and 40 degrees C. The inulinase exhibited an apparent K(m) value of 9.0 mM for inulin. The enzyme also hydrolyzed raffinose, but not bacterial levan.     

Alpha-Chaconine and Alpha-Solanine Content of Potato Peels and Potato Peel Products

Twelve samples of raw and cooked potato peels from commercial potato varieties were analyzed for their α-chaconine and α-solanine content by high-performance liquid chromatography (HPLC). Raw peels contained 1.30–56.67 mg/100g peel (wet weight) α-chaconine and 0.5–50.16 mg/100g peel (wet weight) α-solanine. Raw flesh from the same potatoes contained 0.02–2.32 mg/100g flesh (wet weight) α-chaconine and 0.01–2.18 mg/100g flesh (wet weight) of α-solanine. Peels were cooked by baking, frying and baking-frying. The two types of fried peels contained more α-chaconine (2.18–92.82 mg/100g cooked peel) and α-solanine (1.09–72.09 mg/100g cooked peel). Four commercial potato peel products – wedges, slices, fried peels and baked-fried peels – contained 3.60–13.71 mg α-chaconine/100g cooked product and 1.60–10.48 mg α-solanine/100g cooked product.     

INHIBITION OF RAT CHOLINESTERASE ISOENZYMES IN VITRO AND IN VIVO BY THE POTATO ALKALOID, α-CHACONINE1,2

The inhibition of purified bovine erythrocyte acetylcholinesterase (AchE) and horse serum cholinesterase (ChE) by α-chaconine was found to be a mixed-type with inhibition constants (K_i) for both enzymes of 8.3 × 10^-6 M and 4.0 × 10^-4 M, respectively. Kinetic constants for AchE obtained for the hydrolysis of acetylthiocholine iodide averaged 7.2 × 10^-5 moles/liter/min (V_max) and 6.2 × 10^-5 M (K_m) whereas for ChE these were 3.8 × 10^-4 moles/liter/min (V_max) and 1.3 × 10^-4 M (K_m) for the hydrolysis of butyrylthiocholine iodide. Subcellular acetylcholinesterase activity in nuclear-free rat brain homogenate showed the highest activity to be equally distributed between the mitochondrial and microsomal fractions, with only little activity in the nuclear fraction. Inhibition of the rat brain subcellular enzyme activity in vitro by 0.016 M α-chaconine was 43% for whole homogenate, 55% for nuclear fraction, 35% for mitochondria, and 33% for microsomes. In vivo administration of α-chaconine, (10, 30 and 60 mg/kg) to adult male rats caused a reduction of brain AchE activity to 79, 55 and 18% of the control activity for the respective doses. Heart and plasma AchE activity of treated animals did not show a dose-related inhibition similar to that of brain. Inhibition of heart AchE was 61%, while that of plasma was 51% for rats administered to 10 mg/kg and no further inhibition for rats given 30 mg/kg α-chaconine was noted. Isoenzymes of rat brain cholinesterases, separated by acrylamide gel electrophoresis, were inhibited by 30 and 60 mg/kg α-chaconine administered intraperitoneally. Electrophoretic separation of plasma from the treated animals resulted in five anodally migrating zones that hydrolyzed acetylthiocholine and α-naphthylacetate. Inhibition of the activity of isoenzyme bands 1 and 2 was 40 and 77% respectively for animals given 10 mg/kg α-chaconine, while rats given 30 mg/kg showed inhibition of 100 and 75%, respectively. Isoenzyme bands 3 and 4 were completely inhibited in the plasma of alkaloid treated animals. In vitro inhibition of rat plasma, erythrocyte and brain esterase isoenzymes was estimated by incubating gels with 10^-4 M α-chaconine after electrophoretic separations. The slower-moving isoenzyme bands were inhibited completely, while the faster migrating isoenzyme bands in plasma and erythrocytes were least affected by the concentration of α-chaconine used.     

Filamentous fungi producing ochratoxin a during cocoa processing in Cameroon

Ochratoxin A (OTA) is the main mycotoxin occurring in cocoa. A study was conducted in Cameroon to assess how filamentous fungi and toxigenesis were affected by the type of cocoa post-harvest treatment (boxes or heaps). The filamentous fungi isolated were almost identical when fermentation was carried out in boxes or heaps, with the presence of abundant black Aspergillus filamentous fungi: A. niger and A. carbonarius. Filamentous fungi were more abundant at the end of the harvesting season. Factors affecting bean integrity (poor handling, deferred processing) resulted in a qualitative and quantitative increase in contamination, when the total number of filamentous fungi could reach a maximum value of 5.5+/-1.4x10(7) CFU g(-1) and black Aspergilli a maximum value of 1.42+/-2.2x10(7) CFU g(-1). A toxigenesis study showed that Aspergillus carbonarius was the main OTA-producing strain isolated. Its maximum production could reach 2.77 microg g(-1) on rice medium. Aspergillus niger strains did not always produce OTA and their toxigenesis was much lower. Fermented dried cocoa from poor quality pods was the most contaminated by OTA: up to 48 ng g(-1).     

木瓜蛋白酶水解马铃薯蛋白水解条件的研究

采用马铃薯渣为原料提取马铃薯蛋白,用木瓜蛋白酶对其进行水解。研究水解时间、温度、pH和酶与底物浓度比(E/S)四个因素对水解过程的影响。以水解度(DH)为评价指标,在单因素实验的基础上,采用Box-Benhnken响应面分析法,优化木瓜蛋白酶水解马铃薯蛋白质的工艺条件。结果表明,在酶与底物浓度比为6000U/g时,最佳的水解条件为:温度为64.70℃、pH为7.41、水解反应时间3.12h。在此条件下,水解度为20.19%。     

Glycoalkaloids (α-chaconine and α-solanine) contents of selected Pakistani potato cultivars and their dietary intake assessment

Glycoalkaloids (α-solanine and α-chaconine) are naturally occurring toxic compounds in potato tuber (Solanum tuberosum L.) that cause acute intoxication in humans after their consumption. Present research was conducted to evaluate α-chaconine, α-solanine, and total glycoalkaloids (TGAs) contents in the peel and flesh portions by high-performance liquid chromatography method in selected Pakistani potato cultivars. The α-solanine content varies 45.98 ± 1.63 to 2742.60 ± 92.97 mg/100 g of dry weight (DW) in peel and from 4.01 ± 0.14 to 2466.56 ± 87.21 mg/100 g of DW in flesh. Similarly, α-chaconine content varied from 4.42 ± 0.16 to 6818.40 ± 211.07 mg/100 g of DW in potato peel and from 3.94 ± 0.14 to 475.33 ± 16.81 mg/100 g DW in flesh portion. The TGA concentration varied from 177.20 ± 6.26 to 5449.90 ± 192.68 mg/100 g of DW in peel and from 3.08 ± 0.11 to 14.69 ± 0.52 mg/100 g of DW in flesh portion of all the potato cultivars tested. All the potato cultivars contained lower concentration of TGA than the limits recommended as safe, except 2 cultivars, that is FD8-3 (2539.18 ± 89.77 mg/100 g of DW) and Cardinal (506.16 ± 17.90 mg/kg). The dietary intake assessment of potato cultivars revealed that Cardinal, FD 35-36, FD 8-3, and FD 3-9 contained higher amount of TGA in whole potato, although FD 8-3 only possessed higher content of TGA (154.93 ± 7.75) in its flesh portion rendering it unfit for human consumption. Practical Application: This paper was based on the research conducted on toxic compounds present in all possible potato cultivars in Pakistan. Actually, we quantify the toxic compounds (glycoalkaloids) of potato cultivars through HPLC and their dietary assessment. This paper revealed safety assessment and their application in food industries especially potato processing.     

Strain-specific differences in the attachment of Listeria monocytogenes to alfalfa sprouts

Contamination of fresh produce with Listeria monocytogenes has resulted in outbreaks of systemic listeriosis and febrile gastroenteritis. Recalls of alfalfa sprouts have occurred due to contamination with L. monocytogenes. Alfalfa sprouts were used as a preharvest model to study the interaction with this human pathogen. Seventeen strains were assessed for their capacity to colonize alfalfa sprouts, and strain-specific differences (not related to source, serotype, or lineage) were revealed when the sprout irrigation water was changed daily. Two of the strains colonized and attached to the sprouts very well, reaching levels of more than 5 log CFU per sprout. The remaining strains varied in their final levels on sprouts between less than 1 to 4.7 log CFU per sprout. All of the L. monocytogenes strains grew to equivalent levels on the sprouts when the irrigation water was not changed, suggesting the differences observed with regular changing of the water resulted from differences in attachment. Further analysis of the best colonizing strains indicated that only between 0.3 and 1 log CFU per sprout could be removed by additional washing of the sprout, and the presence of normal sprout bacteria did not compete with the L. monocytogenes strains on the sprouts. The poorest colonizing strain was able to grow in the irrigation water during the experiment but could not attach to the sprouts. Microscopic examination of the sprouts with L. monocytogenes expressing the green fluorescent protein indicated that L. monocytogenes was associated with the root hairs of the sprouting alfalfa, with few to no cells visible elsewhere on the sprout.     

The influence of French fries processing on the glycoalkaloid content in coloured-fleshed potatoes

French fries made from coloured-fleshed potatoes may be interesting alternative to the traditional snacks for consumers. However, potatoes contain glycoalkaloids (TGA), so potato tubers and obtained fried snacks should be subjected to comprehensive examination. The aim of this study was to determine the effect of different stages of French fries processing on the content of TGA (α-solanine and α-chaconine) in the red- and blue-fleshed potatoes, in semi-products and ready-to-eat products. It was stated that during the processing of French fries prepared from coloured-flesh potato varieties significantly decreased the content of TGA (α-solanine and α-chaconine) in the samples obtained at different stages of the process compared to the raw material. Potatoes with blue-fleshed of Vitelotte variety and red-fleshed of Highland Burgundy Red variety can be used to French fries processing due to their low content of TGA (in unpeeled and peeled potatoes). However, Blue Congo variety with blue-fleshed should not be applied to French fries processing, because of high TGA content in raw material and first of all in peeled potatoes flesh. The peeling process of coloured-fleshed potatoes decreased in TGA content on average by about 50 %, cutting process on average by about 53 %, whereas blanching on average by about 58 % compared with the raw material. The highest decrease in TGA content was caused by frying process. The mean values were about 97.5 % in ready-to-eat French fries. In French fries after I and II steps of frying, the ratio α-solanine to α-chaconine was lower (1.0:2.0) than in unpeeled potatoes (1.0:2.3).     

Purification and characterization of N-Acetylglucosamine 6-phosphate deacetylase from Thermus caldophilus

N-Acetylglucosamine 6-phosphate deacetylase [EC 3.5.1.25] was purified and biochemically characterized from an extreme thermophile, Thermus caldophilus GK24. The optimum temperature and pH of the enzyme were 80 degrees C and 7.5, respectively. The enzyme is a tetramer composed of identical 45 kDa subunits. The N-terminal amino acid sequence of the purified enzyme was determined to be MSVDLKTLHRRHVLTP. It hydrolyzed GlcNAc-6-P, but not GlcNAc-1-P or chitin oligosaccharides. The deacetylase activity was completely inhibited by the addition of 1 mM Cu2+, but moderately activated by that of 1 mM Mn2+ and Co2+. Within 2 h of reaction, 2 mM GlcNAc-6-P was completely hydrolyzed to GlcN-6-P and acetate by the action of the deacetylase.     

The influence of thermal process of coloured potatoes on the content of glycoalkaloids in the potato products

Potato products made from potato varieties with coloured flesh (red and blue-fleshed potatoes) may be an interesting alternative to traditional products. The purpose of this study was to determine the influence of peeling, cooking and frying of red and blue-fleshed potato tubers on the content of glycoalkaloids (α-solanine and α-chaconine) in processed potato products. The material taken for the study consisted of seven coloured potato varieties. French fries and crisps were prepared from two potato variety: Rosalinde and Blue Congo. The content of total glycoalkaloids (TGA) in raw material before and after peeling, in cooked unpeeled and peeled potatoes and also in fried potato products have been determined by HPLC method.     

An Improved Method of the Purification of Bacillus stearothermophilus Exo-α-1,4-glucosidase Active on Soluble Starch by the Use of an Immunosorbent

A p-nitrophenyl-α-D-glucopyranoside-hydrolyzing exo-α-1,4-glucosidase (α-D-glucoside glucohydrolase, EC.3.2.1.20) highly active on soluble starch of Bacillus stearothermophilus ATCC 12016 was 366-fold purified to homogeneity with a yield of 31% by a simple five-step procedure containing as the most efficient step the enzyme elution from immunosorbent with a pH 11 medium including 50% (w/v) glycerol. The enzyme specific activity was 148 μmol p-nitrophenyl-α-D-glucopyranoside hydrolyzed/min/mg protein at 60°C and pH 6.8. The enzyme cleft 71% of glucosidic linkages in α-limit dextrin (average polymerization degree = 9) made from potato amylopectin with Bacillus subtilis α-amylase. The products of α-limit dextrin were hardly hydrolyzed by Bacillus thermoglucosidasius oligo-1,6-glucosidase (dextrin 6-glucano-hydrolase, EC.3.2.1.10).     

Synergistic cytotoxicity induced by α-solanine and α-chaconine

α-Solanine and α-chaconine are well-known potato toxins, but the mechanism of the synergistic cytotoxic effect of these alkaloids has been little clarified. This study confirmed their synergistic cytotoxic effects on C6 rat glioma cells by three different cell viability tests, namely WST-1 (water-soluble tetrazolium) assay sensitive to intracellular NADH concentration, menadione-catalysed chemiluminescent assay depending on both NAD(P)H concentration and NAD(P)H:quinone reductase activity, and LDH (lactate dehydrogenase) assay sensitive to the release of LDH from damaged cells. The maximum cytotoxic effect was observed at a ratio of 1:1 between α-solanine and α-chaconine at micromolar concentrations. The cytotoxic effects of these alkaloids were observed immediately after incubation and were constant after 30 min, suggesting that rapid damage of plasma membrane causes the lethal disorder of metabolism.     

华莱士瓜采后丝状真菌的分离及rDNA ITS区序列分析

从采后贮藏期间自然发病的华莱士瓜上分离到12 株丝状真菌,将分离菌经纯化后回接到健康无损的华莱士瓜上,发病特征与分离发病果实一致,确定其为华莱士瓜的病原菌。结合形态学观察和rDNA内转录间隔区（internal transcribed spacer,ITS）序列分析,结果表明：分离到1 株黑附球菌（Epicoccum nigrum）、1?株草酸青霉菌（Penicillium oxalicum）、2 株链格孢属（Alternaria Nees ex Wallr.）真菌、3 株曲霉属（Aspergillus Micheli ex Fr.）真菌和5 株镰刀属（Fusarium LK. ex Fr.）真菌。其中,黑附球菌（E. nigrum）为甜瓜中首次发现的病原菌,而草酸青霉菌（P. oxalicum）、细极链格孢菌（Alternaria tenuissima）、泡盛曲霉菌（Aspergillus awamori）、木贼镰刀菌（Fusarium equiseti）、轮状镰刀菌（F. verticillioides）和燕麦镰刀菌（F. avenaceum）分别为甜瓜上分离到的青霉属、链格孢属、曲霉属和镰刀属的新菌株。     

Liquid Chromatography–Hybrid Linear Ion Trap–High-Resolution Mass Spectrometry (LTQ-Orbitrap) Method for the Determination of Glycoalkaloids and Their Aglycons in Potato Samples

Potatoes produce biologically active secondary metabolites like glycoalkaloids and their aglycons, which may have both adverse and beneficial effects in the diet. A new analytical method that uses liquid chromatography–mass spectrometry (LTQ-Orbitrap) has been developed for the analysis of glycoalkaloids and their aglycons in potato samples. Two glycoalkaloids, α-solanine and α-chaconine, and two aglycons, demissidine and solasodine, were quantified in potato samples. Samples were extracted using methanol, purified on an SPE Strata C₁₈ cartridge, and then analyzed in HPLC–mass spectrometry (LTQ-Orbitrap) with the FTMS operating in full scan at a resolving power of 30,000 (FWHM), enabling the detection and accurate mass measurement and with the ITMS mode operating in MRM (multiple reaction monitoring) for glycoalkaloids and their aglycons using the [M + H]⁺ ions and their optimized collision energies. After validation, the method was applied to screen different type of potatoes, and some cooking experiment were conducted.     

Labeling of Starch Granules by Bombardment with Tritium Atoms

Starch granules were labeled by exposure to tritium atoms produced by thermal dissociation of tritium gas at a tungsten filament. Activity was shown by α-amylase etching experiments to be confined to the surface of the granule. Dextrins and low molecular weight compounds could be partially resolved on Sephadex G-75. The dextrin produced during tritiation had an elution volume corresponding to dextrin of 20–35 glucose units and was hydrolyzeable by α-amylase. Labeled starch was hydrolyzed first with ß-amylase and the limit dextrin hydrolyzed further with α-amylase. A comparison of the specific activities of the maltoses produced by the two enzymes demonstrated that labeling was more heavily concentrated in outer brunches than in inner branches. Amylose obtained from labeled potato starch was purified by crystallization and on DEAE-Sephadex columns. The ion-exchange purified potato amylose was hydrolyzed with ß-amylase with 50% of the activity retained in maltose. Tritium was found to be concentrated at the nonreducing ends of the amylose molecules. These results suggest that starch molecules are organized in granules with nonreducing ends oriented to the surface.     

Inhibitory activity of plumbagin produced by Drosera intermedia on food spoilage fungi

BACKGROUND: The aim of this study was to investigate the growth‐inhibiting efficacy of Drosera intermedia extracts (water, methanol and n‐hexane) against four food spoilage yeasts and five filamentous fungi strains responsible for food deterioration and associated with mycotoxin production, in order to identify potential antimycotic agents. RESULTS: The n‐hexane extract showed a broad activity spectrum against all tested microorganisms, followed, in activity, by the methanol and water extracts. The major component of the n‐hexane extract was purified using a solid‐phase extraction column and identified as plumbagin. Results show that high‐purity plumbagin can be produced from D. intermedia cultures following a simple and effective isolation procedure. A sample of purified plumbagin was tested against the same panel of microorganisms and high growth‐inhibiting capacity was observed. Minimum inhibitory concentrations less than 2 µg mL^?1 were obtained against the filamentous fungi. In the case of the species Aspergillus fumigatus, A. niger and A. flavus , activities comparable to miconazole were obtained. CONCLUSION: The results obtained provided evidence of the antimycotic activity of plumbagin, suggesting that D. intermedia could be the source of an interesting compound for the food industry as an alternative to preservatives. Copyright © 2011 Society of Chemical Industry