Anthocyanin Analysis as a Measure of Glycosidase Activity in Enzymes for Juice Processing

A screening procedure combining HPLC and spectrophotometric analyses was developed to measure glycosidase activity of enzyme preparations used for juice processing. Enzyme preparations (27) were evaluated; several contained β-galactosidase activity which can decompose cranberry juice pigments. β-galactosidase and α-arabinosidase activities were also determined using standard procedures (nitrophenol glycosides as substrates). Comparative results showed inconsistencies between the two procedures. Cranberry juice processing demonstrated that some enzyme preparations could decompose anthocyanin pigments under processing conditions. Pigment loss was much higher when enzymes were used with juice than with crushed fruit.     

The effect of weather damage on wheat enzymes

Sound (ungerminated) and germinated wheat kernals (cv Banks) were milled into wholemeal flours, and the activities of a range of enzymes important in baking were determined. Germination of the wheat gave increases in α-amylase (EC 3.2.1.1), protease (azocaseinase), endo-arabinoxylanase (EC 3.2.1.55), endo-carboxymethylcellulase (EC 3.2.1.4) and β-D-glucopyranosidase (EC 3.2.1.21) activities, and a slight increase in activity was detected for β-D-xylopyranosidase (EC 3.2.1.37). β-Amylase (EC 3.2.1.2) glutathione reductase (EC 1.6.2.4) and most of the glycosidase activities tested did not significantly increase on germination.     

高产糖苷酶非酿酒酵母菌株筛选、鉴定及其发酵过程中酶活性变化

为探究发酵过程中本土非酿酒酵母菌株的发酵能力和糖苷酶活性,利用WL培养基、七叶苷培养基从宁夏贺兰山东麓产区自然发酵的葡萄汁中初步分选非酿酒酵母菌株;通过对硝基苯酚法测定β-D-葡萄糖苷酶、β-D-木糖苷酶、α-L-鼠李糖苷酶和α-L-阿拉伯糖苷酶活性,比较筛选高产糖苷酶菌株;并在模拟葡萄汁发酵过程中动态监测菌株的生长动力学和糖苷酶活性。结果表明：经26S rDNA的D1/D2区鉴定为Hanseniaspora opuntiae、Metschnikowia pulcherrima、3 株Hanseniaspora uvarum、Torulaspora delbrueckii共6 株非酿酒酵母菌株具有较高的糖苷酶活性,且不同菌株间表现出一定差异性。在发酵过程中,T. delbrueckii表现出较好的发酵能力和最大的糖苷酶累积活性（94.10～127.70 mU/mL）,是对照菌株的1.96～2.30 倍;供试菌株M. pulcherrima具有较高的α-L-鼠李糖苷酶和α-L-阿拉伯糖苷酶活性;H. opuntiae和H. uvarum-3表现出高水平β-D-木糖苷酶和α-L-阿拉伯糖苷酶活性。本研究优选的本土非酿酒酵母具有较高的糖苷酶活性,具有葡萄酒增香酿造的应用潜力。     

In Vitro Hypoglycemic Activity of the Phenolic Compounds in Longan Fruit (Dimocarpus longan var. Fen Ke) Shell Against α-Glucosidase and β-Galactosidase

The in vitro hypoglycemic effect of longan fruit (Dimocarpus longan var. Fen Ke) shell extracts was evaluated by inhibiting α-glucosidase and β-galactosidase activities. The IC₅₀ values of hot water and 50% ethanol ultrasonic extracts against the α-glucosidase were 9.2 and 13.4 mg/mL, and those against the β-galactosidase were 12.9 and 19.7 mg/mL, respectively. The hot water extracts (20 mg/mL) with aid of ultrasound-assisted extraction may enhance the inhibitory rates of α-glucosidase and β-galactosidase by 10.6 and 12.0% as compared with conventional extraction, respectively. Two phenolic compounds, gallic acid and ellagic acid, were identified as the major phenolic in hot water extracts from longan fruit shell against α-glucosidase and β-galactosidase. Inhibition of α-glucosidase and β-galactosidase were proven to be as a therapeutic approach for decreasing postprandial hyperglycemia. As a result, the longan fruit shell extracts may develop to be as a potential strategy for early treatment of postprandial hyperglycemia.     

The distribution of acyl lipids and tocopherols in flours millstreams

Kernels from a mixed hard wheat grist were dissected into germ, bran (pericarp, testa and aleurone), and starchy endosperm for direct analysis of tocopherols in lipid extracts by high-performance liquid chromatography. α- and β-tocopherols were almost exclusively in the germ, α-tocotrienol was mostly in the bran, and β-tocotrienol was equally distributed between the bran and the starchy endosperm. Acyl lipids and tocopherols were quantified in 23 millstreams obtained from this grist. Components related to germ and bran (triglyceride, diacylphospholipids, ã- and β-tocopherols) and testa (flour colour) showed the highest coefficients of variation whereas endosperm components (glycolipids, N-acylphospholipids and β-T-3) showed exceptionally low variation. The quantities of marker tocopherols in the streams were used to calculate the composition of the lipid transferred to the flour from germ and aleurone, and to predict the composition of the basic endosperm, free of aleurone and germ lipids. Low proportions of diacylphospholipids in the lipid transferred to high-grade millstreams indicated the transfer of spherosome lipid. The low-grade streams exhibited high proportions of phospholipids suggesting additional transfer of germ tissue and aleurone tissue containing membrane lipids. Protein and ash contents of the transferred fraction confirmed that a substantial proportion of the transferred lipid was probably accompanied by protein bodies or by tissue fragments. It is estimated that aleurone contributed less than one-quarter of the transferred lipid in any stream. Hexane-extractable free lipids in four representative streams consisted of almost all the non-polar lipids, 40–67% of the glycolipids, 47–54% of the diacylphospholipids and 30–60% of the lysophospholipids.     

IMPROVED EXTRACTION AND ASSAY OF β-AMYLASE BROM BARLEY AND MALT

β-Amylase was extracted from barley or malt using four physical techniques to break up grists which had been prepared using a Moulinex coffee grinder. Grinding with a Polytron homogeniser apparently completely disrupted all cells, as determined by transmission electron microscopy, and increased the efficiency of extraction of β-amylase from barley by more than 30%. The other treatments tested were without value . The β-amylase activity in extracts of barley or malt was assayed by measuring the production of reducing sugars from reduced soluble starch, using a PAHBAH reagent. α-Amylase, which interferes with the quantitation of β-amylase in extracts of malt, was not totally inactivated by the chelating buffer used for enzyme extraction or by several other chelating agents. α-Amylase activity was quantified specifically using Phadebas. Using purified α-amylase a calibration was developed which related activity, as determined using Phadebas, to reducing power units. Thus the α-amylase activity present in an extract containing β-amylase could be determined using Phadebas and the reducing power equivalent activity subtracted from the total “apparent” activity to give the actual β-amylase activity. α-Glucosidase and limit dextrinase activities are believed to be too low to have a significant effect on the apparent β-amylase . The soluble and bound β-amylase activities were measured in samples taken from micromalting barley (Alexis). Dry weight losses increased to over 10% after 8 days germination. Antibiotics, applied during steeping, were used to control microbes in one experiment. However, their use checked germination and reduced malting losses to 8.4% in 8 days germination. The soluble enzyme present in extracts from steeped barley and early stages of germination was activated (20–40%) by additions of the reducing agent DTT .     

Antioxidant properties of commercial soft and hard winter wheats (Triticum aestivum L.) and their milling fractions

The total phenolic content (TPC), total antioxidant activity (TAA), 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) scavenging capacity, inhibition of coupled oxidation in a β‐carotene–linoleate model system, iron(II) chelation activity and inhibition of copper‐induced oxidation of human low‐density lipoprotein (hLDL) cholesterol of 80% ethanolic extracts of soft and hard winter wheat samples and their milling fractions, namely flour, germ, bran and shorts, were investigated. Soft wheat extracts examined demonstrated higher TPC and TAA compared to those of hard wheat extracts. The germ fraction possessed the highest TPC, followed by bran, shorts, whole grain and flour for both wheat types examined. The TAA of both wheat types showed similar results except that shorts performed better than bran in this assay. Free radical scavenging properties of whole grain and milling fractions of both soft and hard wheat samples were examined against DPPH radical. The germ and flour fractions demonstrated the highest and lowest DPPH radical scavenging activity, respectively, among wheat fractions. Wheat extracts were also efficient in preventing bleaching of β‐carotene, which is also known to be free radical mediated. In the iron(II) chelation assay the flour extracts demonstrated excellent activity, while the germ extracts showed a weak activity. The trends were similar in both soft and hard wheat for the iron(II) chelation assay. Wheat extracts also inhibited copper‐induced oxidation of hLDL. In LDL oxidation assay, wheat extracts performed better than the reference antioxidant, ferulic acid. Thus wheat phenolics may serve as effective antioxidative components as measured by in vitro techniques. Copyright © 2005 Society of Chemical Industry     

Some beta-glycosidases in barley and other cereals

The levels of β-glucosidase and β-galactosidase in barley, wheat, oats and rye were compared. It was found that while the nature of these enzymes in the cereals appeared to be the same, there was a marked difference in the ratio of their occurrence in the cereal seed. A comparison of β-glucosidase and β-galactosidase activities in different varieties of barley growing at different centres showed that enzyme activity was significantly affected both by variety and environment.     

Protein,ash, lutein and tocols distribution in einkorn (Triticum monococcum L. subsp. monococcum) seed fractions

The distribution of protein, ash, lutein, tocopherols and tocotrienols in the germ, bran and endosperm portions was studied in seeds of two einkorn accessions and one bread wheat. The two einkorns showed a higher content of most compounds, but the distribution within the kernel was similar in both species. The germ fraction showed the highest concentration of protein, lutein, α-tocopherol, β-tocopherol and total tocols. Ash, α-tocotrienol and β-tocotrienol levels were highest in the bran fraction, although significant quantities were detected also in the germ and, for tocotrienols, in the flour.     

乳酸菌对发酵豆乳风味成分的影响

以实验室保藏的发酵豆乳感官特性具有差异的5株嗜热链球菌为对象,研究了乳酸菌α-乙酰乳酸脱羧酶、α-半乳糖苷酶、β-半乳糖苷酶酶活和乳酸脱氢酶等关键酶的酶活性、发酵豆乳特性和挥发性风味成分间的相互关系。结果表明,发酵豆乳感官评价与乳酸菌的α-乙酰乳酸脱羧酶和α-半乳糖苷酶呈现显著负相关和正相关,与风味成分2,3-丁二酮、2-庚酮和2-壬酮呈显著正相关,与正己醇、2,4-癸二烯醛和2-戊基呋喃呈显著负相关;发酵豆乳酸度值与乳酸菌的β-半乳糖苷酶和乳酸脱氢酶均呈显著正相关。     

Isolation,purification, and characterization of the globulin from wheat germ

This research optimized the extraction and purification of globulin from wheat germ and assessed the molecular weight distribution and structure properties of the globulin obtained. The results showed that the relative extraction efficiency and purity of wheat germ globulin (WGG) reached 18.0% and 89.1% under the enzymolysis conditions of 0.32‰ α-amylase, pH 6.5, and 55 °C. The maximal precipitation rate of WGG (91.3%) was obtained with pH 4.3 (acid precipitation). Additionally, the molecular weight of WGG was mainly distributed below 70 kDa. FT-IR confirmed that random coils (30.95%), β-sheet (27.02%), α-helix (26.55%), and β-turn (15.48%) were the secondary structures of WGG. Furthermore, LTQ mass spectrometry showed that WGG was rich in variety and high in complexity, which retrieved 1274 proteins belonging to 392 proteomes by inverse peptide analysis. The findings endow a great potential of preparing WGG with superior functionality for food applications.     

The composition of acyl lipids and tocopherols in wheat germ oils from various sources

The compositions of acyl lipids, fatty acids and tocopherols were determined for a series of laboratory-extracted and commercial wheat germ oils and a comparison made of the total carotenoid contents. High-performance liquid chromatography (h.p.l.c.) with fluorescence detection was used to determine tocopherols and tocotrienols without the need for pretreatment of the oils. The laboratory-extracted oils were similar in composition except for the content of free fatty acids and the β-tocotrienol derived from bran in the unpurified mill germ; carotenoid content was lower in oil from rancid or cooked germ. The commercial oils contained very low concentrations of carotenoids and polar lipids and three samples had unusual tocopherol compositions very different from those of the laboratory-extracted oils. One of these samples contained no detectable amount of linolenic acid which is usually present in cereal oils. It is concluded that the quality and composition of commercial wheat germ oil is very variable and that methods of assessing quality and detecting adulteration are essential. H.p.l.c. with fluorescence detection is well-suited to the analysis of tocopherols, tocotrienols and α-tocopherol acetate in wheat germ oil.     

α-半乳糖苷酶基因在大肠杆菌中的表达及酶学性质研究

利用大肠杆菌（Escherichia coli）表达系统,经异丙基-β-D-硫代半乳糖苷（IPTG）诱导,成功异源表达了一株链球菌（Streptococcus）S1的α-半乳糖苷酶基因,重组α-半乳糖苷酶经镍柱纯化后,测定其酶学性质。重组α-半乳糖苷酶最适pH值为6.5,最适温度为50 ℃,在碱性环境中（pH 7.5～10.0）及在40 ℃以下温度条件下该酶较为稳定;酶催化动力学结果显示,该酶在最适条件下水解硝基苯-α-D-半乳糖苷（pNPG）的最大水解速率（Vmax）为508.38 μmol/（min·mg）,米氏常数（Km）值为1.2 mmol/L;通过薄层层析（TLC）法检测到该重组α-半乳糖苷酶可以高效地水解天然底物蜜二糖、棉籽糖和水苏糖中的α-半乳糖苷键。     

Glycosidase activities in bovine milk over lactation

The presence and activity of glycosidases in Irish bovine milk over three months of lactation was investigated. A low level of variation between animals was observed and the highest level of glycosidase activity was present in colostrum, decreasing through transitional milk production to minimal but constant levels in mature milk. N-acetyl-β-d-glucosaminidase, α-l-fucosidase, α-galactosidase and N-acetyl-neuraminidase appear to be the most biologically relevant glycosidases in bovine milk. The elevated levels of enzymatic activity in colostrum suggests the milk glycosidases may play a role in the digestion of bovine milk glycans in the infant mammal, possibly acting as substitutes for bacterial glycosidases prior to colonisation by the gut microflora, which are involved in in vivo oligosaccharide metabolism in the lower gastrointestinal tract.     

芝麻香型白酒微量成分硫化物及吡嗪化合物的分解甘油三酯和α-葡萄糖苷酶抑制活性

目的:测定芝麻香型白酒微量成分硫化物及吡嗪化合物的分解甘油三酯和α-葡萄糖苷酶抑制活性。方法:分别采用对硝基苯酚法及α-糖苷酶抑制活性测试方法,测定3-甲硫基丙醇、3-甲硫基丙酸乙酯、2-甲基吡嗪、2,3-二甲基吡嗪、2,3,5-三甲基吡嗪、2,3,5,6-四甲基吡嗪的分解甘油三酯和α-葡萄糖苷酶抑制活性。结果:芝麻香型白酒中6种主要含硫和含氮成分,均有一定的分解甘油三酯的能力,其中2,3,5-三甲基吡嗪分解甘油三酯的活力最高且高于脂肪酶。而只有2,3-二甲基吡嗪和2,3,5,6-四甲基吡嗪具有一定的α-葡萄糖苷酶抑制活性,分别为28.57%和19.05%。结论:芝麻香型白酒中微量成分硫化物及吡嗪化合物对于人体降血糖、血脂可能发挥一定的作用。     

Carbohydrate and protein sources influence the induction of α- and β-galactosidases in Lactobacillus reuteri

The induction of α- and β-galactosidases in six strains of Lactobacillus reuteri (L. reuteri) by six carbohydrate sources and four protein sources was studied. L. reuteri grown on raffinose had the highest α-galactosidase activity (10.55 Gal U/ml), while lactose exhibited the highest β-galactosidase activity (43.82 Gal U/ml) when compared to other carbohydrate sources. L. reuteri grown on yeast extract exhibited the highest α- and β-galactosidases activity (15.27 and 12.88 Gal U/ml, respectively) when compared to other protein sources. MF14C and SD2112 grown on raffinose had the highest α-galactosidase activity (14.75 and 14.18 Gal U/ml, respectively) followed by CF2-7F (13.38 Gal U/ml). CF2-7F grown on lactose had the highest β-galactosidase activity (82.01 Gal U/ml). SD2112, MM2-3 and CF2-7F grown on yeast extract (20.96, 19.67, 19.67 Gal U/ml, respectively) showed the highest α-galactosidase activity. MM2-3 and CF2-7F grown on yeast extract showed the highest β-galactosidase activity (18.1 and 17.59 Gal U/ml, respectively). Raffinose and lactose were the best carbohydrate sources to produce α- and β-galactosidases, respectively. Yeast extract was the best protein source to produce both enzymes and CF2-7F strain was the best producing strain on all tested conditions.     

A food-grade vector for Streptococcus thermophilus based on the α-complementation of β-galactosidase

Streptococcus thermophilus is a common yogurt starter that consumes lactose as its primary carbon source. The enzyme β-galactosidase is essential for the lactose metabolism and the growth of this species. Streptococcus thermophilus appears to be a promising cell factory. Food-grade vectors have advantages in heterologous protein expression. This study aimed to determine whether the β-galactosidase of S. thermophilus has the α-complementary characteristic and to develop a novel food-grade vector based on this phenomenon. The N-terminal 7 to 36 AA residues of the β-galactosidase in S. thermophilus were deleted. The obtained mutant S. thermophilus Δα lost β-galactosidase activity and growth ability in the lactose medium. Subsequently, plasmids expressing α-fragments with different lengths of 1 to 36 (Sα1), 1 to 53 (Sα2), and 1 to 88 (Sα3) AA were constructed and transformed into S. thermophilus Δα. Recombinant S. thermophilus Δα expressing Sα2 or Sα3 recovered the ability to grow in the lactose medium, and their β-galactosidase activity accounted for 24.5% or 11.5% of the wild strain, respectively. These results indicated that the α-complementation system of β-galactosidase existed in S. thermophilus. Based on the characteristic, a food-grade vector pSEα was constructed. Except for Sα2, vector pSEα expressed the α-donor derived from E. coli β-galactosidase. This facilitated the construction of recombinant plasmids in E. coli DH5α and thus improved the transformation efficiency of S. thermophilus. Green fluorescent protein as a reporter protein could be highly expressed in S. thermophilus using this vector. As a result, pSEα is an efficient and safe vector for S. thermophilus with potential food applications.     

THE USE OF EXTRUDED BARLEY,WHEAT AND MAIZE AS ADJUNCTS IN MASHING

The principles of extrusion cooking are summarised. In small scale trials good extracts were obtained from extruded barley when it was mashed with industrial enzymes, using a programmed temperature cycle. Extruded barley, wheat and maize and wheat flour yielded acceptable levels of extract when mashed with lager malt (70%) using a programme with 1 hour rests at 50°C and 65°C. The extracts obtained from these grists were increased above those obtained from grists of lager malt alone and the viscosities of the worts were reduced when the mashes were supplemented by preparations of bacterial enzymes. Enzyme additions also improved extract recoveries from all-malt mashes and reduced the viscosities of the derived worts. Using a temperature programmed mashing cycle and supplementary enzymes beers were prepared from a lager malt grist and grists in which the lager malt was partly replaced, by 30%, with extruded barley or extruded wheat, or extruded maize or wheat flour pellets. In every case wort was recovered relatively easily, the worts fermented normally and the beers were all fully acceptable, although their flavours did differ. However, in contrast to results of preliminary brewing trials, the head retentions of the beers made with adjuncts were unusually low, possibly because of particular enzyme additions.     

NITROGENOUS COMPONENTS OF WORTS AND BEERS BREWED FROM ALL-MALT AND MALT PLUS WHEAT FLOUR GRISTS

Worts and beers prepared from grists containing malt and wheat flour contain more and larger proteins than those prepared from grists containing only malt. These properties are related to the superior foam stability possessed by malt plus wheat flour beers. Malt solubilizes some of the wheat flour protein during mashing without subjecting it to significant proteolysis. Only very limited enzyme inhibition is exhibited. Finings residues in draught beers increase the precipitability of protein by sodium chloride and are particularly effective with malt plus wheat flour beers. The proteinaceous material isolated in this way has marked foam-stabilizing properties. The relationship between the protein removed by chilling the beer and salt-precipi-table protein and foam stability is also examined. Non-fining hazes from certain malt plus wheat flour beers are related to other beer protein fractions and some recommendations for brewery practice are made.     

MICROBIAL ENZYMES AND THEIR EFFECTS ON EXTRACT RECOVERIES FROM UNMALTED ADJUNCTS

Radial diffusion tests were used to detect β-glucan-, starch-, protein-, pentosan- and triglyceride-degrading enzyme activities in a variety of commercial enzyme preparations. Some implications of the presence of unexpected enzymes in particular preparations are discussed. A series of laboratory mashes were made with grists of milled barley, extruded barley, and extruded wheat, using various enzyme additions and temperature-time programmes. In addition a limited number of experiments were made with pale ale barley malt, and wheat flour pellets. Extract yields varied from acceptable to outstandingly good. The most intensive mashing system, with enzyme supplementation, increased the extract yield of an all-malt mash by 3·9% relative to the control mashing programme. Some worts were evaluated for Total Soluble Nitrogen (T.S.N.), Free Amino Nitrogen (F.A.N.) and viscosity. Slow wort-filtration rates occurred in some mashes although the viscosities of the worts from these mashes were low. This problem did not occur in any of the samples intensively mashed over a 5 h period. The results apparently exclude the possibility that a simple, reliable technique for estimating the extract yields of adjuncts could be devised, using these enzyme preparations.