大麦发芽过程中金属离子对大麦酶系中淀粉酶活力影响研究

在大麦发芽过程中通过浸泡方式添加金属离子K+、Na+、Cu2+、Mg2+、Zn2+来提高制麦酶系中α和β淀粉酶的活力, 使麦汁中的离子构成及含量更加合理,这对于啤酒发酵是非常重要的实验发现,Na+、Cu2+对α和β淀粉酶活力影响较大;Mg2+和Zn2+对α-淀粉酶活力有一定的影响, 对β-淀粉酶活力有影响,但幅度较小;K+对α-淀粉酶活力有影响,而时β-淀粉酶活力有一定的影响;当Na+、K+、Mg2+、Zn2+和Cu2+浓度分别达到80、60、40、20、20mg/kg 时,两种淀粉酶活力都达到最大值。      

大麦发芽过程中金属离子对大麦酶系中淀粉酶活力影响研究

在大麦发芽过程中通过浸泡方式添加金属离子K 、Na 、Cu2 、Mg2 、Zn2 来提高制麦酶系中α和β淀粉酶的活力, 使麦汁中的离子构成及含量更加合理,这对于啤酒发酵是非常重要的实验发现,Na 、Cu2 对α和β淀粉酶活力影响较大;Mg2 和Zn2 对α-淀粉酶活力有一定的影响, 对β-淀粉酶活力有影响,但幅度较小;K 对α-淀粉酶活力有影响,而时β-淀粉酶活力有一定的影响;当Na 、K 、Mg2 、Zn2 和Cu2 浓度分别达到80、60、40、20、20mg/kg 时,两种淀粉酶活力都达到最大值。     

Zn2+对制麦中苹果酸脱氢酶和呼吸消耗的影响

研究了在制麦过程中添加Zn2+对苹果酸脱氢酶(MDH)活性和大麦呼吸消耗的影响.结果表明在制麦过程中添加浓度为0.50mmol/L、0.75mmol/L、1.00mmol/L的Zn2+均可有效的抑制MDH活力,进行动力学分析得出Zn2+大麦MDH的抑制类型为非竞争性抑制;添加浓度为0.75mmol/L、1.00mmol/L的Zn2可降低制麦过程中的呼吸消耗,其中添加1.00mmol/L Zn2+的试验组的呼吸消耗率最低,相对于对照组,两种大麦的呼吸消耗分别降低了5.22％和10.7％.同时研究了Zn2对于麦芽中的α、β淀粉酶和果胶酶活力影响,表明对α、β淀粉酶有促进作用,对果胶酶影响不明显.     

大麦发芽过程中添加金属离子对纤维素酶和蛋白酶活力影响研究

在大麦发芽的过程中蛋白酶和纤维素酶是关系到麦芽和啤酒质量的重要酶类,通过浸泡方式添加金属K+、Na+、Cu2+、Mg2+、Zn2+离子,来提高制麦酶系中纤维素外切酶和蛋白酶的活力,加速大麦胚乳细胞壁的溶解和提高麦汁中α-氨基氮的含量,这对于啤酒发酵是非常重要的。实验发现:Na+、K+、Mg2+、Zn2+和Cu2+对纤维素酶活力有一定的影响,而对蛋白酶活力有很大的影响,当其浓度分别达到60mg/kg、60mg/kg、40mg/kg、20mg/kg、20mg/kg时,两种酶活力达到最大值。     

啤酒大麦制麦过程中淀粉酶活性变化动态的研究

以不同品种的大麦为材料,运用底物分析法检测三种淀粉酶的活性。不同品种的大麦中α-淀粉酶、β-淀粉酶和极限糊精酶活力差异较大,因此实际生产中可对不同品种大麦进行筛选,选用酶活较高的大麦来制备麦芽;在制麦过程中,浸麦可在一定程度上抑制淀粉酶的活力;淀粉酶的总活力在发芽初期缓慢增加,2～3d后急剧增加至最大值;焙焦可使三种酶酶活有不同程度的下降。      

啤酒大麦制麦过程中淀粉酶活性变化动态的研究

以不同品种的大麦为材料,运用底物分析法检测3种淀粉酶的活性。不同品种的大麦中α-淀粉酶、β-淀粉酶和极限糊精酶活力差异较大,因此实际生产中可对不同品种大麦进行筛选,选用酶活较高的大麦来制备麦芽;在制麦过程中,浸麦可在一定程度卜抑制淀粉酶的活力;淀粉酶的总活力在发芽初期缓慢增加,2d～3d后急剧增加至最大值;焙焦可使3种酶酶活有不同程度地下降。     

啤酒大麦制麦过程中淀粉酶活性变化动态的研究

以不同品种的大麦为材料,运用底物分析法检测三种淀粉酶的活性.不同品种的大麦中α-淀粉酶、β-淀粉酶和极限糊精酶活力差异较大,因此实际生产中可对不同品种大麦进行筛选,选用酶活较高的大麦来制备麦芽;在制麦过程中,浸麦可在一定程度上抑制淀粉酶的活力;淀粉酶的总活力在发芽初期缓慢增加,2～3d后急剧增加至最大值;焙焦可使三种酶酶活有不同程度的下降.     

制麦过程中添加金属离子与赤霉素对大麦发芽过程淀粉酶系影响的研究

大麦发芽过程中,添加不同浓度的金属离子Mg2+、Ca2+、Zn2+、K+、Na+和赤霉素(GA3)对α-淀粉酶、β-淀粉酶和极限糊精酶活性有一定的激活和抑制作用;实验发现,添加量分别为:Mg2+50mg/kg,Ca2+50mg/kg,Zn2+20mg/kg,K+60mg/kg,Na+80mg/kg,GA30.5mg/kg时,对上述3种淀粉酶酶活均有一定的激活作用。与单独用金属离子或赤霉素浸麦相比,金属离子和赤霉素的配合使用对3种淀粉酶的酶活提高作用更为显著。     

大麦中酶形成的时间曲线

通过浸麦,发芽和提取浸出物来观察大麦中酶形成的顺序。提取制麦过程中大麦籽粒的不同部分,研究了以下5种酶的形成次序：羧肽酶（Ec3．4．16．1）、内－β1—3,1-4葡聚糖酶（EC3．2．1．73）、内－B1—4－木聚糖酶（EC3．2．1．136）、阿拉伯呋喃糖苷酶（EC3．2．1．551和α－淀粉酶。早期形成的羧肽酶及跟随其后稍微晚一点形成的β－葡聚糖酶和最后形成的仪一淀粉酶,证实了早期关于这些酶合成次序的报道。虽然木聚糖酶在浸麦和出芽早期就形成了,可是其他研究人员发现这个酶在制麦过程中形成得比较晚。除了最终均匀分散于麦粒中的木聚糖酶,其他酶在麦粒近末端有较高水平的活性。在四个效应物的条件下,在不发芽大麦环片中检验酶的形成。水分对照样反映了在全部大麦籽粒中观察到的酶形成方式。赤霉酸促进形成更高的酶总活力并同时形成了所有的酶。脱落酸促进了后期酶（木聚糖酶,阿拉伯呋喃糖苷酶和α－淀粉酶）的形成并且木聚糖酶活力比单独用水处理有明显的增高。赤霉酸和脱落酸的混合物显示了非排他的,复合的更高活力水平的反应以及酶形成起始期的变化。和用赤霉酸或赤霉酸与脱落酸的混合物处理相比,用赤霉酸和氯化钙的混合物处理引起了羧肽酶活力的较大提高和木聚糖酶活力的大幅降低。     

浸麦过程中添加金属离子对麦芽内源抗氧化活力的影响

以大麦为实验材料,通过在浸麦阶段添加不同量(0、20、40、60、80、100mg/kg)金属离子(K+、Mg2+、Zn2+、Ca2+和Cu2+),测定所制得成品麦芽的相关氧化还原酶(SOD、CAT、POD和PPO)活力、总酚含量、DPPH自由基清除率、还原力和TBARs值,以考察浸麦过程中添加的金属离子对麦芽内源性氧化还原酶与抗氧化活力的影响。结果表明:不同添加量的各金属离子对麦芽氧化还原酶类的影响不尽相同;适宜添加量的上述金属离子均有效增强了麦芽的总酚含量、DPPH自由基清除能力和还原力,降低了麦芽的TBARs值。     

THE CHROMATOGRAPHIC PROPERTIES OF BARLEY AND MALT β-AMYLASES

Barley β-amlyase occurs as a heterogeneous, polydisperse enzyme in thiol-free extracts of Conquest barley. During malting, the polydisperse enzyme is altered, resulting in the formation of four distinct enzyme components which increase in activity as germination progresses. Addition of thioglycerol to a thiol-free extract of barley, or initial extraction with thioglycerol, produces extracts containing two discrete β-amylase enzymes. β-amylase I is the major component of the extract; β-amylase II occurs as a minor component. Similarly, malt extracts containing thioglycerol have two β-amylase enzymes, β-amylase III and IV. Barley β-amylase II and malt β-amylase III have similar chromatographic properties on CM-cellulose but it is not known whether these enzymes are identical. During the early stages of germination, barley β-amylase I disappears and cannot be detected in extracts of 1-day malt; β-amylase III is the major β-amylase enzyme in this extract. Malt β-amylase IV cannot be detected in barley extracts. It develops during germination until it becomes the major β-amylase in malt extracts.     

α-GLUCOSIDASE ACTIVITY OF SORGHUM AND BARLEY MALTS

Sorghum malt α-glucosidase activity was highest at pH 3.75 while that of barley malt was highest at pH 4.6. At pH 5.4 employed in mashing sorghum malt α-glucosidase was more active than the corresponding enzyme of barley malt. α-Glucosidase was partly extracted in water but was readily extracted when L-cysteine was included in the extraction buffer, pH 8. Sorghum malt made at 30°C had higher α-glucosidase activities than the corresponding malts made at 20°C and 25°C. Nevertheless, the sorghum malts made at 20°C and 25°C produced worts which contained more glucose than worts of malt made at 30°C. Although barley malts contained more α-glucosidase activity than sorghum malts, the worts of barley had the lowest levels of glucose. The limitation to maltose production in sorghum worts, produced at 65°C, is due to inadequate gelatinization of starch and not to limitation to β-amylase and α-amylase activities. Gelatinization of the starch granules of sorghum malt in the decantation mashing procedure resulted in the production of sorghum worts which contained high levels of maltose, especially when sorghum malt was produced at 30°C. Although the β-amylase and α-amylase levels of barley malt was significantly higher than those of sorghum malted optimally at 30°C, sorghum worts contained higher levels of glucose and equivalent levels of maltose to those of barley malt. It would appear that the individual activities of α-glucosidase, α-amylase and β-amylase of sorghum malts or barley malts do not correlate with the sugar profile of the corresponding worts. In consequence, specifications for enzymes such as α-amylase and β-amylase in malt is best set at a range of values rather than as single values.     

QUANTITATIVE DETERMINATION OF α-AMYLASE ENZYMES IN GERMINATED BARLEY AFTER SEPARATION BY ISOELECTRICFOCUSING1

Quantitative extraction of malt and germinated barley α-amylases from polyacrylamide gels after isoelectricfocusing was achieved using bovine serum albumin (2 mg/ml) in the extracting medium. Sharp bands of activity were obtained when extracts from polyacrylamide gels were re-focused on another gel. This technique demonstrated that α-amylase III was the major component in malt and germinated barley extracts. This enzyme was converted to α-amylase II when such extracts were heated at 70°C.     

Further purification and characterisation of a new amylase found in barley.

An amylase, previously detected in barley and described as a new barley amylase, has been further purified by immuno-affinity and ion exchange chromatography on CM-cellulose Analysis by isoelectricfocusing and immunochemical techniques showed that thses enzyme preparation did not contain the normal α- and β-amylases usually found barley and malt. The enzyme had a very low isoelectric point (ca pH 3.0) and was identified as an α-amylase on the basis of its action pattern on amylose.     

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 .     

ASSESSMENT OF BACILLUS LICHENIFORMIS α-AMYLASE AS A CANDIDATE ENZYME FOR GENETIC ENGINEERING OF MALTING BARLEY1

Bacillus licheniformis α-amylase, a thermostable starch-degrading enzyme, has been assessed as a candidate enzyme for the genetic transformation of malting barley. The temperature optimum, pH optimum and thermostability of B. licheniformis α-amylase were compared with those of barley α-amylase. The bacterial enzyme has a higher pH optimum (?9), a higher temperature optimum (?90°C) and much higher thermostability at elevated temperatures than the barley enzyme. The specific activity of the bacterial enzyme under conditions of pH and temperature relevant to the brewing process (pH 5.5, 65°C) is ?1.5-fold higher than that of the barley enzyme. Measurements of α-amylase activity during a micro-mash showed that the bacterial enzyme is at least as stable as the barley enzyme under these conditions, and that a level of expression for the bacterial enzyme corresponding to ?0.5% of total malt protein would approximately double the α-amylase activity in the mash. B. licheniformis α-amylase activity was rapidly eliminated by boiling following mashing as would occur during brewing. The combined results suggest that barley expressing the bacterial enzyme may be useful in the brewing process.     

Comparative immunological and chromatographic study of some plant β-Amylases

A comparative study has been made of the β-amylases of barley, wheat, rye, oats and sweet-potato by means of exclusion chromatography and immunochemical analysis. The reactivity of barley malt and wheat β-amylase was compared with different anti-barley and anti-wheat sera. In exclusion chromatography on Sephadex G100, barley β-amylase yielded four, and both wheat and rye, two active components, whereas oat and sweet-potato had only one active component. During the storage of barley, wheat and rye β-amylases the large-molecule components were split into smaller ones; no changes occurred in oat and sweet-potato β-amylases. On analysis against a specific barley β-amylase antiserum, wheat and rye β-amylase gave a reaction which indicated that they were immunologically partly identical with barley β-amylase, and identical with each other. This serum induced no reaction in β-amylases of sweet-potato and oats. The rye β-amylase precipitation line did not display enzymic activity after reaction with this antiserum. Analyses with different antisera of barley and wheat confirmed the partial immunological identity of barley malt and wheat β-amylase. With some barley antisera, partial inhibition of wheat β-amylase activity was observed. A similar phenomenon was apparent when barley malt β-amylase was precipitated with some wheat antisera.     

FAILURE OF MERCURIC CHLORIDE TO SELECTIVELY INHIBIT β-AMYLASE IN SORGHUM MALT

Mercuric chloride has been reported to be a suitable reagent for the determination of α-amylase activity in sorghum malt, based on its ability to selectively inhibit β-amylase. In this re-investigation, the α- and β-amylase activities of eight sorghum malts were determined after treatment of malt extracts with various concentrations of mercuric chloride. At a malt: mercuric chloride ratio of 8.3 × 10³: 1, incomplete inhibition of β-amylase activity, as measured by the Betamyl assay, occurred in all extracts. However, this concentration resulted in significant inhibition of α-amylase activity in all extracts, as measured by both the Ceralpha assay and the Phadebas assay. In addition, α-amylase activity was found to be significantly inhibited at malt: mercuric chloride ratios as low as 1.0 × 10⁵: 1, when measured by the AmyloZyme assay. These findings do not support the original report that a malt: mercuric chloride ratio of 4.0 × 10³: 1 will selectively inhibit β-amylase in sorghum malt. Furthermore, in this context it should be emphasised that the original report was based upon inhibition studies conducted on β-amylase derived from barley, not sorghum malt .     

ACTIVITIES OF VARIOUS PEPTIDASES IN BARLEY,GREEN MALT AND MALT

The three groups of malt peptidases (carboxypeptidases, “naphthylamidases” and (amino) peptidases acting on Leu-Tyr and Ala-Gly) are present in unmalted barley; the activities are low and of a similar order of magnitude. On germination the activities of the different carboxypeptidases increase from 10- to 20-fold; the “naphthylamidases” increase only 2-fold, and the peptidase activities increase from 3- to 6-fold. None of these enzymes is inactivated during kilning to any significant extent. There are considerable differences between the carboxypeptidase activities of malts derived from different varieties of barley and the activities are correlated with α-amylase activity.