Effect of supportive enzymes on chemical composition and viscosity of rye mashes obtained by the pressureless liberation of starch method and efficiency of their fermentation

Rye mashes obtained by the pressureless liberation of starch method were treated with supportive enzymes such as pullulanase (Promozyme 200L), endo-1,4-β-xylanase (Shearzyme 500L), cellulase (Cellustar), β-glucosidase (Novozyme) and bacillolysin (Bacillus neutral proteinase) (Neutrase 0.5L). Effects of this treatment on physicochemical properties of these mashes, the course and yield of alcoholic fermentation and concentration of by-products in raw spirits were determined. Treatment with the endo-1,4-β-xylanase and cellulase coupled with β-glucosidase caused the greatest decrease in viscosity of the mashes by 97–99% in relation to the reference mashes (1,051 mPa s). Mashes treated with pullulanase and endo-1,4-β-xylanase contained more reducing sugars and were characterized by the relatively high starch saccharification degree. All the applied enzymatic preparations, in particular Promozyme 200L, Shearzyme 500L and Neutrase 0.5L improved the dynamics and yield of fermentation of the rye mashes. Enzymes contained in the examined preparations had no significant impact on concentration of by-products in raw spirits     

外加酶法酿制低糖啤酒糖化工艺的研究

以普鲁兰酶Ｐｒｏｍｏｚｙｍｅ１２０Ｌ为重点，综合分析了外加酶糖化过程中影响麦汁总还原糖量和糖组成的各种因素，如各酶制剂的用量、糖化温度、料水比、物料比及各因素之间的相互作用等，确定了一套最优的糖化工艺方案。所得麦芽汁浸出率高、色度浅、粘度低、还原糖含量高；经过高效液相色谱分析（ＨＰＬＣ）表明，其糖组成合理；经过十天的发酵，发酵度达８２．２％，酒精分为６．４５５％（Ｗ／Ｗ）。     

The Usefulness of Intermediate Products of Plum Processing for Alcoholic Fermentation and Chemical Composition of the Obtained Distillates

In this study, an evaluation of intermediate products of plum processing as potential raw materials for distillates production was performed. Effects of composition of mashes on ethanol yield, chemical composition and taste, and flavor of the obtained spirits were determined. The obtained results showed that spontaneous fermentations of the tested products of plum processing with native microflora of raisins resulted in lower ethanol yields, compared to the ones fermented with wine yeast Saccharomyces bayanus. The supplementation of mashes with 120 g/L of sucrose caused an increase in ethanol contents from 6.2 ± 0.2 ÷ 6.5 ± 0.2% v/v in reference mashes (without sucrose addition, fermented with S. bayanus) to ca. 10.3 ± 0.3% v/v, where its highest yields amounted to 94.7 ± 2.9 ÷ 95.6 ± 2.9% of theoretical capacity, without negative changes in raw material originality of distillates. The concentrations of volatile compounds in the obtained distillates exceeding 2000 mg/L alcohol 100% v/v and low content of methanol and hydrocyanic acid, as well as their good taste and aroma make the examined products of plum processing be very attractive raw materials for the plum distillates production.     

三种金属离子协同复合酶制剂对木薯酒精产率的影响

为探究金属离子协同复合酶制剂对木薯原料产酒精的影响,选取了Mg2+、Zn2+和Fe3+结合纤维素酶和普鲁兰酶的复合酶制剂进行实验。实验结果表明Mg2+、Zn2+和Fe3+的最适添加量分别为0.8mg/g原料、1.2mg/g原料和0.4mg/g原料。其中Zn2+可以激活和促进发酵过程中的多种酶,效果优于Mg2+和Fe3+,是纤维素酶和普鲁兰酶组成的复合酶制剂的最适辅助金属离子,最适添加量为1.2mg/g原料,出酒率提高了8.44%。     

Model Studies to Understand the Effects of Amylase Additions and pH Adjustment on the Rheological Behaviour of Simulated Brewery Mashes

This paper reports on the application of a previously published laboratory scale rheological method to understand the effects that amylase addition and pH adjustment have on the rheological changes, which are taking place during the brewery mashing process. Clear correlations were found between the level of amylase present in mashes and the rheological data points representing primary grain/starch swelling (PVG, R² = 0.9821) and subsequent starch digestibility (BR44, R² = 0.9751). In addition, secondary starch gelatinisation rheological data representing viscosity increases due to the presence of smaller starch granules were clearly correlated with the level of mash amylase (VG2D, R² = 0.9882). The influence of mash pH on starch gelatinisation/breakdown and amylase activities could be quantified from the rheological data. Overall, the studies clearly show how grain components, amylolytic enzymes and mash pH collectively influence the viscosity profiles during the mashing process. In addition, application of the method can provide the maltster/brewer/distiller with more functional information regarding raw materials and process conditions.     

Characterisation of fermentation of high-gravity maize mashes with the application of pullulanase,proteolytic enzymes and enzymes degrading non-starch polysaccharides

The aim of the research was to assess the possibility of the fermentation productivity rising through the increase in corn mashes extract from 16–17 to 20–21°Balling, yet keeping a 3-day fermentation period. The second goal was to obtain the highest possible utilization of starch in the raw material through deep enzymatic degradation and utilization of available sugars and simultaneous maintenance of high quality spirit. It was found that fulfilling the above during the 3-day fermentation period was possible with the application of pullulanase as an additional amylolytic enzyme. Adding pullulanase resulted in the acceleration of the starch hydrolysis degree, which led to lower amounts of unhydrolyzed dextrins and higher ethanol yield. When the supportive enzymes complex (pullulanase, protease and cellulase) was used, the final ethanol concentration reached 10.86 ± 0.04% v/v, with ethanol yield at 68.41 ± 0.23 dm³ of absolute ethanol (A₁₀₀) per 100 kg of starch, which was 95.25 ± 0.32% at the theoretical value. The acceleration of starch enzymatic degradation and the application of a proteolytic preparation visibly shortened both initial and main fermentation phases. This in turn increased the time of the final fermentation phase and resulted in more extensive utilization of substrates by yeasts with simultaneous reduction of the final concentration of acetaldehyde (26.0 ± 0.5 mg/dm³A₁₀₀) and diethyl acetal of acetaldehyde (2.5 ± 0.5 mg/dm³A₁₀₀). The quality of spirit obtained was positively verified also in terms of relatively low concentration of higher alcohol (3912.2 ± 9.8 mg/dm³A₁₀₀). Preliminary analysis of costs (without raw-material) of 1 l distillate production indicated the possibility to reduce the costs by 18–20%.     

SMALL SCALE MASHING EXPERIMENTS WITH GRISTS CONTAINING HIGH PROPORTIONS OF RAW SORGHUM

Small scale mashes (50 g total grist) with grists containing high proportions of raw sorghum (50%–80% malt replacement) showed high values of extract recovery and produced worts of lower total nitrogen, free amino nitrogen, viscosity and colour but higher values of pH compared to worts produced from all malt mashes. Increasing the proportion of raw sorghum in the grist relative to malt resulted in a decline in extract recovery, wort total nitrogen, free amino nitrogen and an increase in wort pH. Addition of industrial enzyme preparations to mashes containing raw sorghum resulted in higher values of extract recovery (enzyme preparations containing α amylase and β glucanase), higher values of wort total nitrogen and free amino nitrogen (enzyme preparations containing a neutral proteinase) and decreased wort viscosity (enzyme preparations containing β glucanase or cellulases) compared to worts produced from untreated mashes. Worts and beers were produced on a pilot brewery scale from 50% malt and 50% polished (whole) sorghum (single decoction mashing regime) and 20% malt and 80% raw sorghum supplemented with an industrial enzyme preparation (double mashing regime). Mashes comprising 50% malt and 50% polished sorghum showed comparable wort filtration behaviour (lautering) to that of control mashes (70% malt and 30% maize grists) whereas wort produced from 20% malt and 80% raw sorghum filtered slowly. Worts produced from grists containing sorghum were of high fermentability and showed lower levels of total nitrogen and free amino nitrogen compared to control worts. Analysis of worts produced from small scale mashes containing raw sorghum and a pilot brewery scale mash comprising 20% malt and 80% raw sorghum demonstrated that the levels of total nitrogen and free amino nitrogen were higher than expected from the reduction in the malt content of the mash, consistent with the release of nitrogenous components (polypeptides, peptides and amino acids) derived from sorghum into the wort. Beers produced from 50% malt and 50% polished sorghum and 20% malt and 80% raw sorghum were filtered without difficulty and were of sound flavour. Beers produced from 50% malt and 50% polished sorghum contained lower levels of isobutanol, 2-methylbutanol, dimethylsulphide and higher levels of n propanol and diacetyl compared to control beers.     

无蒸煮生淀粉酒精发酵研究

从自然界分得野生型菌株黑曲霉n-11(Aspergillus niger n-11),进行了无蒸煮生淀粉酒精发酵研究。结果表明原料的种类、用酶量和醪液的浓度对酒精发酵有较大影响,酒母的接种量影响不大。该酒精发酵工艺砍掉了蒸煮工序,可以大大节约能耗等。50立升罐无蒸煮生淀粉酒精发酵试验已通过省级技术鉴定,结果如下。(1)薯干面原料:醪液酒度10.7％(v/v,20℃)。淀粉利用率88.6％。原料出酒率36.5％。(2)玉米面原料:醪液酒度12.4％(v/v,20℃),淀粉利用率90.7％,原料出酒率35.8％。     

嗜冷普鲁兰酶制备玉米抗性淀粉工艺优化及其表征

以玉米淀粉为原料,采用嗜冷普鲁兰酶脱支处理和压热处理相结合的方式制备玉米抗性淀粉,考察了玉米淀粉乳质量分数、耐高温α-淀粉酶添加量、嗜冷普鲁兰酶添加量、嗜冷普鲁兰酶作用时间对抗性淀粉得率的影响,采用正交试验对压热-酶解法制备玉米抗性淀粉的工艺参数进行了优化。采用扫描电子显微镜、X-射线衍射和差示扫描量热仪对玉米抗性淀粉形貌、晶体结构、热特性进行了观察与分析。结果表明,制备玉米抗性淀粉的最佳工艺条件为：玉米淀粉乳质量分数18%、耐高温α-淀粉酶添加量7 U/g、嗜冷普鲁兰酶添加量10 U/g、嗜冷普鲁兰酶作用时间9 h。在最佳条件下,玉米抗性淀粉得率为16.84%。玉米淀粉经复合酶法处理后,抗性淀粉形成了致密的层状晶体结构,表面形态结构呈现出不同于玉米原淀粉A型晶体结构的V型晶体结构;玉米抗性淀粉的起始温度、峰值温度、终止温度和相变焓值分别为117.07、140.69、153.03 ℃和1 858.12 J/g,均高于玉米原淀粉。     

STARCH HYDROLYSIS IN MALTING AND MASHING

Starch hydrolysis in malting and mashing processes was studied by gel chromatography on Sepharose Cl 4B. The final molecular weight distribution of starch components in malt differs from those in barley. During malting a molecular weight fraction close to the void volume appears as an intermediate product. Minor differences in starch hydrolysis in mashes with different malts is obtained, though the final dextrin composition of the worts is similar.     

Volatile composition of raw spirits of different botanical origin

The main purpose of the research was to determine the volatile composition of raw spirits, on the basis of trace compound isolation from their volatile fraction, as well as a comparison of the obtained volatile profiles of agricultural distillates of different botanical origins (maize, wheat, triticale, rye). This approach was chosen to improve methods of quality control and quality assurance in the spirit industry. Volatile composition of all raw spirit samples derived from different raw materials (rye, triticale, maize and wheat) were formed using headspace solid‐phase microextraction (HS‐SPME) and capillary gas chromatography–mass spectrometry (GC‐MS). Performed studies indicated the presence of over 100 compounds in the raw spirit volatile fraction. The richest volatile profile was obtained from samples produced from wheat and the poorest from those produced from corn. Moreover, on the basis of performed discriminant analysis (using the variables 2‐methylbutanol + 3‐methylbutanol, 3‐methylbutyl hexanoate, ethyl dodecanoate, ethyl heptanoate and 3‐methylbutyl octanoate), it was possible to distinguish between agricultural distillates originating from triticale and distillates produced from wheat. Copyright © 2013 The Institute of Brewing & Distilling     

Optimisation of the Mashing Procedure for 100% Malted Proso Millet (Panicum miliaceum L.) as a Raw Material for Gluten‐free Beverages and Beers

Proso millet is a gluten‐free cereal and is therefore considered a suitable raw material for the manufacturing of foods and beverages for people suffering from celiac disease. The objective of this study was to develop an optimal mashing procedure for 100% proso millet malt with a specific emphasis on high amylolytic activity. Therefore, the influence of temperature and pH on the amylolytic enzyme activity during mashing was investigated. Size exclusion chromatography was used to extract different amylolytic enzyme fractions from proso millet malt. These enzymes were added into a pH‐adjusted, cold water extract of proso millet malt and an isothermal mashing procedure was applied. The temperatures and pH optima for amylolytic enzyme activities were determined. The α‐amylase enzyme showed highest activity at a temperature of 60°C and at pH 5.0, whereas the β‐amylase activity was optimum at 40°C and pH 5.3. The limit dextrinase enzyme reached maximum activity at 50°C and pH 5.3. In the subsequent mashing regimen, the mash was separated and 40% was held for 10 min at 68°C to achieve gelatinisation. The next step in the mashing procedure was the mixture of the part mashes. The combined mash was then subjected to an infusion mashing regimen, taking the temperature optima of the various amylolytic enzymes into account. It was possible to obtain full saccharification of the wort with this mashing regimen. The analytical data obtained with the optimised proso millet mash were comparable to barley wort, which served as a control.     

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.     

酱油酿造过程中微生物及生物酶的研究进展

酱油是以大豆或豆粕、小麦粉或麸皮为原料,依靠微生物发酵而生产的一种液态调味品。在发酵过程中,微生物对原料中的蛋白、淀粉等营养物质进行分解,此过程起主导作用的是微生物所分泌产生的生物酶。当前国产酱油采用米曲霉沪酿3.042（Aspergillus oryzae 3.042）进行发酵,利用其产生的碱性和中性蛋白酶把原料中的蛋白分解为 氨基酸和多肽,为酱油提供以鲜味为主的多种滋味,但仅以米曲霉单菌种酿造的酱油存在原料利用率低、风味相对差等问题。随着消费者对酱油品质要求的提高,学术界和生产企业正在通过微生物诱变、多菌种发酵、生物酶制剂应用等多种方式改善发酵过程中生物酶的种类和活性,以进一步提升酿造酱油的品质。该文重点综述了酱油酿造过程中的关键微生物、生物酶及其研究进展和在酱油中的应用,以期对利用微生物、生物酶制剂提升酱油品质提供理论指导。     

Variation in maltose in sweet wort from barley malt and rice adjuncts with differences in amylose structure

Starch from malt and solid adjuncts provides the majority of fermentable sugars for fermentation. However, there is no current data on the variation in starch structure (particularly long chained amylose) and its impact on the final wort composition of fermentable sugars, specifically maltose. This is the first study to report variation in amylose structure from barley malt and rice used as an adjunct and how this impacts the production of maltose. We compared four commercial malts with two rice adjuncts mashes, in solid and liquid additions, with an all‐malt mash used as a control. All combinations of malt and rice adjuncts were tested under two grist‐to‐liquor (G:L) ratios (1:3 and 1:4) in a 65°C ramped mash. After mashing, the wort original gravity and maltose concentration were measured. The commercial malts had different malt quality but very similar gelatinisation temperatures (~65°C). The malts varied in starch and amylose contents but had only minor variations in average amylose chain lengths. The two rice adjuncts also had similar average amylose chains lengths, but quite different amylose contents, and hence different gelatinisation temperatures. The results showed that liquid adjunct mashes had higher original gravity and maltose concentration for both G:L ratios. However, there was no consistent result in original gravity or maltose between G:L ratio or adjunct type, suggesting interactions between each malt and rice adjunct. Knowing amylose chain length could improve understanding of the potential maltose levels of the sweet wort prior to fermentation. © 2018 The Institute of Brewing & Distilling     

Pilot Scale Production of a Lager Beer from a Grist Containing 50% Unmalted Sorghum

Pilot scale (1000 L) brews were carried out with a grist comprising of unmalted sorghum (50% of total wet weight of grain) (South African variety) and malted barley (50% of total wet weight of grain) grist using a mashing program with rests at 50°C, 95°C and 60°C. Mashes were supplemented with a high heat stable bacterial α‐amylase, a bacterial neutral protease and a fungal α‐amylase. A control brew containing 100% malted barley was also carried out. Saccharification difficulties were encountered during mashing, and extraction of the grist was lower for the sorghum mashes. The sorghum mashes showed comparable lautering behaviour to that of the control mash. At mashing off the sorghum worts were starch positive. Apparent degree of fermentation of the sorghum gyles were less than the control gyles. Green beer filtration proved unproblematic. The sorghum beers compared quite closely with the control beer with regard to colour, pH and colloidal stability. Foam stability deficiencies were apparent with the sorghum beer. However, the fermentability of the sorghum worts were lower. Hence the sorghum beers were lower in total alcohol. Sensory analysis indicated that no significant differences existed between the sorghum beer and both the control beer and a commercial malted barley beer with regard to aroma, mouth‐feel, after‐taste and clarity. However, the sorghum beer was found to be significantly different to both of the other beers with regard to colour, initial taste and foam stability.     

Effect of mashing procedures on brewing

The effect of the double decoction mashing method (method A) and the single decoction plus infusion mashing method (method B) on brewing were compared. The trials were carried out with the same raw material (malt and a minor amount of corn as adjunct) on an industrial-scale plant. The effects of mashing methods A and B were evaluated in wort and beer samples obtained with the high gravity system. The analytical parameters of the worts and beers produced and the economic aspects of production (yield, beer quality, time and energy) were discussed. The results showed no considerable differences in beer quality, while a significant difference was observed in the composition of fermentable sugars of worts. Method B gave a wort with a higher content of fermentable sugars which were converted to alcohol during fermentation; therefore, it allowed to obtain a higher beer volumetric yield of the same quality while saving time and energy.     

The influence of proteolytic and cytolytic enzymes on starch degradation during mashing

The objective of this research was to investigate the impact of proteolysis and cytolysis on starch degradation over the course of the mashing process. A proteolytic enzyme (Neutrase 0.8L, Novozymes) and a number of cytolytic enzymes (barley β‐glucanase from Megazyme, Shearzyme 500L and Ultraflo Max from Novozymes) were used to test their efficiency on starch degradation during mashing. The proteolytic and cytolytic enzymes had positive effects on the levels of starch‐degrading enzymes and starch solubilization during mashing, resulting in higher levels of wort sugar compared with the control, indicating that proteins and residual non‐starchy polysaccharides limited the digestibility of starch during mashing. Moreover, the proteolytic enzyme showed a significantly greater improvement than the cytolytic enzymes, yielding a 57% increase in β‐amylase, a 173% increase in limit dextrinase, rapid starch solubilization during mashing and a higher percentage of fermentable sugars in resultant wort. The increases in limit dextrinase and β‐amylase promoted by the protease suggest that sufficient proteinaceous inhibitor existed in the wort during mashing to inhibit their activities, leading to the unavailability of β‐amylase and especially limiting dextrinase. Furthermore, Ultraflo Max, which contained a β‐glucanase–xylanase mixture, showed a greater improvement than either the individual β‐glucanase or the xylanase on starch hydrolysis. These findings suggest that β‐glucanase is the major enzyme responsible for the degradation of cell walls, and that the complete hydrolysis of the residual cell walls depends on the synergistic effect of β‐glucanase and xylanase. The results suggest that brewers should adjust the degradation of the cell walls and correct the degree of protein modification in order to obtain the desired wort composition. Copyright © 2014 The Institute of Brewing & Distilling     

利用D-最优混料设计优化浓香型白酒生物发酵液的配方

以黄水澄清液、酒尾、尾水为主要原料,通过添加生物酶制剂和控制发酵条件得到己酸乙酯含量较高的浓香型白酒生物发酵液。利用D-最优混料设计试验,研究不同黄水澄清液、酒尾和尾水配比对生物发酵液己酸乙酯生成量的影响,建立了配比与生物发酵液中己酸乙酯含量之间的回归模型,探讨了配方中各原料的互作效应,获得了浓香型白酒生物发酵液的最佳配方为：黄水澄清液26.20%、酒尾36.34%、尾水37.46%。在此最佳条件下,生物发酵液己酸乙酯含量达16.32 g/L,总酸含量为152.30 mg/100 mL,总酯（以己酸乙酯计）含量为17.66 g/L。所得生物发酵液风味成分种类丰富,黄色清亮,具有典型的发酵液复合香味。     

Characterization of Dextrins obtained by Enzymatic Treatment of Cationic Potato Starch

The substitution pattern of cationic potato starches was studied using starch hydrolyzing enzymes and a characterization of the hydrolysis products. Native and cationic starch samples were hydrolyzed with pullulanase, isoamylase, and α‐amylase and the molecular‐weight distributions of the resulting dextrins were studied using gel permeation chromatography. Isoamylase hydrolyzed the native potato starch sample readily, whereas hydrolysis with pullulanase was incomplete. Pullulanase hydrolyzed, however, cationic starch with higher DS (degree of substitution) more efficiently than isoamylase. The hydrolysis products obtained with pullulanase were separated according to charge using cation‐exchange chromatography into one unbound and two bound fractions. The unbound fraction possessed an increasing number of short chains from amylopectin with increasing DS of the starch sample. The bound material contained amylose and dextrins with sizes corresponding to the long B‐chains. The high portion of uncharged dextrins after α‐amylolysis suggested that the substitution pattern, on the molecular level, was non‐random. The composition of the unbound and bound material, obtained by ion‐exchange chromatography of α‐amylase treated starches, suggested a more intense fragmentation with increasing DS of the sample. Possibly, the substituents influence substrate conformation and thereby alter the hydrolysis patterns. It is concluded that a thorough understanding of the enzymatic hydrolysis patterns is of ultimate importance in structural studies of modified starch.