The impact of treatment of cereal mashes with hydrolases of non‐starch polysaccharides and pullulanase on the chemical composition of the obtained distillates

The distilling industry has been looking for more efficient technologies for spirit production. The pressureless starch liberation method (PLS) is an energy‐saving alternative to high‐pressure treatment of starch raw materials. Also, one of the promising solutions is the use of supportive enzymatic preparations during the mashing of starch raw materials. Rye and corn mashes obtained by the PLS method were treated with enzymes (pullulanase, xylanase, cellulases and cellobiase). The composition of volatile compounds in the distillates of the fermentation mashes was determined. Acetaldehyde was the dominant aliphatic carbonyl compound found in the raw distillates. Rye‐based fermentation distillates contained small amounts of propionaldehyde, isobutyraldehyde and isovaleraldehyde, which were not found in any of the corn distillates. The treatment of mashes with cellulolytic preparations caused an increase in the methanol content of the obtained distillates. All of the mashes treated with auxiliary enzymes yielded raw distillates with much higher levels of higher alcohols. The application of hydrolases of non‐starch polysaccharides, or pullulanase preparations, during the mashing of the starch raw materials affects the chemical composition of the distillates. Xylanase and pullulanase can be used in the processing of rye and corn mashes for subsequent use in distilled ethanol beverage production. Copyright © 2014 The Institute of Brewing & Distilling     

Production of Cellulases through Solid State Fermentation Using Kinnow Pulp as a Major Substrate

A study was conducted to appraise the potential of using kinnow pulp for production of cellulases by Trichoderma reesei Rut C-30. Out of the different combinations tried out, dried kinnow pulp supplemented with wheat bran in the ratio of 4:1 resulted in the highest filter paper cellulase (FPase) activity of 13.4 IU/gds whereas endo-1,4-β-glucanase (CMCase) activity was found to be best when kinnow pulp was supplemented with wheat bran in the ratio 3:2 using Mandel Weber (MW) medium. β-glucosidase activity of 18 IU/gds was again found to be maximum in treatment involving 3:2 ratio of kinnow pulp to wheat bran in MW medium. However, supplementing kinnow pulp with wheat bran in 3:2 using water as medium resulted in an FPase:β-glucosidase ratio of nearly 1:1 which is considered to be most appropriate for achieving ideal saccharification efficiency in case of pretreated lignocellulosic material. Thus, this study involved the utilisation of kinnow pulp for production of cellulases and demonstrated that a substrate which does not find any commercial significance and causes environmental pollution due to its poor disposal holds promise as a substrate for production of cellulases.     

Screening of different agroindustrial by-products for industrial enzymes production by fermentation processes

Agroindustrial by-products are an abundant source of biocompounds that contain valuable nutrients, which are not exploited. In this work, lignocellulosic wastes (LW) were used in submerged fermentation (SmF) and solid-state fermentation (SSF) by Aspergillus niger NRRL3 to obtain valuable enzymes required in industries. SmF using soya bean hulls (SH), wheat bran (WB) and a by-product of wheat flour (F) produced the highest activities of endo-1,4-β-xylanase (Xyl) and endo-1,4-β-D-glucanase (EG) being at least 3 times lower than those obtained by SSF. The highest ratio of Xyl to EG was obtained in SmF with F. Xyl obtained by SmF with WB was the most thermally resistant. The enzymatic extract obtained in SmF using SH presented a high power of saccharification. The production of enzymes for further application such as bioethanol generation process revalue these LW and can help offset growing environmental problems.     

Kombinierte enzymatische Stärkehydrolyse

Combined Enzymatic Starch Hydrolysis. From researches so far there comes out that glucoamylase AMG 300 L® and pullulanase Promozyme 200 L® when used in quantities the same as in preparation of Dextrozyme 225/75 L® Novo at an action on liquified starch by means of α-amylase after 48 h of saccharification already (similarly like Dextrozyme®) are able to get up to 98 DE. Chromatographic analysis proved that glucoamylase AMG 300 L Novo® and succouring it pullulanase Promozyme 200 L® are working most effectively when both enzymes are added to the liquified starch medium simultaneously. From this comes out that pullulanase hydrolyzes better α-1,6 bonds in lowmolecular dextrins than in oligosaccharides G₄ to G₇ formed at previous action of glucoamylase. At an optimum ratio of glucoamylase and pullulanase in relation to the dissolved starch after 8 h of the hydrolysis there are neither iso-sugars (isomaltose, panose), no oligosaccharides higher than G₅ and no dextrins. At the solution of the starch by α-amylase and its hydrolysis by enzymatic preparation Fungamyl 800 L Novo®, at doses 0,02–0,08% to d. s. of starch, already after 8 h the reaction of hydrolysis contents of 36–62% maltose in dry substance of hydrolyzates are reached with only traces of glucose.     

Enzymatic production of linear long-chain dextrin from sago (Metroxylon sagu) starch

Pullulanase (EC.3.2.1.41) was used to generate more linear-chain dextrin from sago starch (24.9% amylose) such that the resulting product could act as a high amylose starch. A starch suspension of 5.0% (w/v) sago starch was heated at 100 °C for 45 min and, after cooling, the gelatinized sago starch was hydrolyzed with 2.0% (v/dry weight starch) pullulanase (Promozyme 400L, Novozymes A/S, Denmark) for 24 h. The linear long-chain dextrin (LLD) content of the hydrolysate after drying, was then compared with the initial LLD content. The surface morphology of the starch granules was observed with a scanning electron microscope (SEM). The effects of gelatinization, time of reaction, pretreatment with different strengths of hydrochloric acid prior to enzyme hydrolysis, and starch and enzyme concentrations were studied. Raw sago starch was resistant to the action of pullulanase, but caused an increase in the LLD of that sago starch from an initial concentration of 24.9–33.2% following gelatinization. The best conditions to maximize the amount of LLD were 5.0% (w/v) sago starch, 2.0% (v/w) enzyme and 12 h reaction time. Acid pretreatment of the sago starch did not cause greater improvement in the accessibility and susceptibility of pullulanase as the LLD content, following pullulanase action did not change significantly. Shrinkage on the surface of the starch granules was observed with the SEM.     

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%.     

Changes of Carbohydrates and Molecular Structure of Dextrins During Enzymatic Hydrolysis of Starch with Maltogenase Participation

Investigations of the potato starch hydrolysis during bacterial α-amylase “BAN 240 L” liquefaction to 19.5 DE and then saccharification with exo-acting α-amylase “Maltogenase 1000 L” alone and together with pullulanase “Promozyme 200 L”, were carried out. Under adequate conditions at different enzyme dosages hydrolyzates of the maltose, content of 60 to about 80% in DS at significantly lower glucose and minimal maltotriose content were obtained. Investigations comprised both carbohydrate contents in hydrolyzates, changing with hydrolyse course and dextrin molecular structure in hydrolyzates. It was found that decreasing dextrin molecular weight and the number of branchings in dextrin molecules was accompanied by characteristic changes of the viscosity of hydrolyzate solutions.     

Statistical Optimization of Pullulanase Production by Raoultella planticola DSMZ 4617 Using Sago Starch as Carbon and Peptone as Nitrogen Sources

Response surface methodology based on central composite design was employed for statistical optimization of medium components for the production of pullulanase by Raoultella planticola DSMZ 4617. The combined effects of three independent variables, i.e., concentrations of sago starch and peptone and initial culture pH were analyzed using a reduced cubic model, which was developed by central composite design in predicting the optimum yield of pullulanase activity. Under these conditions, the model predicted a maximum production of pullulanase (1.94 U/mL) at 6.12 g/L sago starch, 15.34 g/L peptone, and at initial culture pH of 7.23. By applying the statistical design, the pullulanase production was enhanced to about nearly two times higher (1.70 U/mL) as compared with the fermentation using nonoptimized medium (0.95 U/mL). It was observed that the experimental values obtained were in good agreement with the values predicted from the models with relatively small errors between the values.     

Enhanced Oil Recovery by Pre-treatment of Mustard Seeds Using Crude Enzyme Extract Obtained from Mixed-Culture Solid-State Fermentation of Kinnow (Citrus reticulata) Waste and Wheat Bran

Solid-state fermentation (SSF) of kinnow (Citrus reticulata) waste supplemented with wheat bran was used for production of cellulase, protease and pectinase with individual and mixed cultures of Trichoderma reesei and Aspergillus niger. Mustard seeds were pre-treated with crude filtrate extract (CFE) obtained from A. niger and T. reesei independently, and combination of the two cultures, prior to solvent extraction. Mixed-culture fermentation resulted in higher enzyme activity for filter paper cellulase (FPU), carboxymethyl cellulase (CMCase), β-glucosidase, and exoploygalacturonase (exo-PG) in comparison to fermentation with individual cultures. This study indicated that pre-treatment using crude enzyme obtained with mixed cultures enhanced the oil recovery by 11% as compared to control where no enzyme was used. Mustard seeds pre-treated with CFE obtained from mixed cultures having ratio of 2:1:1 for exo-polygalacturonase, FPU, and protease resulted in highest oil recovery. About 7–10% more oil was recovered when mustard seeds were pre-treated with CFE obtained from individual cultures, compared with control. Enzymatic pre-treatment also improved some of the quality attributes of mustard oil. To the best of our knowledge, this is the first study where mixed-culture SSF was attempted to produce enzyme consortium for pre-treatment of mustard seeds for enhanced oil recovery.     

The impact of coupled acid or pullulanase debranching on the formation of resistant starch from maize starch with autoclaving–cooling cycles

Maize starch was treated by autoclaving–cooling cycles, coupled with acid or pullulanase hydrolysis to prepare resistant starch (RS). Debranching of retrograded or gelatinized maize starch with acid or pullulanase was studied to show the corresponding impact on RS formation. When maize starch was treated with three autoclaving–cooling cycles and retrograded maize starch was hydrolyzed at room temperature, with 0.1 mol L⁻¹ citric acid for 12 h, analysis results showed that debranching of citric acid was helpful in RS formation for RS yield increased from 8.5 to 11%. Debranching of gelatinized or retrograded maize starch at 60 °C with pullulanase at addition level of 3 PUN g⁻¹ starch showed a more favorable effect on RS formation. When gelatinized maize starch was hydrolyzed by pullulanase for 12 h and then treated with two autoclaving–cooling cycles, RS yield increased to 23.5%. If retrograded maize starch subjected to one autoclaving–cooling cycle was hydrolyzed by pullulanase for 10 h and then followed by two autoclaving–cooling cycles, RS yield elevated to 32.4%. The debranching effect of pullulanase on retrograded maize starch to help RS formation is obvious and most effective, indicating this treatment is applicable in RS preparation to increase the RS yield.     

响应面法分析复合酶对扔糟淀粉出酒率的影响

该试验对提高衡水老白干清渣制酒工艺淀粉出酒率进行了研究,利用糖化酶、纤维素酶、普鲁兰酶制剂处理扔糟,响应面法分析各酶制剂的添加量、作用温度、作用时间以达到最优糖化效果。结果表明,最优糖化条件为：糖化酶1.01%、纤维素酶1.26%、普鲁兰酶0.95%、温度47.41 ℃、时间48.59 h,pH 4.0,最优糖化条件下,扔糟中还原糖含量为4 564.14 mg/100 g,扔糟中淀粉水解率为49.8%。为验证优化结果,在此条件下接种0.05%活性干酵母进行15 d固态发酵,每100 g（湿基）扔糟可得到乙醇3.06%vol,扔糟淀粉出酒率为44.5%,一定程度上提高了衡水老白干发酵工艺中的淀粉出酒率,使扔糟中的淀粉得到进一步的利用。     

Stability of β-glucosidase Activity Produced by Bifidobacterium and Lactobacillus spp. in Fermented Soymilk During Processing and Storage

ABSTRACT: Microorganisms possess endogenous enzymes, however the stability of these enzymes during storage in soymilk has not been studied. β-glucosidase is an important enzyme that could be used in the bioconversion of the predominant soy isoflavone glucosides to their bioactive aglycone forms. Fifteen probiotic microorganisms including bifidobacterium, Lactobacillus acidophilus , and Lactobacillus casei were screened for β-glucosidase activity using p-nitrophenyl-β-d-glucopyranoside as a substrate. Six strains were selected on the basis of β-glucosidase activity produced during fermentation of soymilk. The stability of the enzyme activity was assessed during incubation for up to 48 h and storage for 8 wk at frozen (-80°C), refrigerated (4°C), room (24.8°C), and incubation (37°C) temperatures. L. casei strains showed the highest β-glucosidase activity after 24 h of incubation followed by L. acidophilus strains, whereas bifidobacterium strains showedleast activity. However, p-glucosidase from Bifidobacterium animalis BB12 showed the best stability during the 48 h fermentation. Lower storage temperatures (-80°C and 4°C) showed significantly higher ( P < 0.05) β-glucosidase activity and better stability than that at higher temperatures (24.8°C and 37°C). The stability of β-glucosidase from these microorganisms should be considered for enzymic biotransformation during storage of isoflavone β-glucosides to bioactive isoflavone aglycone forms with potential health benefits.     

响应面法优化巨大芽孢杆菌Y103产普鲁兰酶的培养条件

在玉米秸秆腐殖质土壤中筛选分离得到一株巨大芽孢杆菌（Bacillus megaterium）Y103,采用单因素试验、响应面法优化巨大芽孢杆菌Y103产普鲁兰酶的培养条件。结果表明,最佳培养基配方为糯米淀粉8.5 g/L,酵母膏13.3 g/L,蛋白胨 26.7 g/L,KH2PO4 0.5 g/L,MgSO4·7H2O 0.1 g/L,MnSO4·7H2O 0.05 g/L,NaCl 2.0 g/L。最佳发酵条件为初始pH8.8,发酵温度21 ℃,发酵时间55 h。在最优条件下,普鲁兰酶酶活力为（0.77±0.03） U/mL,是优化前的3.21倍。该酶的最适作用温度为50 ℃,最适pH为8.0,其水解普鲁兰糖的产物中有大量麦芽三糖和麦芽六糖,表明其为一种新颖的II型普鲁兰酶,在洗涤剂、高麦芽糖浆和麦芽六糖的生产中有着潜在的巨大利用价值。     

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%。     

Inclusion interactions and molecular microcapsule of <Emphasis Type="Italic">Salvia sclarea</Emphasis> L. essential oil with β-cyclodextrin derivatives

The inclusion interactions of β-cyclodextrin (β-CD), heptakis (2,6-di-methyl)-β-CD (DM-β-CD), mono[2-O-(2-hydroxyethyl)]-β-CD (2-HE-β-CD), and mono[2-O-(2-hydro-xypropyl)]-β-CD (2-HP-β-CD) with Salvia sclarea L. essential oil (SEO) were investigated by spectrofluorimetry, and the various factors affecting the inclusion process were examined in detail. At the same time, the formation constants at different temperatures and the thermodynamic parameters (ΔH, ΔS and ΔG) were calculated. The molecular microcapsule of SEO with β-CD was prepared by the method of saturated aqueous solution, and the stability of the microcapsule was determined. The results suggest that the stoichiometry of the SEO–CDs inclusion complexes was 1:1 (molar ratio), the formation constants of CDs with SEO decreased with the increasing of temperature, and the order that the capability associated with SEO was β-CD > DM-β-CD > 2-HE-β-CD > 2-HP-β-CD. The thermodynamic measurements showed that the inclusion process was an exothermic and enthalpy-driven process accompanied with a negative entropic contribution, and Van der Waals force plays an importance role in the process. In addition, the content of SEO in the microcapsule was 0.14 g/g and its stabilization was obviously improved.     

普鲁兰酶协同α-葡萄糖苷酶降低青稞快消化淀粉含量酶解工艺优化

以青稞粉为原料,通过普鲁兰酶协同α-葡萄糖苷酶降低青稞快消化淀粉（RDS）含量。通过单因素试验和响应面试验确定降低青稞快消化淀粉含量的最优酶解工艺条件,并测定α-葡萄糖苷酶的抑制率评价其体外降糖活性。结果表明,最佳酶解工艺条件为：普鲁兰酶添加量200 U/g、α-葡萄糖苷酶添加量80 U/g、料液比1∶15(g∶mL)、酶解时间3 h、酶解温度55℃。在此优化条件下,青稞粉快消化淀粉含量为54.95%,比未处理过的青稞快消化淀粉含量降低了20.22%。体外降糖活性测定结果表明,与原粉相比,酶解粉的α-淀粉酶和α-葡萄糖苷酶的抑制率分别增加了68.1%和50.4%,表明经过双酶协同酶解后,青稞淀粉的体外降血糖活性明显提高。     

Saccharification of Tapioca Starch Residue with a Multienzyme Preparation of Aspergillus ustus

Enzyme preparation obtained cultivating Aspergillus ustus on rice straw-wheat bran mixture (7:3) possessed cellulase, D -xylanase, β-D -glucosidase, α-amylase, amyloglucosidase and pectinase activities. Used at 2% level with tapioca starch residue (TSR) slurry gelatinized at 80°C or pressure cooked, it yielded 45–60% reducing sugar and degraded 52–65% of the fiber material. Enhanced saccharification (72%), fiber degradation (75%) could be achieved by pretreating the substrate with mineral acid. Fermentation of the hydrolysates with Saccharomyces cerevisiae produced 29–36 ml alcohol per 100 g of sun dried TSR. Data on TSR hydrolysis by α-amylase and amyloglucosidase, A. ustus enzyme preparation individually and in combination with amyloglucosidase, in acid pretreated or untreated TSR are presented.     

Isolation and identification of Di-D-fructose dianhydrides resulting from heat-induced degradation of inulin

Dry heating of inulin up to 200 °C for 30 min resulted in a wide range of degradation products, from which five quantitatively dominating compounds were isolated using flash chromatography and semi-preparative HPLC–RI. It was possible to identify four different DFDAs (di-d-fructose dianhydrides) by means of one- and two-dimensional NMR, namely DFA III (α-D-Fruf-1,2′:2,3′-β-D-Fruf), DFA I (α-D-Fruf-1,2′;2,1′-β-D-Fruf), DFA VII (α-D-Fruf-1,2′;2,1′-α-D-Fruf), and β-D-Fruf-1,2′;2,1′-β-D-Fruf. For the fifth compound, the structure of the difructan α-D-Fruf-1,2′-β-D-Fruf was proposed. Using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC–PAD), it was possible to quantify those isolated DFDAs in inulin caramels to a total amount of up to 25%, indicating that dry heating of inulin may be a suitable tool to enrich caramels with DFDAs.     

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     

Bioethanol production from ball milled bagasse using an on-site produced fungal enzyme cocktail and xylose-fermenting Pichia stipitis

Sugarcane bagasse is one of the most promising agricultural by-products for conversion to biofuels. Here, ethanol fermentation from bagasse has been achieved using an integrated process combining mechanical pretreatment by ball milling, with enzymatic hydrolysis and fermentation. Ball milling for 2 h was sufficient for nearly complete cellulose structural transformation to an accessible amorphous form. The pretreated cellulosic residues were hydrolyzed by a crude enzyme preparation from Penicillium chrysogenum BCC4504 containing cellulase activity combined with Aspergillus flavus BCC7179 preparation containing complementary β-glucosidase activity. Saccharification yields of 84.0% and 70.4% for glucose and xylose, respectively, were obtained after hydrolysis at 45 °C, pH 5 for 72 h, which were slightly higher than those obtained with a commercial enzyme mixture containing Acremonium cellulase and Optimash BG. A high conversion yield of undetoxified pretreated bagasse (5%, w/v) hydrolysate to ethanol was attained by separate hydrolysis and fermentation processes using Pichia stipitis BCC15191, at pH 5.5, 30 °C for 24 h resulting in an ethanol concentration of 8.4 g/l, corresponding to a conversion yield of 0.29 g ethanol/g available fermentable sugars. Comparable ethanol conversion efficiency was obtained by a simultaneous saccharification and fermentation process which led to production of 8.0 g/l ethanol after 72 h fermentation under the same conditions. This study thus demonstrated the potential use of a simple integrated process with minimal environmental impact with the use of promising alternative on-site enzymes and yeast for the production of ethanol from this potent lignocellulosic biomass.