Sweet sorghum: broth clarification with enzymatic treatment increases the quality of the fermentation wort for ethanol production

Sweet sorghum is currently being evaluated throughout the world as a raw material for biofuel production because its stem juices are rich in sugars that can be directly fermented to ethanol. In this work, the fermentative efficiency of three sweet sorghum genotypes was evaluated, aiming at ethanol production, harvested in two seasons, clean and whole stems, and the treatment of the juice and broth with amylolytic enzymes in order to use the present starch to increase the production of ethanol. The experiment was carried out in the 2013/2014 harvest, in the municipality of Jaboticabal, São Paulo, Brasil, located at 21°14’05’’S and 48°17’09’’W. The experimental design was completely randomized, with sub-subdivided plots and four replications. The primary treatments were the sweet sorghum genotypes (CV147, CV198, and BRS508), the secondary treatments, the type of harvest (whole stems and clean stems); the tertiary the two sampling times (102 and 116 days after sowing - d.a.s) and the quaternary the application of enzymes. In the fermentation process, the yeast PE-2 was used, at the end, the wine was recovered and characterized. Fermentation efficiency and liters of ethanol per ton of sorghum were calculated. The clarification of the juice with enzymatic treatment increases the quality of the fermentation broth and makes it possible to obtain wines with lower levels of RRTs and Brix. Fermentation efficiency is not affected by the genotype; however, it is influenced by the time of harvest and the technological quality of the juice. The use of amylolytic enzymes makes it possible to obtain wines with lower levels of RRTS and Brix. The best period of industrialization was at 102 d.a.s., and the processing of whole stalks resulted in less ethanol production.

The use of wheat hydrolyzate in the technology of extruded gluten-free cereal snacks

Разработана технология экструдированных безглютеновых снеков с использованием в качестве частичной замены безглютенового сырья - рисовой муки ферментализатом пшеницы, глютен которой предварительно прогидролизован комплексом протеолитических и амилолитических ферментных препаратов. Иммуноферментным методом анализа подтверждено соответствие содержания глютена в сырье и гидролизате требованиям Технического регламента Таможенного союза 027/12, предъявляемым к безглютеновой продукции. Полученный гидролизат насосом-дозатором подавался в камеру экструдера в количестве до 35 % к массе перерабатываемой сухой смеси на основе рисовой муки. Было изучено влияние дозировки ферментолизата пшеницы на режимные параметры экструзии, изменение структурно-механических, цветовых и органолептических характеристик полученных образцов снеков. Установлено, что с увеличением содержания гидролизата в перерабатываемой смеси возрастает влажность экструдата, отобранного после режущего устройства, в процессе экструдирования снижаются значения момента сдвига, давления, температуры экструзии, а также удельного расхода. Изменение количества вносимого ферментолизата пшеницы значимо изменяло структурно-механические показатели экструдатов. В снеках без гидролизата значение твердости составляло 6,7 Н, а с добавлением ферментолизата отмечалось увеличение до 14,6 Н. Обратная тенденция установлена для показателей коэффициента взрыва и количества микроразломов, значения которых с ростом дозировки гидролизата снижались с 8,6 до 4,1 и с 14,3 до 6,0 соответственно. Отмечено увеличение значений хроматических составляющих цвета снеков при добавлении ферментолизата в экструдируемую смесь, при этом варьирование его дозировкой не оказывало значимого влияния на изменение цветовых характеристик. По результатам дегустации более высокие оценки получили образцы снеков с добавлением 15-20 % гидролизата пшеницы. Whole wheat flour has been hydrolyzed by a complex of proteolytic and amylolytic enzymes in order to eliminate gluten and allergenic peptides. The resulting hydrolysate was tested in gluten-free snack extrusion technology as a substitute for rice flour. The enzyme-linked immunosorbent assay confirmed the compliance of the gluten content in raw materials and hydrolysate with the requirements of the Technical Regulations of the Customs Union 027/12 for gluten-free products. The resulting hydrolysate was fed by a metering pump into the extruder chamber in an amount up to 35 % by weight of the processed dry mixture based on rice flour. The effect of the dosage of wheat hydrolysate on the operating parameters of extrusion, the changes in the structural, color and organoleptic characteristics of the snacks was studied. It has been established that the moisture content of the extrudate taken out after the cutting device increases with an increase in the content of hydrolysate in the processed mixture. At the same time, the values of torque, pressure, extrusion temperature, as well as specific mechanical energy decreased. The content of hydrolysate in the mixture significantly influenced the structural properties of snacks. The hardness value of the snacks without hydrolysate was 6.7 N. It increased to 14.6 N with the addition of hydrolysate up to 35 %. The opposite trend was established for sectional expansion index and the number of fractures, the values of which decreased from 8.6 to 4.1 and from 14.3 to 6.0, respectively. Adding wheat hydrolysate was significant in changing the color characteristics of the extrudates, while the dosage value did not have a significant effect on the color of snacks. According to the results of sensory analysis, samples of the extruded snacks with the addition of 15-20 % wheat hydrolysate received higher scores.

Amylolytic enzymes - focus on the alpha-amylases from Archae and plants

Amylolytic enzymes represent a group of starch hydrolases and related enzymes that are active towards the α-glycosidic bonds in starch and related poly- and oligosaccharides. The three best known amylolytic enzymes are α-amylase, β-amylase and glucoamylase that, however, differ from each other by their amino acid sequences, three-dimensional structures, reaction mechanisms and catalytic machineries. In the sequence-based classification of all glycoside hydrolases (GHs) they have therefore been classified into the three independent families: GH13 (α-amylases), GH14 (β-amylases) and GH15 (glucoamylases). Some amylolytic enzymes have been placed to the families GH31 and GH57. The family GH13 together with the families GH70 and GH77 constitutes the clan GH-H, well-known as the α-amylase family. It contains more than 6,000 sequences and covers 30 various enzyme specificities sharing the conserved sequence regions, catalytic TIM-barrel fold, retaining reaction mechanism and catalytic triad. Among the GH13 α-amylases, those produced by plants and archaebacteria exhibit common sequence similarities that distinguish them from the α-amylases of the remaining taxonomic sources. Despite the close evolutionary relatedness between the plant and archaeal α-amylases, there are also specific differences that discriminate them from each other. These specific differences could be used in an effort to reveal the sequence-structural features responsible for the high thermostability of the α-amylases from Archaea.

Understanding the Role of Prevotella Genus in the Digestion of Lignocellulose and Other Substrates in Vietnamese Native Goats’ Rumen by Metagenomic Deep Sequencing

Bacteria in rumen play pivotal roles in the digestion of nutrients to support energy for the host. In this study, metagenomic deep sequencing of bacterial metagenome extracted from the goats’ rumen generated 48.66 GB of data with 3,411,867 contigs and 5,367,270 genes. The genes were mainly functionally annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) Carbohydrate-Active enZYmes (CAZy), and HMMER database, and taxonomically classified by MEGAN. As a result, 65,554 genes encoding for 30 enzymes/proteins related to lignocellulose conversion were exploited, in which nine enzymes were seen for the first time in goat rumen. Prevotella was the most abundant genus, contributing 30% hemicellulases and 36% enzymes/proteins for lignocellulose pretreatment, and supporting 98.8% of feruloyl esterases and 71.7% acetylxylan esterases. In addition, 18 of the 22 most lignocellulose digesting- potential contigs belonged to Prevotella. Besides, Prevotella possessed many genes coding for amylolytic enzymes. One gene encoding for endoxylanase was successfully expressed in E. coli. The recombinant enzyme had high Vmax, was tolerant to some salts and detergents, worked better at pH 5.5–6.5, temperature 40–50 °C, and was capable to be used in practices. Based on these findings, we confirm that Prevotella plays a pivotal role for hemicellulose digestion and significantly participates in starch, cellulose, hemicellulose, and pectin digestion in the goat rumen.

Rapid sanitary and technological control of culturing the producers of amylolytic enzymes.

It has been established that the biotechnological process of culturing bacillary microbial producers of amylolytic enzymes can be express-controlled by determining their ATP bioluminescence. The advantages of the method have been shown. The analytical review of producers of hydrolytic enzymes has made it clear how practical it is to use bacillary microorganisms for targeted secretion of amylolytic enzymes in biotechnological production. After monitoring bacillary microorganisms, it has been found advisable to choose Bacillus subtilis ATCC 6633 as the working culture due to its time of production of amylolytic enzymes and its biosynthetic activity. Reasons have been given for using the rapid ATP control method, which is based on the principle of bioluminescence. Different growth media have been compared and evaluated in order to intensify the quantitative biosynthetic activity of the microbial culture in the technological process of culturing bacillary microorganisms. The experiments have proved that growth media can be modified by introducing a number of carbohydrate–protein substrates as inducers of amylolytic complex gene expression. The latter manifests itself in the amylolytic activity accelerated by 12–24 hours, and causes an increase in the number of microorganisms (1.87–3.99 times as many as in the reference culture). Two methods of control (rapid bioluminescent and classic microbiological) have been used for correlative determination of the quantitative growth of Bacillus subtilis cells. Mathematical straight-line correlations have been obtained in a semilogarithmic system for the number of cells of the bacillary producer of the amylolytic enzyme complex. These correlations allow carrying out rapid control in a production environment. Along with the traditional rapid sanitary control in biotechnological production, which includes controlling the contamination of the equipment, personnel’s hands, and water, it has been suggested to perform proprietary technological express control of amylolytic enzyme biosynthesis using the culture Bacillus subtilis ATCC 6633

Potato Starch Extrusion and Roasting with Apple Distillery Wastewater as a New Method for Resistant Starch Production

Potato starch was extruded and roasted with apple distillery wastewater to produce starch esters substituted with malic acid residues. The starch esterification degree was higher at the higher roasting temperatures. Starch modification contributed to its darker color, its increased resistance to the action of amylolytic enzymes, and its decreased solubility and heat of phase transition. The changes in the other starch properties examined depended on the extrusion and roasting temperatures. The process, which was conducted without a chemical agent—in this case, the process of starch extrusion and roasting with apple distillery wastewater—should be deemed a novel method for resistant starch production.

A Modified Brewing Procedure Informed by the Enzymatic Profiles of Gluten-Free Malts Significantly Improves Fermentable Sugar Generation in Gluten-Free Brewing

The mashing step underpins the brewing process, during which the endogenous amylolytic enzymes in the malt, chiefly β-amylase, α-amylase, and limit dextrinase, act concurrently to rapidly hydrolyze malt starch to fermentable sugars. With barley malts, the mashing step is relatively straightforward, due in part to malted barley’s high enzyme activity, enzyme thermostabilities, and gelatinization properties. However, barley beers also contain gluten and individuals with celiac disease or other gluten intolerances should avoid consuming these beers. Producing gluten-free beer from gluten-free malts is difficult, generally because gluten-free malts have lower enzyme activities. Strategies to produce gluten-free beers commonly rely on exogenous enzymes to perform the hydrolysis. In this study, it was determined that the pH optima of the enzymes from gluten-free malts correspond to regions already typically targeted for barley mashes, but that a lower mashing temperature was required as the enzymes exhibited low thermostability at common mashing temperatures. The ExGM decoction mashing procedure was developed to retain enzyme activity, but ensure starch gelatinization, and demonstrates a modified brewing procedure using gluten-free malts, or a combination of malts with sub-optimal enzyme profiles, that produces high fermentable sugar concentrations. This study demonstrates that gluten-free malts can produce high fermentable sugar concentrations without requiring enzyme supplementation.

Effect of Pre-Sowing Magnetic Field Treatment on Enzymes and Phytohormones in Pea (Pisum sativum L.) Seeds and Seedlings

The aim of the presented studies was to evaluate the magnetic field (MF) effect on changes in some enzymes and phytohormones that takes place in the process of seed germination and growth of seedlings. Studies were led in the climatic chambers HERAEUS, on Petri dishes during six consecutive days. Pea seeds were divided into three groups from which one was the control (without stimulation) and two were treated with different doses of magnetic field (30 and 85 mT, respectively). Contents of amylolytic enzymes (AE) and phytohormones were determined at seven terms (0; 24; 48; 72; 96; 120 and 144 h) after placing them on the dishes. A favorable effect of seed stimulation with MF was found on the biochemical processes in the germinating seeds and pea seedlings. The size of changes in enzyme concentration was dependent mainly on the advancement of germination process (i.e., on length of time in which the seeds were on the dishes) and the MF treatment. The use of MF also had an effect on the increase in hormone content in the seeds and organs of seedlings, but values from objects treated with different doses did not differ significantly.