An innovative multi-enzymatic system for gluconic acid production from starch using Aspergillus niger whole-cells.

The use of multi-enzymatic systems for the industrial production of chemical compounds is currently considered an important green tool in synthetic organic chemistry. Gluconic acid is a multi-functional organic acid widely used in the chemical, pharmaceutical, food, textile, and construction industries. Its industrial production from glucose by fermentation using Aspergillus niger has drawbacks including high costs related to cell growth and maintenance of cell viability. This study presents an innovative one-step multi-enzymatic system for gluconic acid production from starch using Aspergillus niger whole-cells in association with amylolytic enzymes. Using soluble starch as substrate, the following results were achieved for 96 h of reaction: 134.5 ± 4.3 g/L gluconic acid concentration, 98.2 ± 1.3 % gluconic acid yield, and 44.8 ± 1.4 gGA/gwhole-cells biocatalyst yield. Although the process has been developed using starch as raw material, the approach is feasible for any substrate or residue that can be hydrolyzed to glucose.

High-solids enzymatic saccharification of starch-rich raw herbal biomass residues for producing high titers of glucose.

The bioresource utilization of herbal biomass residues (HBRs) has been receiving more attention. Herein, three different HBRs from Isatidis Radix (IR) and Sophorae Flavescentis Radix (SFR) and Ginseng Radix (GR) were subjected to batch and fed-batch enzymatic hydrolysis to produce high-concentration glucose. Compositional analysis showed the three HBRs had substantial starch content (26.36-63.29%) and relatively low cellulose contents (7.85-21.02%). Due to their high starch content, the combined action of cellulolytic and amylolytic enzymes resulted in greater release of glucose from the raw HBRs compared to using the individual enzyme alone. Batch enzymatic hydrolysis of 10% (w/v) raw HBRs with low loadings of cellulase (≤ 10 FPU/g substrate) and amylolytic enzymes (≤ 5.0 mg/g substrate) led to a high glucan conversion of ≥ 70%. The addition of PEG 6000 and Tween 20 did not contribute to glucose production. Furthermore, to achieve higher glucose concentrations, fed-batch enzymatic hydrolysis was conducted using a total solid loading of 30% (w/v). After 48-h of hydrolysis, glucose concentrations of 125 g/L and 92 g/L were obtained for IR and SFR residues, respectively. GR residue yielded an 83 g/L glucose concentration after 96 h of digestion. The high glucose concentrations produced from these raw HBRs indicate their potential as ideal substrate for a profitable biorefinery. Notably, the obvious advantage of using these HBRs is the elimination of the pretreatment step, which is typically required for agricultural and woody biomass in similar studies.

Detection and quantification of zearalenone and its modified forms in enzymatically treated oat and wheat flour.

An analytical method for the analysis of the mycotoxin zearalenone (ZEN) and its modified forms was developed. Sample preparation was performed based on a modified QuEChERS method combined with liquid chromatography coupled to a triple quadrupole mass spectrometry detection. The method was tested for linearity, precision, limits of detection and quantification and recoveries. The evaluation of the above-mentioned parameters was performed on oat flour. The method was applied to oat and wheat flours that were submitted to an amylolytic treatment (α-amylase and amyloglucosidase), similar to the one used in the cereal-based baby food production process. A decrease in ß-zearalenol (ß-ZEL) and ß-ZEL-14-sulfate of approximately 40% after 90 min incubation was observed, the other analytes did not show any significant changes. To our knowledge, this is the first method that approaches the identification and assessment of ZEN-sulfate derivates in a cereal matrix. Supplementary Information: The online version contains supplementary material available at 10.1007/s13197-023-05683-6.

Exo-type, endo-type and debranching amylolytic enzymes regulate breadmaking and storage qualities of gluten-free bread.

Starch is crucial to gluten-free bread (GFB) quality. Amylolytic enzymes modified starch in situ during breadmaking, impacting the quality of both fresh and stored bread. Here, the breadmaking and storage properties of GFB with added endo-amylases, exo-amylases and debranching enzymes were compared. The results demonstrated that the addition of amylolytic enzymes, especially exo-amylases and debranching enzymes, facilitated interactions between the enzymatically modified starch segments and structural agents to improve the structure and texture of fresh GFB. Exo-type ß-amylase produced lower crumb firming rate than endo-type α-amylase, although they produced similar retrogradation enthalpies and relative crystallinities. Maltooligosaccharide-forming enzymes with a non-strict endo-/exo-mode of cleavage and debranching enzymes markedly reduced the crumb firming rate and retrogradation enthalpy. Yet, maltooligosaccharide-forming enzymes produced a similar relative crystallinity as α-amylase, indicating that enzymes with endo-/exo-modes depended less on the reduction of starch recrystallization to retard crumb firming compared with debranching enzymes.

Highly efficient glucose production from raw non-pretreated Chinese medicinal herbal residues via the synergism of cellulase and amylolytic enzymes.

Available literature on Chinese medicinal herbal residues (CMHRs) bioconversion highlights pretreatment prior to saccharification with cellulase without considering the presence of starch constituent. Herein, four commonly found CMHRs were tested for starch content, and it was found they all contained starch with content ranging from 4.74% to 16.78%. Hydrolysis of raw CMHRs with combined cellulase and amylolytic enzymes yielded increments of 16.85% to 26.51% in 48-h glucan conversion compared to cellulase alone. Further study showed 48-h glucan conversion of raw CMHRs outperformed that pretreated by water-ethanol successive extraction, ultrasound and acid, but underperformed alkali-pretreated CMHRs. Although increasing 48-h glucan conversion in the range of 7.40% to 24.10% compared to raw CMHRs, alkaline pretreatment demonstrated low glucose recovery and incurred additional cost, making it unfavorable. Saccharification of the four raw CMHRs with combined enzymes seems like a preferred option considering the elimination of high-cost pretreatment step.

Making Traditional Japanese Distilled Liquor, Shochu and Awamori, and the Contribution of White and Black Koji Fungi.

The traditional Japanese single distilled liquor, which uses koji and yeast with designated ingredients, is called "honkaku shochu." It is made using local agricultural products and has several types, including barley shochu, sweet potato shochu, rice shochu, and buckwheat shochu. In the case of honkaku shochu, black koji fungus (Aspergillus luchuensis) or white koji fungus (Aspergillus luchuensis mut. kawachii) is used to (1) saccharify the starch contained in the ingredients, (2) produce citric acid to prevent microbial spoilage, and (3) give the liquor its unique flavor. In order to make delicious shochu, when cultivating koji fungus during the shochu production process, we use a unique temperature control method to ensure that these three important elements, which greatly affect the taste of the produced liquor, are balanced without any excess or deficiency. This review describes in detail the production method of honkaku shochu, a distilled spirit unique to Japan and whose market is expected to expand worldwide, with special attention paid to the koji fungi cultivation step. Furthermore, we describe the history of the koji fungi used today in the production of shochu, and we provide a thorough explanation of the characteristics of each koji fungi. We also report the latest research progress on this topic.

A detailed in silico analysis of the amylolytic family GH126 and its possible relatedness to family GH76.

The glycoside hydrolase (GH) family 126 was established based on the X-ray structure determination of the amylolytic enzyme CPF_2247 from Clostridium perfringens genome. Its original identification as a putative carbohydrate-active enzyme was based on its low, yet significant sequence identity to members of the family GH8, which are inverting endo-ß-1,4-glucanases. As the family GH8 forms the clan GH-M with GH48, the CPF_2247 protein also exhibits similarities with members of the family GH48. The original screening of the CPF_2247 on carbohydrate substrates demonstrated its activity on glycogen and amylose, thus classifying this protein as an "α-amylase". It should be pointed out, however, there are apparent inconsistencies concerning the exact enzyme specificity of the "amylase" CPF_2247, since it exhibits both the endo- and exo-fashion of action. The family GH126 currently counts ~1000 amino acid sequences solely from Bacteria; all belonging to the phylum Firmicutes. The present study delivers the first detailed bioinformatics study of 117 selected amino acid sequences from the family GH126, featuring the insightful sequence-structure comparison with the aim to define seven conserved sequence regions and elucidate the evolutionary relationships within the family. In addition, a comparative structural analysis of the GH126 members with representatives of other GH families adopting the same (α/α)6-barrel catalytic domain fold indicates the possible sharing a catalytic residue between the families GH126 and GH76.

Starch-binding domains as CBM families-history, occurrence, structure, function and evolution.

The term "starch-binding domain" (SBD) has been applied to a domain within an amylolytic enzyme that gave the enzyme the ability to bind onto raw, i.e. thermally untreated, granular starch. An SBD is a special case of a carbohydrate-binding domain, which in general, is a structurally and functionally independent protein module exhibiting no enzymatic activity but possessing potential to target the catalytic domain to the carbohydrate substrate to accommodate it and process it at the active site. As so-called families, SBDs together with other carbohydrate-binding modules (CBMs) have become an integral part of the CAZy database (http://www.cazy.org/). The first two well-described SBDs, i.e. the C-terminal Aspergillus-type and the N-terminal Rhizopus-type have been assigned the families CBM20 and CBM21, respectively. Currently, among the 85 established CBM families in CAZy, fifteen can be considered as families having SBD functional characteristics: CBM20, 21, 25, 26, 34, 41, 45, 48, 53, 58, 68, 69, 74, 82 and 83. All known SBDs, with the exception of the extra long CBM74, were recognized as a module consisting of approximately 100 residues, adopting a ß-sandwich fold and possessing at least one carbohydrate-binding site. The present review aims to deliver and describe: (i) the SBD identification in different amylolytic and related enzymes (e.g., CAZy GH families) as well as in other relevant enzymes and proteins (e.g., laforin, the ß-subunit of AMPK, and others); (ii) information on the position in the polypeptide chain and the number of SBD copies and their CBM family affiliation (if appropriate); (iii) structure/function studies of SBDs with a special focus on solved tertiary structures, in particular, as complexes with α-glucan ligands; and (iv) the evolutionary relationships of SBDs in a tree common to all SBD CBM families (except for the extra long CBM74). Finally, some special cases and novel potential SBDs are also introduced.

Cold hydrolysis of cassava pulp and its use in simultaneous saccharification and fermentation (SSF) process for ethanol fermentation.

The present study investigated cold hydrolysis of cassava pulp (CP) and the use of cold hydrolysis with simultaneous saccharification and fermentation (SSF) for ethanol production. Cold hydrolysis of 100 g-CP/L at 50 °C for 2 h, followed by at 30 °C for 72 h resulted in the production of 71.5 ± 1.8 g/L of reducing sugar, with a yield of 0.72 g/g-CP. A mathematical model describing the cold hydrolysis process was subsequently developed. The model proved to be applicable for other cold hydrolysis systems with satisfactory results. The sequential process of cold hydrolysis at 50 °C for 2 h, followed by SSF at 30 °C for 72 h gave 27.4 g-ethanol/L, with a productivity of 0.37 g/(L h) and a fermentation efficiency of 57.58%. Based on the results, a bioconversion process for CP to ethanol was proposed. In this process, 1 kg of ethanol could be produced from 3.65 kg of CP without any nutrient supplementation.

Properties and applications of starch modifying enzymes for use in the baking industry.

Enzyme technology has many potential applications in the baking industry because carbohydrate-active enzymes specifically react with carbohydrate components, such as starch, in complex food systems. Amylolytic enzymes are added to starch-based foods, such as baking products, to retain moisture more efficiently and to increase softness, freshness, and shelf life. The major reactions used to modify the structure of food starch include: (1) hydrolysis of α-1, 4 or α-1, 6 glycosidic linkages, (2) disproportionation by the transfer of glucan moieties, and (3) branching by formation of α-1, 6 glycosidic linkage. The catalytic reaction of a single enzyme or a mixture of more than two enzymes has been applied, generating novel starches, with chemical changes in the starch structure, in which the changes of molecular mass, branch chain length distribution, and the ratio of amylose to amylopectin may occur. These developments of enzyme technology highlight the potential to create various structured-starches for the food and baking industry.

Extracellular Proteome Analysis and Flavor Formation During Soy Sauce Fermentation.

Aspergillus oryzae is an excellent strain for soy sauce fermentation because of its complicated enzyme system, especially protease. The aim of this study was to investigate the key enzymes and flavors during soy sauce fermentation, and a comparative assessment of extracellular enzymes during various fermentation stages at the proteomic level via iTRAQ analysis is presented. Many important enzymes related to the amino acid and glucose metabolisms participated in the material decomposition under high-salt stress. Dipeptidase, dipeptidyl aminopeptidase, leucine aminopeptidase, aspartic protease pep1, and extracellular metalloproteinase played positive roles during the early stage of soybean mash fermentation, whilst leucine aminopeptidase A and extracellular metalloproteinase NpI were the dominant proteolytic enzymes during the later period of fermentation. At the same time, ß-glucosidase and ß-xylanase exerted great effects upon glucose metabolism throughout the fermentation process. The results show that protease and amylolytic enzymes are complementary in the formation of flavors such as alcohols, acids, esters, aldehydes, furans, and pyrazines during soy sauce fermentation.

Saccharification of Spirulina platensis biomass using free and immobilized amylolytic enzymes.

We aimed to use physical methods of microalgal biomass rupture to study saccharification strategies using free and immobilized amylolytic enzymes. The biomass of Spirulina platensis, which consists of 50-60% carbohydrates, was exposed to physical cell rupture treatments, with better results obtained using freeze/thaw cycles following by gelatinization. In saccharification tests, it was possible to hydrolyze Spirulina biomass with hydrolysis efficiencies above 99% and 83%, respectively, using 1% (v/v) of free enzymes or 1% (m/v) of amylolytic enzymes immobilized together. The use of free and immobilized enzymes yielded high levels of conversion of polysaccharides to simple sugars in Spirulina biomass, showing that these processes are promising for the advancement of bioethanol production using microalgal biomass.

Propriedades físico-químicas do amido isolado, estudo de parâmetros enzimáticos durante o armazenamento e caracterização de enzimas amilolíticas em raízes de maca (Lepidium meyenii Walp) / Physico-chemical properties of isolated starch, enzymatic parameters during storage, and characterization of amylolytic enzymes of maca (Lepidium meyenii Walp. ) root

A maca (Lepidium meyenii Walpers) é uma planta herbácea bienal da família Brassicae, cultivada principalmente na região dos Andes da América do Sul. A parte subterrânea vem sendo consumida por muito tempo devido a seu valor nutricional e energético, mas é mais conhecida no mercado peruano e internacional por alegadas propriedades terapêuticas. Esta raiz apresenta até 76% de carboidratos, dos quais 30% é amido. Este trabalho teve como objetivos estudar: as propriedades físico-químicas e funcionais do amido isolado; os parâmetros enzimáticos durante o armazenamento e a purificação parcial de enzimas amilolíticas. Em relação às propriedades do amido, este apresentou um teor de amilose de 20% valor semelhante aos encontrados em raízes e tubérculos similares. A turbidez das suspensões de amido apresentou estabilidade durante o armazenamento. A temperatura de gelatinização e a viscosidade da pasta foram a 45,7° e 46°C, respectivamente. Com base nos dados obtidos, o amido de maca seria indicado para alimentos que requeiram temperaturas moderadas no processamento, não sendo apropriado para o emprego em alimentos congelados. Os parâmetros enzimáticos medidos tais como teor de amido total, teor de açúcares solúveis, atividade amilolítica total, atividade de α e β amilases, não mostraram diferenças significativas entre as medidas durante um período de armazenamento de 16 dias. As microscopias eletrônicas de varredura (MEV) dos grânulos de amido mostraram grãos íntegros com superfícies lisas, com algumas depressões ao redor dos grânulos os quais poderiam indicar o inicio de ataque enzimático, ou fraturas na purificação. Em relação à purificação de enzimas amilolíticas, foi possível separar uma fração ativa com a carboximetilcelulose (CMC) seguida de cromatografia liquida de alta resolução (CLAE) que permitiu a separação de duas frações protéicas, analisadas por eletroforese SDS-PAGE e eletroforese bidimensional (2D). Os polipeptídeos identificados no gel 2D apresentaram...

Maca (Lepidium meyenii Walpers) is a biennial herbaceous plant from Brassicae family, grown mainly in the Andes of South America. The underground part has been consumed for a long time due to its nutritional value and energy, but is best known in the Peruvian and international market for alleged therapeutic properties. This root has up to 76% carbohydrates, of which 30% is starch. This work aimed to study: the physico-chemical properties of isolated starch, the enzymatic parameters during storage and partial purification of amylases. In relation to the properties of starch, the amylose content showed a 20% value similar to those found in roots and tubers alike. The turbidity of starch suspensions was stable during storage. The gelatinization temperature and viscosity of the paste were 45.7 ° and 46 ° C, respectively. Based on data obtained from the starch of litter would be given to foods that require moderate temperatures in processing and is not suitable for use in frozen foods. The enzymatic parameters measured such as total starch content, soluble sugars, total amylolytic activity, activity of α and β amylases, showed no significant differences between the measures over a storage period of 16 days. Electronic microscopy (SEM) of starch granules showed grains with smooth surfaces, with some depressions around the granules which could indicate the beginning of enzymatic attack, or fractures in the purification. Regarding the purification of amylases was possible to separate an active fraction with carboxymethylcellulose (CMC) followed by high-resolution liquid chromatography (HPLC) which allowed the separation of two protein fractions, analyzed by SDS-PAGE and two-dimensional electrophoresis (2D ). The polypeptides had a molecular mass between 22 and 27 kDa and isoelectric points ranging from 4.8 to 7.3.

Potencial amilolítico do grão de milho maltado no processo de sacarificação do mesmo cereal / Amylolytic potential of maize malt in the process of sacarification of the same cereal

Objetivou-se avaliar o potencial de sacarificação do malte de milho para produção de álcool deste cereal. Para tal, foi realizada germinação do grão de milho a 20 ºC por 5 dias. O potencial amilolítico do malte de milho foi otimizado por meio de um PlanejamentoFatorial Completo 22 com 3 pontos centrais, sendo as variáveis de estudo a temperatura de incubação e concentração de substrato [S]. A sacarificação foi realizada por meio de um Planejamento de Mistura com três pontos centrais, sendo as variáveis de estudo a concentração de malte e concentração de substrato, durante 6 horas. A fermentação foi conduzida com a melhor condição de sacarificação, em reator de 5 L e volume útil de 2 L. O pH foi fixado em 4,5-5. O Brix foi acertado para o valor de 14º. O mosto hidrolisado foi esterilizado e inoculado com 5,0 g.L-1 de Saccharomyces cerevisiae proveniente de fermento comercial liofilizado. O sistema foi submetido a 2 horas de aeração inicial (170 LO2.h -1) e mantido na temperatura de 30 °C. Durante o processo de fermentação, foram realizadas determinações da concentração de células (g.L-1), açúcares redutores (mg.mL-1) e álcool (g.L-1). As variáveis temperatura de incubação e concentração de substrato foram significativos (p< 0,05) na condição de estudo de 20 % de substrato e incubação a 70 °C. Os experimentos centrais com 50 % de malte apresentaram os melhores parâmetros cinéticos com taxa volumétrica de formação de produto em etanol de 2,81 getanol.L-1.h-1 que corresponde a 8,0 ºGL e conversão de substrato em células 0,127 gcélula.gglicose-1.

The aim of this research was the production of maize malt and the evaluation of its use for alcohol production. The maize grain was allowed to germinate at 20°C for five days. The amylolitic potential of malt was optimized through a Complete Factorial Planning 22 with three central points, where the study variables were the temperature of incubation and the substrate concentration [S]. The sacarification was performed through a Planning of Mixture with three central points, where the study variables were the malt concentration and [S] during six 6 hours. The fermentation was carried out using the best sacarification condition, in a five-liter reactor and a two-liter working volume. The pH was maintained at 4.5 – 5.0. The Brix was corrected to 14º. The sterile hydrolyzedproduct was inoculated with 5.0 g.L-1 Saccharomyces cerevisiae. The system was submitted to two hours of initial aeration (170 LO2.h-1) and kept at 30°C. During the fermentation process, analyses were made to determine the cell concentrations (g.L-1), reducingsugars (mg.mL-1), alcohol (g.L-1) and °Brix. The temperature of incubation and substrate concentration were significant factors (p<0.05) in the determination of the amylolytic potential under the conditions of study utilizing 20% of substrate and incubation at 70°C. The central experiments with 50% malt presented the best kinetic parameters with a volumetric rate of ethanol formation of 2.81 gethanol.L-1.h-1, which correspond to 8.0 ºGL, and a substrate conversion in cells of 0.127gcells.gglucose -1.

Evaluation of the types of starch for preparation of LDPE/starch blends

This study evaluated in relation the growth, and the amylolytic activity of mixed and isolated cultures of Phanerochaete chrysosporium and Talaromyces wortmanni on different types of starch. The thermal and mechanical properties in polyethylene/starch blends (proportion: 80/20 (w/w) before and after inoculation of the mixed cultures were evaluated. The regular starch Amidex 3 and the modified starch Fox5901 stood out in relation to the cellular growth and production of the amylase enzyme. In spite of the short time that the blends were exposed to the fungi, the microorganisms promoted physical and chemical changes in the structure of the blend, modifying its thermal and mechanical properties. The alteration of the degree of crystallinity and mechanical properties of the blends could be indications of the modification caused by the biodegradation process.

Nesse trabalho foi realizado um estudo sobre diferentes tipos de amido quanto ao crescimento, e a atividade amilolítica de culturas mistas e isoladas dos fungos Phanerochaete chrysosporium e Talaromyces wortmannii. Avaliaram-se também as propriedades térmicas e mecânicas das blendas de polietileno/amido anfótero (na proporção 80/20 (m/m)) antes e apos a inoculação das culturas mistas desses fungos.O amido regular Amidex 3 e o amido modificado Fox5901 foram os que se destacaram quanto ao crescimento celular e produção da enzima amilase. Apesar do pouco tempo de exposição dos filmes com os fungos, pode-se concluir que os microrganismos promovem mudanças físicas e químicas na estrutura da blenda, modificando suas propriedades térmicas e mecânicas. A alteração do grau de cristalinidade e das propriedades mecânicas das blendas podem ser indícios da modificação provocada pelo processo de biodegradação.

Alterações pós-colheita em raízes de mandioquinha-salsa (Arracacia xanthorrhiza Bancroft): atividade enzimática, identificação de contaminante e caracterização parcial do amido / Alterations after harvest in peruvian carotovora (arracacia santhorrhiza bancroft) roots: enzymatic activity, pollutant identification and partial characterization of the starch

Este trabalho teve como principais objetivos verificar as alterções na atividade de enzimas amilolíticas, pectinolíticas e celulásicas em raízes de mandioquinha-salsa durante o período pós-colheita, sob diferentes condições de armazenamento, visando avaliar os mecanismos de deterioração das raízes, bem como identificar o microrganismo possivelmente responsável pela alta perecibilidade das raízes. Além disso, foram estudadas características físico-químicas e reológicas do amido de mandioquinha extraído em laboratório. Para a detecção de atividade pectinesterásica (PE) e poligalacturonásica (PG) nas raízes, os parâmetros de extração destas enzimas foram otimizados através de metodologia de superfície de resposta (MSR)...

The aim of this work was to verify the changes in amylolytic, pectinolytic and cellulasic activity in Peruvian carrot roots after harvest, under different storage conditions, in order to evaluate the deteriorative mechanisms of the roots, as well as to identify the microorganism possible responsible to its low conservation time. In addition, physico-chemical and rheological characteristics of Peruvian carrot starch were studied. For pectinesterase (PE) and poligalacturonase (PG) detection on the roots, the extraction parameters of both enzymes were optimized by response surface methodology. The enzymes presented the optimum pH values at 7.5 and 4.0 for PE and PG, respectively. Extraction time and NaCI concentration were considered non-significant by the model. Pectic enzymes seams to be related to the deterioration process of Peruvian carrot, that is associated to the root softening. Considering the high volume of gas under specific packing and temperature, the presence of microorganisms soft rot promoters could be the main cause of the high perecibility of the roots. The amylolytic enzymes present an important role on Peruvian carrot deterioration related to the starch hydrolysis and the releasing of reducing sugars, substrate for opportunistic microorganisms. The cellulasic activity was not significant during storage time. Best conditions for roots conservation occurred at 4°C and under vacuum package. The bacteria isolated from the roots were identified by biochemical reactions as Erwinia carotovora subsp. odorifera. Peruvian carrot...