Addition Of Enzyme Research Articles

Production efficiency of young Acehnese cattle by giving fermented of water hyacinth (Eichhornia crassipes), palm fronds, and sago stems with addition of digestive enzymes

Production efficiency of young Acehnese cattle by giving fermented of water hyacinth (Eichhornia crassipes), palm fronds, and sago stems with addition of digestive enzymes

Gut health benefits and associated systemic effects provided by functional components from the fermentation of natural matrices.

Recently, the role of the gut microbiota in metabolic health, immunity, behavioral balance, longevity, and intestine comfort has been the object of several studies from scientific communities. They were encouraged by a growing interest from food industries and consumers toward novel fermented ingredients and formulations with powerful biological effects, such as pre, pro, and postbiotic products. Depending on the selected strains, the operating conditions, the addition of suitable reagents or enzymes, the equipment, and the reactor configurations, functional compounds with high bioactivity, such as short-chain fatty acids, gamma-aminobutyric acid, bioactive peptides, and serotonin, can be enhanced and/or produced through fermentation of several vegetable matrices. Otherwise, their formation can also be promoted directly in the gut after the dietary intake of fermented foods: In this case, fermentation will aim to increase the content of precursor substances, such as indigestible fibers, polyphenols, some amino acids, and resistant starch, which can be potentially metabolized by endogenous gut microorganisms and converted in healthy molecules. This review provides an overview of the main functional components currently investigated in literature and the associated gut health benefits. The current state of the art about fermentation technology as a promising functionalization tool to promote the direct or indirect formation of gut-health-enhancing components was deepened, highlighting the importance of optimizing microorganism selection, system setups, and process conditions according to the target compound of interest. The collected data suggested the possibility of gaining novel functional food ingredients or products rich in functional molecules through fermentation without performing additional extraction and purification stages, which are needed when conventional culture broths are used.

A sequential bioconversion-purification process to produce high purity xylo-oligosaccharides (XOS) from Nothophagus pumilio sawdust wastes

Xylo-oligosaccharide (XOS) production from lignocellulosic biomass (LCB) is an ongoing technology in which the best raw material, catalyst, and reaction conditions remain unclear. This study proposed a three-step sequential operation to produce prebiotic Xylo-oligosaccharide (XOS) from lignocellulosic biomass (LCB) Nothophagus pumillio sawdust. First, a commercial (Enzeco Xylanase S) and recombinant (GtXyl10A) xylanase were evaluated in XOS production from pre-extracted xylan. Then, the effect of auxiliary enzyme addition (α-glucuronidase) was evaluated in xylan hydrolysis, and finally, XOS were purified by three steps of membrane filtrations: an ultrafiltration (UF) followed by two nanofiltrations (NF). From the above, XOS production yield reach 333 [mgxos∙gxylan−1] of highly purified XOS (95 %) when the process was carried out without auxiliary enzyme addition and 473 [mgxos∙gxylan−1] of 96 % purified XOS when auxiliary enzyme was added, resulting in an improvement of the XOS production yield in 42 %.This shows the feasibility to produce high purity prebiotics from sawdust wastes of the forestry industry.

Gestational supplementation of Bifidobacterium, Lactobacillus, and Streptococcus thermophilus attenuates hepatic steatosis in offspring mice through promoting fatty acid β-oxidation.

Currently, Bifidobacterium, Lactobacillus, and Streptococcus thermophilus (BLS) are widely recognized as the crucially beneficial bacteria in the gut. Many preclinical and clinical studies have shown their protective effects against non-alcoholic fatty liver disease (NAFLD). However, whether gestational BLS supplementation could alleviate NAFLD in the offspring is still unknown. Kunming mice were given a high-fat diet (HFD) for 4weeks before mating. They received BLS supplementation by gavage during pregnancy. After weaning, offspring mice were fed with a regular diet up to 5 weeks old. Gestational BLS supplementation significantly increased the abundance of Actinobacteriota, Bifidobacterium, and Faecalibaculum in the gut of dams exposed to HFD. In offspring mice exposed to maternal HFD, maternal BLS intake significantly decreased the ratio of Firmicutes to Bacteroidetes as well as the relative abundance of Prevotella and Streptococcus, but increased the relative abundance of Parabacteroides. In offspring mice, maternal BLS supplementation significantly decreased the hepatic triglyceride content and mitigated hepatic steatosis. Furthermore, maternal BLS supplementation increased the glutathione content and reduced malondialdehyde content in the liver. In addition, mRNA and protein expression levels of key rate-limiting enzymes in mitochondrial β-oxidation (CPT1α, PPARα, and PGC1α) in the livers of offspring mice were significantly increased after gestational BLS supplementation. Thus, gestational BLS supplementation may ameliorate maternal HFD-induced steatosis and oxidative stress in the livers of offspring mice by modulating fatty acid β-oxidation.

Effect of addition of yeasts and enzymes during fermentation on physicochemical quality of fine aroma cocoa beans

Theobroma cacao is a recognized crop worldwide within the market for raw materials or processed products. Cocoa beans quality, industrial transformation and health benefits have been widely studied, but there are few studies on the fermentation process and even less on Ecuadorian cacao. In Latin America, this crop plays a very important role from an ecological and social viewpoint. Production is usually carried out by local farmers who also perform the natural fermentation of the beans, without specific management, which represents a challenge in terms of reliability and batch-to-batch homogeneity. This drawback can be effectively addressed by controlling the fermentation conditions. In addition, certain enzymes have the potential to improve the fermentation process. The aim of this work was to study the effect of using yeasts and/or enzymes during fermentation on the physicochemical quality characteristics of cocoa beans. Two trials were carried out, the first used yeasts (Saccharomyces cerevisiae), with and without molasses, and the second used enzymes (Polyphenoloxidase and Pectintranseliminase). During the fermentation process there was not external temperature control, although cocoa beans mass temperature was assessed. After the assay, the cutting test and physicochemical variables (percentage of testa, testa and cotyledon pH, moisture, ash, acidity, protein and fat contents) were evaluated. The yeasts trial stood out as the most favorable option, showing better fermentation in the cutting test, a slight increase in protein content and lower moisture. The inclusion of enzymes in the trials resulted in higher fermentation temperatures and fat content of the beans, but the samples showed lower fermentation percentage, higher moisture and mold presence, as enzymes make it difficult to completely dry the samples. It can be concluded that the addition of yeasts and/or enzymes influences fermentation and could improve the process in order to obtain high quality cocoa beans.

Construction of a soybean protein isolate/polysaccharide-based whole muscle meat analog: Physical properties and freeze-thawing stability study

A growing interest has arisen in recreating real meat by mimicking its texture characteristics and muscle fiber structure. Our previous work successfully created meat analog fiber based on soybean protein isolate (SPI) and sodium alginate (SA) with the wet-spinning method. In this work, we analyzed the microstructure, texture profile, and water retainability of the assembled plant-based whole muscle meat analog (PMA) made of SPI/SA-based meat analog fiber and systematically studied the effect of different combinations and contents of transglutaminase (TG), salt, and soybean oil on the rheological behavior of the formulated adhesive. The estimated optimal condition that has the most similar texture characteristic with real chicken breast meat is: for every 1:1 mass ratio of simulated plant meat fibers to the adhesive, add 0.1 % TG enzyme addition in the adhesive and 100 mM NaCl addition. The physical behavior of PMA during cryopreservation was investigated through freeze-thaw cycles and freezing times. The addition of a small amount of oil and salt can efficiently prevent the PMA through freezing conditions which is comparable with the addition of D-Trehalose (TD). Overall, this study not only created a plant-based whole muscle meat analog product that is similar in texture to real chicken breast meat but also provided a new direction for constructing fiber-rich structure protein-based muscle meat analogs and their further commercialization.

Detection of terminal deoxynucleotidyl transferase activity based on self-mediated nucleic acid elongation and elemental labeling inductively coupled plasma-mass spectrometry

Terminal deoxynucleotidyl transferase (TdT), a specialized DNA polymerase, is recognized as a promising biomarker for acute leukemia. Herein, taking the advantage of the self-mediated strand elongation property of TdT, a simple and sensitive method for TdT activity assay was developed based on gold nanoparticles (AuNPs) labeling inductively coupled plasma mass spectrometry (ICP-MS). In the presence of TdT, the primer DNA on magnetic beads is elongated with an adenine-rich single stranded long chain that can label poly-thymine modified AuNPs. After acid elution, the labeled AuNPs were detected by ICP-MS, and the signal intensity of 197Au reflected the TdT activity. Under the optimal conditions, the limit of detection for TdT activity is down to 0.054 U mL−1, along with good selectivity and strong tolerance to other interfering proteins. Furthermore, it achieves a straightforward and accurate detection of TdT activity in acute lymphoblastic leukemia cells without sample pre-processing and tool enzyme addition. Therefore, the proposed method shows great promise as a valuable tool for TdT-related biological research and leukemia therapeutics.

Characterization of a Fermented Beverage from Discarded Bread Flour Using Two Commercial Probiotics Starters.

The aim of this study was to develop a plant-based fermented beverage from discarded bread flour and to analyze its characteristics as a novel functional product. Eight cereal-based probiotic beverages were produced by inoculating discarded bread flour with a monoculture of Lactobacillus rhamnosus or a co-culture consisting of lactic acid bacteria and Bifidobacterium. Two additional factors, namely, the addition of amylolytic enzymes and matrix desalting, were studied alongside the type of culture. The organic acid content and microbial growth were monitored during fermentation and storage (15 and 21 days). Proximal composition, gamma-aminobutyric acid, and volatile compounds were measured in the final product. Sensory analysis was only conducted on the enzymatically treated samples. The estimated shelf life of the bread beverage was 15 days. The variables studied significantly influenced the amountof organic acids and specific volatile compounds responsible for the aroma of fermented beverages. The beverage produced via co-culturing was preferred by consumers in the sensory test.

Metabolomics-based study of chemical compositions in cellulase additives derived from a tobacco-origin Bacillus subtilis and their impact on tobacco sensory attributes.

To enhance the quality of tobacco leaves and optimize the smoking experience, diverse strains of functional bacteria and their associated metabolites have been used in tobacco aging. Exogenous cellulase additives are frequently employed to facilitate the degradation of cellulose and other macromolecular matrices and enhance the quality of the tobacco product. However, little is known about how microbial metabolites present in exogenous enzyme additives affect tobacco quality. In this study, crude cellulase solutions, produced by a tobacco-originating bacterium Bacillus subtilis FX-1 were employed on flue-cured tobacco. The incorporation of cellulase solutions resulted in the reduction of cellulose crystallinity in tobacco and the enhancement of the overall sensory quality of tobacco. Notably, tobacco treated with cellulase obtained from laboratory flask fermentation demonstrated superior scent and flavor attributes in comparison to tobacco treated with enzymes derived from industrial bioreactor fermentation. The targeted and untargeted metabolomic analysis revealed the presence of diverse flavor-related precursors and components in the cellulase additives, encompassing sugars, alcohols, amino acids, organic acids, and others. The majority of these metabolites exhibited significantly higher levels in the flask group compared to the bioreactor group, probably contributing to a pronounced enhancement in the sensory quality of tobacco. Our findings suggest that the utilization of metabolic products derived from B. subtilis FX-1 as additives in flue-cured tobacco holds promise as a viable approach for enhancing sensory attributes, establishing a solid theoretical foundation for the potential development of innovative tobacco aging additives.

Effects of Phytase Enzyme Supplementation on Protein Digestibility, Efficiency of Feed Utilization, Growth and Carcass Protein Content of Tambaqui (Colossoma macropomum)

The success of intensive cultivation of tambaqui (Colossoma macropomum) is largely determined by the availability of high-quality feed. High-quality fish diets consist of fish meal produced from animal-based proteins in order to provide fish with the protein requirement. In intensive farming systems, feed costs account for 60–70% of total production costs. Due to fish meal's increasing cost; hence, less expensive ingredients are required to substitute fish meal. Soybean meal can be used as an alternative source of protein to substitute for fish meal due to its lower price and high protein content in fish diet. Phytate acid, one of the antinutritional materials contained within soybean meal, may decrease the nutrient content of feed by affecting with the fish's ability to absorb nutrients. The research aimed to examine the effect of adding the phytase enzyme to diet feed on increasing protein digestibility, efficiency of feed utilization and growth of tambaqui fish. The fingerlings of tambaqui used as test fish had an average weight of 3.85 ± 0.08 g/fish. The diet treatments in this study were phytase enzyme addition in diet with different doses, namely: A (0 FTU/kg feed), B (250 FTU/kg feed), C (500 FTU/kg feed), D (750 FTU/kg feed) and E (1000 FTU/kg feed). The results showed that phytase enzyme addition in diet significantly increased protein digestibility, efficiency of feed utilization, growth, and carcass protein content of tambaqui fish. The most effectiveness phytase enzyme dose for tambaqui fish was 1000 FTU/kg feed, which resulted in highest values of ADCp, PER, FCR, and RGR at 68.46±0.28%, 2.27±0.05, 1.53±0.04, 74.59±0.45%, and 6.01±0.19%/day, respectively.

Metabolizable Energy and Amino Acid Digestibility of Soybean Meal from Different Sources for Broiler Chickens Supplemented with Protease.

This study investigated the effect of the serine protease on metabolizable energy and amino acids' digestibility of different soybean meal for broilers. A total of 684 broilers chickens form 14 to 23 d age were distributed with nineteen treatments, six replicates, and six birds per replicate. Nine samples of soybean meal from different regions in Brazil were used, with some samples supplemented with the protease enzyme and others without addition. Apparent and corrected-for-nitrogen-balance metabolizable energy were evaluated, as well as the coefficients of amino acid digestibility. All collected data were submitted to ANOVA at a significance level of 5% and Tukey's test was applied. The results showed that the addition of the protease enzyme significantly increased the values of AME and AMEn in all soybean meal samples. The soybean meal of different origins has significant variations in AME and AMEn. The addition of the protease improved the digestibility of essential amino acids compared to soybean meal without enzyme addition. These results indicate that supplementation with serine protease can improve the metabolizable energy and amino acid digestibility of soybean meal from different regions in the diet of broilers, potentially being an effective strategy to enhance nutrient utilization and animal performance.

Comparison study between single enzyme and multienzyme complex in distiller's dred grains with soluble supplemented diet in broiler chicken.

Upregulation of the nutritional value of feed is the major target of various studies in the livestock industry, and dietary enzyme supplementation could aid in digesting the nondegrading nutrients of grains in feed ingredients. Dried distillers' grains with solubles (DDGS) is a byproduct of the fermentation process in the beverage industry and can be used as a large supply source of fiber in feed. Therefore, we conducted an experiment with male broiler chickens to investigate the effect of various types of enzymes on DDGS and compare the efficacy of single enzyme and multienzyme complexes on growth performance and gut environments in broiler chickens. We used 420 1-day-old broiler chickens (Ross 308), and they were allotted into 4 dietary treatments with seven replications (CON, corn-soybean meal [SBM] diet; NC, DDGS supplemented diet; SE, 0.05 % of mannanase supplemented DDGS-based diet; MC, 0.10% of multienzyme complex (mannanase and xylanase, glucanase) supplemented DDGS-based diet. The dietary exogenous enzyme in the DDGS-supplemented diet could improve growth performance as much as the growth of the control group, and digestibility of dry matter, crude protein, and gross energy were significantly increased by enzyme addition in groups of chicks fed DDGS-supplementation diet. Moreover, the populations of pathogenic bacteria, coliforms, and Bacteroidetes were significantly decreased by enzyme supplementation, which might lead to improved gut mucus-secreting cells and inflammatory cytokines in the jejunum. Collectively, dietary single enzyme and multienzyme complexes could improve gut environments, including intestinal immune responses and gut microbial population, and lead to improvement of growth performance in broiler chickens.

Cysteine protease inhibitor 1 promotes metastasis by mediating an oxidative phosphorylation/MEK/ERK axis in esophageal squamous carcinoma cancer

Cysteine protease inhibitor 1 (CST1) is a cystatin superfamily protein that inhibits cysteine protease activity and is reported to be involved in the development of many malignancies. Mitochondrial oxidative phosphorylation (OXPHOS) also plays an important role in cancer cell growth regulation. However, the relationship and roles of CST1 and OXPHOS in esophageal squamous cell carcinoma (ESCC) remains unclear. In our pilot study, CST1 was shown the potential of promoting ESCC migration and invasion by the activation of MEK/ERK pathway. Transcriptome sequencing analysis revealed that CST1 is closely associated with OXPHOS. Based on a real-time ATP rate assay, mitochondrial complex I enzyme activity assay, immunofluorescence, co-immunoprecipitation, and addition of the OXPHOS inhibitor Rotenone and MEK/ERK inhibitor PD98059, we determined that CST1 affects mitochondrial complex I enzyme activity by interacting with the GRIM19 protein to elevate OXPHOS levels, and a reciprocal regulatory relationship exists between OXPHOS and the MEK/ERK pathway in ESCC cells. Finally, an in vivo study demonstrated the potential of CST1 in ESCC metastasis through regulation of the OXPHOS and MEK/ERK pathways. This study is the first to reveal the oncogenic role of CST1 in ESCC development by enhancing mitochondrial respiratory chain complex I activity to activate the OXPHOS/MEK/ERK axis, and then promote ESCC metastasis, suggesting that CST1/OXPHOS is a promising target for ESCC treatment.

Effects of enzyme addition on the expansion properties and dietary fiber composition of barley flour-blended breads

Effects of enzyme addition on the expansion properties and dietary fiber composition of barley flour-blended breads

Natural diversity screening, assay development, and characterization of nylon-6 enzymatic depolymerization

Successes in biocatalytic polyester recycling have raised the possibility of deconstructing alternative polymers enzymatically, with polyamide (PA) being a logical target due to the array of amide-cleaving enzymes present in nature. Here, we screen 40 potential natural and engineered nylon-hydrolyzing enzymes (nylonases), using mass spectrometry to quantify eight compounds resulting from enzymatic nylon-6 (PA6) hydrolysis. Comparative time-course reactions incubated at 40-70 °C showcase enzyme-dependent variations in product distributions and extent of PA6 film depolymerization, with significant nylon deconstruction activity appearing rare. The most active nylonase, a NylCK variant we rationally thermostabilized (an N-terminal nucleophile (Ntn) hydrolase, NylCK-TS, Tm = 87.4 °C, 16.4 °C higher than the wild-type), hydrolyzes 0.67 wt% of a PA6 film. Reactions fail to restart after fresh enzyme addition, indicating that substrate-based limitations, such as restricted enzyme access to hydrolysable bonds, prohibit more extensive deconstruction. Overall, this study expands our understanding of nylonase activity distribution, indicates that Ntn hydrolases may have the greatest potential for further development, and identifies key targets for progressing PA6 enzymatic depolymerization, including improving enzyme activity, product selectivity, and enhancing polymer accessibility.

Decreasing acid value of fatty acid ethyl ester products using complex enzymes.

Recently, enzymatic method has been used to prepare biodiesel using various oils. But the high acid value of the biodiesel product using enzyme as a catalyst has been one issue. In this work, an attempt to reduce the acid value of fatty acid ethyl ester (FAEE) product to satisfy the specified requirement (AV ≤ 0.5mgKOH/g), a complex enzyme-catalyzed method was used for the ethanolysis of Semen Abutili seed oil (SASO) (AV = 5.5 ± 0.3mgKOH/g). The effects of various variables (constituents of complex enzyme, type and addition of water removal agent, time, temperature, enzyme addition load, substrate ratio) on the enzymatic reaction were investigated. The optimal reaction conditions were: 1% addition of liquid lipase Eversa® Transform 2.0% and 0.8% of enzyme dry powder CALB, reaction temperature 35°C, alcohol-oil ratio 9:1 (mol/mol), 0.8g/g of 4A-MS and reaction time 24h. Under the optimal reaction conditions, the FAEE yield was 90.8% ± 1.5% and its acid value was decreased from 12.0 ± 0.2mgKOH/g to 0.39 ± 0.10mgKOH/g. In further evaluating the feasibility of preparing FAEE from SASO, the FAEE products obtained under the optimal reaction conditions were purified and evaluated with reference to the ASTM D6751 standard for the main physicochemical indexes. The results obtained were in accordance with the requirements except for the oxidative stability.

ULTRASOUND-ENZYME ASSISTED EXTRACTION OF POLYPHENOL AND AMYLASE ENZYME FROM VIETNAMESE RAMIE LEAVES: PREDICTIVE MODELING DEVELOPMENT AND OPTIMIZATION STUDY

Ramie leaves are rich in antioxidant polyphenols and amylase enzyme, which could be a good source for food applications. This study investigated the impact of combined ultrasound-enzyme treatment (U-EAT), including ultrasound intensity (10-30%), content of Viscozyme L. enzyme addition (0.5-1%) and extraction time (5-15 min) on polyphenol recovery, antioxidant activity, and amylase activity. The result revealed that U-EAT could enhance the efficiency of extraction, with a significantly higher yield of bioactive compounds and antioxidant capacity, and amylase enzyme activity compared to an untreated sample. Multiple regression analysis was applied to predict the efficacy of extraction through the values of total phenolic content, antioxidant activity and enzyme activity. The established models shown high accuracy in predicting the effect of the extracted conditions on the yield of the extraction of antioxidant and enzyme amylase. It was found that the maximum yield of extraction could be obtained when the operation conditions were ultrasound intensity of 26.5%, enzyme concentration of 0.87% and extraction time of 10.88 min. Under these optimal conditions, the highest values were found (TPC of 99.047 mgGAE/100 g, antioxidant activity of 65.02% and amylase activity of 45.36 U/mL). In summary, the findings indicate that the utilization of ultrasound combined with the enzymatic extraction process of polyphenol and amylase enzyme from ramie leaves holds potential for enhancing both the extraction yield and bioactivity of the extract. Further study should be concerned with the process of producing food ingredients or products from this extract.

Protease-Mediated T1 Contrast Enhancement of Multilayered Magneto-Gadolinium Nanostructures for Imaging and Magnetic Hyperthermia.

In this work, we constructed a multifunctional composite nanostructure for combined magnetic hyperthermia therapy and magnetic resonance imaging based on T1 and T2 signals. First, iron oxide nanocubes with a benchmark heating efficiency for magnetic hyperthermia were assembled within an amphiphilic polymer to form magnetic nanobeads. Next, poly(acrylic acid)-coated inorganic sodium gadolinium fluoride nanoparticles were electrostatically loaded onto the magnetic nanobead surface via a layer-by-layer approach by employing a positively charged enzymatic-cleavable biopolymer. The positive-negative multilayering process was validated through the changes occurring in surface ζ-potential values and structural characterization by transmission electron microscopy (TEM) imaging. These nanostructures exhibit an efficient heating profile, in terms of the specific absorption rates under clinically accepted magnetic field conditions. The addition of protease enzyme mediates the degradation of the surface layers of the nanostructures with the detachment of gadolinium nanoparticles from the magnetic beads and exposure to the aqueous environment. Such a process is associated with changes in the T1 relaxation time and contrast and a parallel decrease in the T2 signal. These structures are also nontoxic when tested on glioblastoma tumor cells up to a maximum gadolinium dose of 125 μg mL-1, which also corresponds to a iron dose of 52 μg mL-1. Nontoxic nanostructures with such enzyme-triggered release mechanisms and T1 signal enhancement are desirable for tracking tumor microenvironment release with remote T1-guidance and magnetic hyperthermia therapy actuation to be done at the diseased site upon verification of magnetic resonance imaging (MRI)-guided release.

The use of biological catalyst (enzyme) for enhanced oil recovery in Niger Delta

This study is aimed at using bio-catalyst (enzymes) for enhanced oil recovery in Niger Delta. Three different enzymes produced from microbial isolates with code ID; LPE1, LPE2 and AME were use in analyzing enzymes effect on the rock-fluid and fluid-fluid interaction test to simulate the conditions in the reservoir. The tests carried out include; wettability, interfacial tension (IFT) and adhesion test. The results from these tests show that addition of enzymes to rock-fluid interface has the potential of enhancing recovery of crude oil. From the result, the use of enzyme caused an alteration in the wettability of the rock from oil-wet to water-wet with a 33° difference. The selected enzyme LPE1 showed a significant difference in reduction of interfacial tension by a factor of 4, while LPE2 and AME did not show any significant difference. In conclusion, the wettability and IFT tests results were the basis for choosing LPE1 for core flooding process. Enzyme (LPE1) flooding after waterflood was able to give an additional 11.5 % oil recovery. From the results, the percentage recovery is a pointer to the fact that when the primary and secondary oil recovery methods are no longer viable in terms of economics, the Enyme enhanced oil recovery is the best method of mobilizing trapped crude oil in the Secondly, there is no negative effect on the crude oil quality using the enzyme-EOR because the enzymes only increase the rate of the chemical reaction on the rock media without undergoing any permanent chemical change.

Enzyme-Mediated Exponential Glucose Release: A Model-Based Strategy for Continuous Defined Fed-Batch in Small-Scale Cultivations.

Miniaturized cultivation systems offer the potential to enhance experimental throughput in bioprocess development. However, they usually lack the miniaturized pumps necessary for fed-batch mode, which is commonly employed in industrial bioprocesses. An alternative are enzyme-mediated glucose release systems from starch-derived polymers, facilitating continuous glucose supply. Nevertheless, while the glucose release, and thus the feed rate, is controlled by the enzyme concentration, it also strongly depends on the type of starch derivative, and the culture conditions as well as pH and temperature. So far it was not possible to implement controlled feeding strategies (e.g., exponential feeding). In this context, we propose a model-based approach to achieve precise control over enzyme-mediated glucose release in cultivations. To this aim, an existing mathematical model was integrated into a computational framework to calculate setpoints for enzyme additions. We demonstrate the ability of the tool to maintain different pre-defined exponential growth rates during Escherichia coli cultivations in parallel mini-bioreactors integrated into a robotic facility. Although in this case study, the intermittent additions of enzyme and dextrin were performed by a liquid handler, the approach is adaptable to manual applications. Thus, we present a straightforward and robust approach for implementing defined continuous fed-batch processes in small-scale systems, where continuous feeding was only possible with low accuracy or high technical efforts until now.