Commercial Enzyme Preparations Research Articles

LexA, an SOS response repressor, activates TGase synthesis in Streptomyces mobaraensis

Transglutaminase (EC 2.3.2.13, TGase), an enzyme that catalyzes the formation of covalent cross-links between protein or peptide molecules, plays a critical role in commercial food processing, medicine, and textiles. TGase from Streptomyces is the sole commercial enzyme preparation for cross-linking proteins. In this study, we revealed that the SOS response repressor protein LexA in Streptomyces mobaraensis not only triggers morphological development but also enhances TGase synthesis. The absence of lexA significantly diminished TGase production and sporulation. Although LexA does not bind directly to the promoter region of the TGase gene, it indirectly stimulates transcription of the tga gene, which encodes TGase. Furthermore, LexA directly enhances the expression of genes associated with protein synthesis and transcription factors, thus favorably influencing TGase synthesis at both the transcriptional and posttranscriptional levels. Moreover, LexA activates four crucial genes involved in morphological differentiation, promoting spore maturation. Overall, our findings suggest that LexA plays a dual role as a master regulator of the SOS response and a significant contributor to TGase regulation and certain aspects of secondary metabolism, offering insights into the cellular functions of LexA and facilitating the strategic engineering of TGase overproducers.

Effect of commercial fibrolytic enzymes application to normal- and slightly lower energy diets on lactational performance, digestibility and plasma nutrients in high-producing dairy cows.

The inclusion of fibrolytic enzymes in the diet is believed to have positive effects on animal production. Hence, the objective of this study was to investigate the impact of supplementing diets with a commercial fibrolytic enzyme preparation (Vistamax; mixture of xylanase and cellulase) derived from Trichoderma reesei on lactational performance, digestibility, and plasma nutrient levels in high-producing dairy cows. Two dietary energy levels were considered: a normal energy diet (metabolizable energy = 2.68 Mcal/kg) and a slightly lower energy diet (metabolizable energy = 2.55 Mcal/kg). A total of 120 lactating Holstein cows (parity = 2; Days in Milk = 113 ± 23) were randomly assigned to four treatment groups using a 2 * 2 factorial arrangement. The dietary treatments consisted of: (1) normal energy diet without enzyme supplementation (NL); (2) normal energy diet with enzyme supplementation (NLE); (3) slightly lower energy diet without enzyme supplementation (SL); and (4) slightly lower energy diet with enzyme supplementation (SLE). The amount of enzyme added to the diets was determined based on previous in vitro studies and supplier recommendations. The enzyme and premix were mixed prior to the preparation of the total mixed ration, and the trial lasted for a duration of 42 days. The results indicated that the application of the fibrolytic enzyme did not have a significant effect on dry matter intake (DMI), but it did enhance the digestibility of dry matter (DM), neutral detergent fiber (NDF), potentially digestible NDF (pdNDF), organic matter (OM), milk production, milk urea nitrogen (MUN), and blood urea nitrogen (BUN). On the other hand, the slightly lower energy diet resulted in a decrease in DMI, milk production, milk protein yield, plasma free amino acids (FAA), and an increase in plasma B-hydroxybutyrate (BHBA). In conclusion, the inclusion of the fibrolytic enzyme in the diets of dairy cows led to improvements in the digestibility of DM, NDF, pdNDF, OM, milk production, and feed efficiency. Furthermore, the application of the enzyme to the slightly lower energy diet resulted in milk production levels comparable to those observed in cows fed the untreated normal energy diet.

Transformation of agricultural wastes into functional oligosaccharides using enzymes and emerging technologies.

Pectin-oligosaccharides (POS) serve diverse purposes as a food ingredient, antimicrobial and biostimulant in plants, and their functionality is linked to the degree of esterification. Grape and broccoli wastes emerge as environmentally friendly alternatives to obtaining pectin, serving as a sustainable source to producing POS. For example, microwaves have proven to be an effective and sustainable method to extract polysaccharides from plant matrices. This work aims to use grape and broccoli wastes as alternative sources for obtaining pectin by microwave-assisted extraction and biotransformation into POS, which possess biological properties. The extraction conditions were identified at a power of 400 W, 300 s for the extraction of pectin from grape pomace and broccoli waste. Biotransformation of pectins into POS, using commercial enzyme preparations (Viscozyme L and Pectinase). Characterisation was carried out by Fourier-transform infrared spectroscopy, thermogravimetric analysis, and scanning electron microscopy. Physicochemical analysis indicated grape pomace and broccoli waste pectins had galacturonic acid content of 63.81 ± 1.67 and 40.83 ± 2.85 mg 100 mg-1, low degree of esterification of 34.89% and 16.22%, respectively. Biotransformation of pectins into POS resulted in a 20% hydrolysis rate. The main enzymatic activity was polygalacturonase for the degradation of the main structure of the pectin. Production of POS from agro-industrial wastes by emerging technologies, such as the combined use of microwave-assisted extraction and enzymatic processes, represents an alternative method for the generation of bioactive compounds with distinctive properties suitable for different applications of interest.

Green and sustainable techniques to increase the extraction of phenolic compounds from turnip greens by‐products

AbstractThe work was aimed to optimize extraction and recovery of phenolics from turnip greens by‐products using eco‐sustainable techniques based on the use of microwaves, ultrasound, or enzymes and of water as solvent. The optimization of ultrasound‐ and microwave‐assisted extractions was performed using a 4‐factors/3‐levels and a 3‐factors/3‐levels Box–Behnken Design (BBD), respectively and the results of the corresponding trials were fitted to a second order polynomial model. The enzymatic extractions were performed using three different commercial enzymatic preparations, optionally combined with an ultrasound treatment carried out according to the central values of the BBD design. The best results obtained from each green technique were compared with those of the conventional solid–liquid extraction performed with a 70% ethanol aqueous solution. Regarding ultrasounds, the conditions able to maximize the total phenolic concentrations of the extracts (solid–water ratio, 50 g/L; T, 45°C; t, 40 min; power, 24 W) increased the extraction efficiency by about 75% and reduced the extraction times from 2 h to 10 min with respect to the conventional extraction. Concerning microwaves, the optimized conditions (solid‐water ratio, 75 g/L; t, 3 min; power, 160 W) allowed the increase of the extraction efficiency by about 48% and the decrease of the extraction times to just a few minutes. For both techniques, the factor that mainly affected extraction yield and antioxidant activity were “solids‐to‐solvent ratio” and “power,” respectively. According to the results of the enzymatic treatments, the best extraction was that performed with the commercial mix of pectinlyase, polygalacturonase, pectinesterase, and arabinase, which had the same extraction efficiency of microwaves but required longer times (90 min). Furthermore, the combination with ultrasounds did not improve its performances. Extraction technique also affected the phenolic profiles in terms of number and concentration of compounds identified. In conclusion, ultrasounds and microwaves are the best treatments to maximize extraction yield (the first) and minimize extraction time (the second).Practical applicationsThis study presents an approach to the sustainable recovery of phenolics contained in vegetable by‐products that can be adapted to a variety of food waste. It also supplies an overview of the extraction technique efficiency. The industrial adoption of our findings can lead to the following advantages: reduction food waste and/or remove from it compounds that can alter the ecosystem balance; reduction of disposal cost; production of precious ingredients for functional foods/natural cosmetics/natural supplements; obtainment of a new source of income by creating a further activity that can run in parallel with the main production of the company and can serve to diversify investments; improvement of the green image of the company, especially if the energy necessary to the energy needed to power the new process is obtained from renewable sources.

Determination of malvidin-3,5-diglucoside content in wines from Cabernet Cortis and three Italian-resistant hybrids, with preliminary evaluation of oenological glycosidases for enzyme-induced breakdown of malvidin-3,5-diglucoside and malvidin-3-glucoside

ABSTRACT The content of malvin (malvidin-3,5-diglucoside) was determined in a set of wines obtained from Cabernet Cortis and other three recently introduced grape-resistant hybrids (Cabernet Volos, Merlot Khantus and Merlot Khorus) produced in South Tyrol (Italy), to establish their conformity to legal requirements and suggest winemaking approaches for their production and use. Different skin maceration times and ageing periods were considered for the wines investigated. Malvin content exceeded the limit of 15 mg/L, imposed by the Italian government for the commercialization of wines, in 18 out of 21 samples and showed a significant decrease during wine ageing (by about 40–50% every 12-month period) with the scarce effect of maceration time and fermentation temperature. Single-cultivar wines became suitable for commercialization after ca. 7 years ageing, following traditional winemaking approaches. The use of two commercial enzymatic pectolytic preparations indicated for aroma enhancement and possessing β-glucosidase activity was also investigated at two different dosages, to increase the degradation rate of malvin in wines after winemaking. They are unsuitable for significant malvin content decrease within a 6-month time lapse.

Optimization of the recovery yield of the enzymatic aqueous extraction of oil from wet açaí decocts using Design of Experiment

These last decades, açaí oil has been extensively studied for its biological properties and has gained interest from the health industry. It has thus become necessary to develop eco-friendly extraction techniques. The main objective of this study was the use of experimental designs for the maximization of the recovery yield of the enzymatic aqueous extraction process of açaí oil from wet decocts. A Simplex-Lattice Mixture Design was employed for the optimization of the proportion of three commercial enzymatic preparations. Subsequently, a Central Composite Design was used to identify the optimal values for total enzymatic concentration (0.5-4.5%) and extraction time (2-12h). The “Response Surface Methodology” (RSM) revealed that the maximum yield (60.55 ± 5.98%) was obtained using a 0.49:0.25:0.28 ternary mixture of Celluclast 1.5 L, Viscozyme L and Ultrazym AFP-L at a total enzymatic concentration of 2.85% for 10.9 hours. This study concluded that the enzymatic aqueous extraction of açaí oil is an efficient and sustainable process.

Selective Wine Aroma Enhancement through Enzyme Hydrolysis of Glycosidic Precursors.

Selective enhancement of wine aroma was achieved using a broad spectrum of exogenous glycosidases. Eight different enzyme preparations were added to Verdejo wine, resulting in an increase in the levels of varietal volatile compounds compared to the control wine after 15 days of treatment. The enzyme preparations studied were robust under winemaking conditions (sulfur dioxide, reducing sugars, and alcohol content), and no inhibition of β-glucosidase activity was observed. Significant differences were detected in four individual terpenes (α-terpineol, terpinen-4-ol, α-pinene, and citronellal) and benzyl alcohol in all the treated wines compared to the control wine, contributing to the final wine to varying degrees. In addition, a significant increase in the other aromatic compounds was observed, which showed different patterns depending on the enzyme preparation that was tested. The principal component analysis of the data revealed the possibility of modulating the different aromatic profiles of the final wines depending on the enzyme preparation used. Taking these results into account, enhancement of the floral, balsamic, and/or fruity notes of wines is possible by using a suitable commercial enzyme preparation.

Enzymatic Method of Bioactive Peptides Production from Milk Protein Raw Materials: A Scoping Review

Introduction: For 14 years, interest in the use of dairy raw materials for the production of bioactive peptides (BPs) with antihypertensive, antioxidant and antidiabetic effects has been in a phase of rapid growth because of the need to prevent different diseases. Particular attention is being paid to enzymatic production method, as it allows for a targeted process of BP extraction with specified properties.The purpose of this scoping review was to summarize and systematize the experience of international research over the last 14 years in the field of FG as a method to extract BPs from milk protein substrates.Materials and Methods: This scoping review was performed in accordance with the PRISMA-ScR guidelines. Scientific papers were searched in the Google Scholar database, because allows searching for scientific sources without restrictions on their format and type of scientific data presentation (book, dissertation, article), as well as includes scientific sources from other databases and performs relevance sorting.Results: The search identified 72 sources published between 2007 and 2021 in English. Included sources are devoted to hydrolysis by starter bacteria enzymes (SBE) - 50% and by commercial enzyme preparations (CEP) - 50%.Conclusion: There are a number of challenges that need to be addressed in order to increase the level of commercialization of BP production through directed hydrolysis. Creation of databases; building up the research base on co-fermentation by microorganisms and commercial enzymes; conducting PCR studies of microbial protease genes will increase the practical applicability of research and the validity of conducting more evidence-based in vivo and clinical studies.

In vitro evaluation of enzymatically derived blackcurrant extract as prebiotic cosmetic ingredient: extraction conditions optimization and effect on cutaneous microbiota representatives

BackgroundBlackcurrant is a rich source of polyphenols with proven physiological activity, that have lately been classified as emerging prebiotics, making its extracts suitable for application in functional food and bioactive cosmetics. However, these biomolecules are trapped in cell wall structures and nowadays, enzymatic hydrolysis is recognised as powerful tool for extraction efficiency improvement in an environmentally sound manner. This study aimed to optimize enzyme-assisted extraction to maximize yields of blackcurrant polyphenols. In addition, their prebiotic potential was tested by examining their influence on typical skin microbiota representatives as model microorganisms—beneficial coagulase-negative Staphylococcus epidermidis and two opportunistic pathogens, Staphylococcus aureus and Cutibacterium acnes.ResultsAmong four examined commercial enzyme preparations, Viscozyme® L and Rohapect® MC, demonstrated the highest potential as extraction enhancers by increasing antioxidant activity, total polyphenol, and flavonoid contents. Furthermore, their synergetic effect enabled 95.3% increase in polyphenol concentration compared to conventional extraction with a maximum of 27.6 mg gallic acid equivalents (GAE)/g dry matter (DM) at a total enzyme concentration of 0.05 mL/g DM (Viscozyme® L to Rohapect® MC ratio 2:1) after 60 min. HPLC profiles of two samples showed differences in polyphenol composition indicating simultaneous extraction–biotransformation. At a concentration of 0.025 mg GAE/mL, PAS (prebiotic activity score) values were 0.096 and 0.172 for conventionally and enzymatically obtained extract, respectively. Moderate inhibitory effect of optimal extract concentration on Cutibacterium acnes was shown, as well. Hydrogel prepared with optimal extract concentration showed improved hydration effect, decreased irritability and increased dermatological compatibility comparing to basic formulation.ConclusionsOverall results demonstrated that blackcurrant extract obtained under optimized conditions using enzymes possesses an emerging prebiotic potential for selective skin microbiota stimulation.Graphical

Antifungal peptides from faba bean flour fermented by Levilactobacillus brevis AM7 improve the shelf-life of composite faba-wheat bread

Faba bean is an important crop from an ecological, nutritional, and economical perspective, hence its incorporation in plant-based products can play a key role in the sustainability of the whole food system. In this study, the antifungal properties of faba bean flour were investigated employing three bioprocessing treatments including hydrolysis with a commercial proteolytic enzyme preparation, and fermentation with selected or commercial mixed sourdough starters. Faba bean flour fermented by Levilactobacillus brevis AM7 showed the broadest inhibitory spectrum against the fungal species tested and antifungal activity ranging from 25 to 73 %. Bioactive proteins and peptides were found to be responsible for the antifungal activity. Polypeptides sequences corresponding to defensin-like and non-specific lipid-transfer proteins and seven antifungal peptides having 11–22 amino acid residues and mass from 1240.7 to 2624.2 Da, were identified via Liquid Chromatography-Electrospray Ionisation-Mass Spectra/Mass Spectra (nano-LC-ESI-MS/MS). To assess the antifungal effect in food processing conditions, fermented faba bean was used as ingredient in breadmaking at a substitution level of 30 % of the dough weight. Wheat composite-bread containing faba bean sourdough, which could be claimed as “source of proteins”, according to the current EC Regulation, delayed the molds contamination, lasting up to 10 days before mold appearance, and behaving similarly to wheat bread containing calcium propionate.

Short-Chain Fructooligosaccharide Synthesis from Sugarcane Syrup with Commercial Enzyme Preparations and Some Physical and Antioxidation Properties of the Syrup and Syrup Powder.

Short-chain fructooligosaccharides (sc-FOS) are prebiotics beneficial to human health, which can be synthesized from raw material containing a high sucrose content. Sugarcane syrup (SS) without molasses removal contains sucrose as a major sugar and is a rich source of several bioactive compounds. The aim of this study is to investigate factors affecting sc-FOS synthesis from SS using commercial enzyme preparations containing fructosyltransferase activity as biocatalysts. sc-FOS content increased significantly as the sucrose concentration of SS in the reaction mixture increased up to 40% (w/v). Changes in carbohydrate compositions during the transfructosylating reaction of a pure sucrose solution and SS prepared from the two sugarcane cultivars Khon Kaen 3 and Suphanburi 50, catalyzed by Pectinex Ultra SP-L and Viscozyme L, showed similar profiles. Both enzymes showed a high ability to transfer fructosyl moieties to produce sc-FOS and a plateau of the total sc-FOS concentration was observed after 4 h of reaction time. For the pure sucrose solution and SS (Suphanburi 50), Viscozyme showed a superior ability to convert sucrose to Pectinex, with a higher sc-FOS yield (g FOS/100 g of initial sucrose), GF2 or 1-kestose yield (g GF2/g of initial sucrose) and GF3 or nystose yield (g GF3/g of initial sucrose). sc-FOS syrup (FOS SS) and the foam-mat-dried syrup powder prepared from SS and FOS SS, respectively, contained a high total phenolic content and possessed higher antioxidant activities than those prepared from pure sucrose, but contained lower calories.

Protein preparations from rapse processing waste: A review of the current status and development prospects of existing technologies

The demand for protein products is increasing due to the demographic growth of the world’s population. As an alternative to traditional sources of protein, waste from plant raw material processing is becoming increasingly popular. An important place in the global economy is occupied by oilseeds, in particular rapeseed, which production volumes are increasing in the Russian Federation every year. Rapeseed (Brassicaceae napus) is of great interest due to its high oil content (39.80–46.00%) and rich fatty acid composition, while cake and meal formed in the process of oil production are characterized by a significant content of crude protein (35.00–45.00%) and crude fiber (8.20–17.50%); however, they are used mainly as a feed additive. Recent studies on the processing of rapeseed waste indicate the value of this raw material as a source of dietary protein, which has a balanced amino acid profile and a high degree of digestibility (up to 85%). To obtain protein, rapeseed processing is envisaged: cleaning, grinding, cold pressing at a temperature of ≤ 40 °C, fat extraction with a solvent. At the next stages, the protein is extracted with 0.1–0.5 M NaCl at pH 5.3–12.0 and a temperature of 5–30 °C for 1 hour. The extracted protein is precipitated at the isoelectric point (pH 4.0) with HCl, separated from the mixture and neutralized. The result is a protein isolate with a protein content of 90.0–98.7%. It is possible to increase the quality and yield of the protein product due to the additional stage of processing the defatted cake with cellulolytic enzyme preparations. In this case, additional studies are required to determine the substrate specificity of commercial cellulase enzyme preparations and the optimal hydrolysis conditions. The parameters of extraction and precipitation of the protein in the case of using the stage of enzymatic lysis should also be specified.

The effect of different wheat varieties and exogenous xylanase on bird performance and utilization of energy and nutrients

The aims of the present study were to first, determine the xylan fractions of 10 different wheat cultivar samples and their response to treatment by the same commercial xylanase enzyme preparation. Second, use information obtained to select 5 of the wheats for use within a feeding experiment to determine whether the rate of xylan release can be used to predict the feeding value of the wheats when diets have been supplemented with xylanase. Treatment of 10 different wheat varieties by the same enzyme resulted in varying levels of hydrolysis. Soluble xylan content ranged from 7.85 to 14.40 and 3.20 to 5.13 (mg/g) when treated with and without xylanase, respectively. Oligosaccharide content ranged from 0.34 to 1.58 and 0.05 to 0.54 (mg/g) when treated with and without xylanase, respectively. Five of the 10 wheats were then selected based on the determined xylan fractions to use within a feeding experiment. A total of 360 male Ross 308 broilers were randomly allocated to 60 raised floor pens. A soybean meal (SBM) balancer feed was formulated to contain 12.07 MJ/kg apparent metabolizable energy (AME) and 392.9 g/kg crude protein (CP). Five diets were prepared by mixing 630 g/kg of each of the 5 experimental wheats with 370 g/kg of the balancer. Each diet was split into 2, one of which was supplemented with 100 g/MT of Econase XT (223,000 BXU/g), resulting in a total of 10 diets. The birds were fed the diets from 0 to 28 d of age. Wheat cultivar had an effect (P=0.044) on feed intake (FI), while the addition of xylanase increased (P < 0.05) weight gain (WG) and improved feed conversion ratio (FCR). Various interactions were observed (P < 0.05) between wheat cultivars and xylanase for AME and nutrient utilization. This study suggests that wheats treated with the same xylanase, differ in their susceptibility to release soluble xylan and oligosaccharides, which may partially explain the varying performance and nutrient digestibility responses noted in the literature.

Recent advances in substrate-enzyme interactions facilitating efficient biodegradation of lignocellulosic biomass: A review

Lignocellulosic biorefinery has long been recognized as a potential strategy for sustainable production of biofuels, materials and chemicals. These productions in many cases rely on pretreatment and enzymatic hydrolysis of lignocellulosic carbohydrates, whereas the low degradation efficiency and high enzyme dosage are challenging bottlenecks of economically industrial production. The complex recalcitrant nature of lignocellulose requires a well-matched enzyme cocktail to achieve complete and efficient degradation. Numerous studies have investigated the relationship between biomass components, structural properties and enzymes synergistic action in the past decades. This article aims to present a comprehensive review on this subject by introducing recent advances in substrate modification techniques, underlying mechanisms of enzyme synergism and governing factors in structure-activity interaction. In addition, newly developed modellings and multi-omic analysis techniques for preparing commercial enzyme preparations are introduced, revealing possible avenues for achieving economic viability of lignocellulose bioconversion.

Nanomolar clodronate induces adenosine accumulation in the perfused rat mesenteric bed and mesentery-derived endothelial cells.

The vesicular nucleotide transporter (VNUT) is critical for sympathetic co-transmission and purinergic transmission maintenance. To examine this proposal, we assessed whether the bisphosphonate clodronate, claimed as a potent in vitro VNUT blocker, modified spontaneous and/or the electrically evoked overflow of ATP/metabolites and NA from mesentery sympathetic perivascular nerve terminals. Additionally, in primary endothelial cell cultures derived from this tissue, we also evaluated whether clodronate interfered with ATP/metabolite cell outflow and metabolism of N6-etheno adenosine 5'-triphosphate (eATP), N6-etheno adenosine (eADO), and adenosine deaminase enzyme activity. Rat mesenteries were perfused in the absence or presence of .01-1,000nM clodronate, 1-1,000nM Evans blue (EB), and 1-10µM DIDS; tissue perfusates were collected to determine ATP/metabolites and NA before, during, and after perivascular electrical nerve terminal depolarization. An amount of 1-1,000nM clodronate did not modify the time course of ATP or NA overflow elicited by nerve terminal depolarization, and only 10nM clodronate significantly augmented perfusate adenosine. Electrical nerve terminal stimulation increased tissue perfusion pressure that was significantly reduced only by 10nM clodronate [90.0 ± 18.6 (n = 8) to 35.0 ± 10.4 (n = 7), p = .0277]. As controls, EB, DIDS, or reserpine treatment reduced the overflow of ATP/metabolites and NA in a concentration-dependent manner elicited by nerve terminal depolarization. Moreover, mechanical stimulation of primary endothelial cell cultures from the rat mesentery added with 10 or 100nM clodronate increased adenosine in the cell media. eATP was metabolized by endothelial cells to the same extent with and without 1-1,000nM clodronate, suggesting the bisphosphonate did not interfere with nucleotide ectoenzyme metabolism. In contrast, extracellular eADO remained intact, indicating that this nucleoside is neither metabolized nor transported intracellularly. Furthermore, only 10nM clodronate inhibited (15.5%) adenosine metabolism to inosine in endothelial cells as well as in a commercial crude adenosine deaminase enzyme preparation (12.7%), and both effects proved the significance (p < .05). Altogether, present data allow inferring that clodronate inhibits adenosine deaminase activity in isolated endothelial cells as in a crude extract preparation, a finding that may account for adenosine accumulation following clodronate mesentery perfusion.

Cyclic Production of Galacto-Oligosaccharides through Ultrafiltration-Assisted Enzyme Recovery

Galacto-oligosaccharides (GOS) are prebiotics manufactured enzymatically from lactose as substrate. The growing GOS market facilitates the valorization of dairy by-products which represent cheap and abundant sources of lactose. Large-scale GOS production typically employs soluble enzymes in batch reactors that are commonly associated with low enzyme usability and, therefore, high operational expenditures. In this study, we investigate the possibility of recovering enzymes by ultrafiltration (UF) and reusing them in repeated reaction steps. The proposed process scheme included 24 h batch reaction steps with Biolacta N5, a commercial enzyme preparation of Bacillus circulans origin. The reaction steps were followed by UF steps to separate the carbohydrate products from the enzymes by applying a volume concentration factor of 8.6. Then, the collected biocatalysts were reused for repeated cycles by adding fresh lactose. Enzyme losses were quantified with a direct method by analyzing the underlying relationship between reaction rates and enzyme dosage obtained from additional experiments conducted with known enzyme loads. Within five cycles, the enzyme activity declined gradually from 923 to 8307 U·kg−1, and the half-life was estimated as ca. 15.3 h. The outcomes of this study may serve as a basis for further optimization of the reported process scheme with enhanced enzyme usability.

Determination of vitamin B12 in commercial pepsin preparations using immunoaffinity chromatography and LC‐MS/MS

AbstractVitamin B12 is a critical micronutrient in a vegan diet and thus dietary sources are limited and often low in the vitamin. Analytical determination of vitamin B12 requires laborious sample preparations including the proteolytic breakdown of the (food) matrix with proteases and the enrichment of the vitamin via immunoaffinity methods. Proteases like pepsin have an animal origin and thus might contain significant amounts of vitamin B12. We hypothesize that pepsin preparations potentially transfer residual vitamin B12 into the sample and falsify results. Hence the aim of our study was to determine trace amounts of cyano‐cobalamin by LC‐MS/MS in commercial protease enzyme preparations. The use of a stable 15N‐labeled cyano‐cobalamin as internal standard enabled us to detect vitamin B12 with a LOQ as low as 0.7 µg/l. Significant amounts of the vitamin were found in pepsin whereas the vitamin B12 content of papain preparations was below the detection limit. We conclude that sample preparation with pepsin bears the risk of false positive results, especially for samples with low vitamin B12 content, and we therefore recommend papain as an alternative protease for food sample preparation.

Continuous production of fructo-oligosaccharides using selectively immobilized fructosyltransferase from Aspergillus aculeatus onto Purolite® A109

Within this study, the investigation of potential selective immobilization of fructosyltransferase (FTase) derived from Aspergillus aculeatus (from commercial enzyme preparation Pectinex® Ultra SP-L) onto the polystyrenic macroporous resin Purolite® A109 and its further utilization for the synthesis of a highly valuable group of prebiotic compounds named fructo-oligosaccharides (FOS), was performed. The obtained results suggest that partial purification of fructosyltransferase from complex enzyme mixture with predominant pectinase activity (ratio of activity immobilization yields around 20.0) occurred by performing a simple immobilization protocol, provided that immobilization lasted for 5 h using an enzyme concentration of 50 mg g−1 of support under slightly acidic conditions (pH 6.0). Immobilized preparation was thereafter successfully utilized for FOS synthesis (56–59 % FOS of total sugars) in both a batch and air-lift reactor system. Operational stability study proved the exceptionally high potential of immobilized preparation for reproducible and cost-effective application, since 90 % of initial activity was preserved after ten consecutive reaction cycles. Thereafter, a prospectus for continuous production of FOS in the air-lift reactor was assayed. Achieved yields, 53 % FOS of total sugars for at least 9 h, indicated the great suitability of the obtained immobilized preparation for application in large-scale systems.

T-cells in human trigeminal ganglia express canonical tissue-resident memory T-cell markers

BackgroundTrigeminal ganglia (TG) neurons are the main site of lifelong latent herpes simplex virus type 1 (HSV-1) infection. T-cells in ganglia contribute to long-term control of latent HSV-1 infection, but it is unclear whether these cells are bona fide tissue-resident memory T-cells (TRM). We optimized the processing of human post-mortem nervous tissue to accurately phenotype T-cells in human TG ex vivo and in situ.MethodsPeripheral blood mononuclear cells (PBMC; 5 blood donors) were incubated with several commercial tissue digestion enzyme preparations to determine off-target effect on simultaneous detection of 15 specific T-cell subset markers by flow cytometry. Next, optimized enzymatic digestion was applied to ex vivo phenotype T-cells in paired PBMC, normal appearing white matter (NAWM) and TG of 8 deceased brain donors obtained < 9 h post-mortem by flow cytometry. Finally, the phenotypic and functional markers, and spatial orientation of T-cells in relation to neuronal somata, were determined in TG tissue sections of five HSV-1-latently infected individuals by multiparametric in situ analysis.ResultsCollagenase IV digestion of human nervous tissue was most optimal to obtain high numbers of viable T-cells without disrupting marker surface expression. Compared to blood, majority T-cells in paired NAWM and TG were effector memory T-cells expressing the canonical TRM markers CD69, CXCR6 and the immune checkpoint marker PD1, and about half co-expressed CD103. A trend of relatively higher TRM frequencies were detected in TG of latently HSV-1-infected compared to HSV-1 naïve individuals. Subsequent in situ analysis of latently HSV-1-infected TG showed the presence of cytotoxic T-cells (TIA-1+), which occasionally showed features of proliferation (KI-67+) and activation (CD137+), but without signs of degranulation (CD107a+) nor damage (TUNEL+) of TG cells. Whereas majority T-cells expressed PD-1, traits of T-cell senescence (p16INK4a+) were not detected.ConclusionsThe human TG represents an immunocompetent environment in which both CD4 and CD8 TRM are established and retained. Based on our study insights, we advocate for TRM-targeted vaccine strategies to bolster local HSV-1-specific T-cell immunity, not only at the site of recurrent infection but also at the site of HSV-1 latency.

Digestion of protein and toxic gluten peptides in wheat bread, pasta and cereal and the effect of a supplemental enzyme mix.

There has been an increasing interest in the relationship between wheat digestibility and potential toxicity to the host. However, there is a lack of understanding about temporal profile of digestion of wheat proteins from different food matrices under physiologically relevant conditions. In this study, digestion of three wheat-based foods (bread, pasta and cereal) was conducted based on the INFOGEST semi-dynamic protocol in the absence and presence of a commercial supplemental enzyme preparation (a Glutalytic® based supplement, which will be marketed as Elevase®). Protein hydrolysis (OPA- ortho-phthalaldehyde - assay), molecular weight distribution (SEC-HPLC) and potential toxicity (R5 antibody-based competitive ELISA), were assessed. Our results demonstrated that under normal conditions, the complexity of the food influenced the temporal profile of protein hydrolysis and gluten breakdown throughout simulated gastric and intestinal digestion. However, treatment with the enzyme supplement significantly and acutely increased protein hydrolysis and gluten degradation in the gastric stage, and this enhanced digestion was maintained into the intestinal environment. These findings highlight the limitations of temporal gastric proteolysis and gluten degradation under normal conditions to different food types. They also show that supplemental enzyme mixes can effectively accelerate the breakdown of protein and hydrolysis of toxic gliadin fractions from the early stages of gastric digestion, thereby reducing intestinal exposure and potentially limiting the sensitization of the host.