An overview on fermentation strategies to overcome lignocellulosic inhibitors in second-generation ethanol production using cell immobilization.

The development of technologies to ferment carbohydrates (mainly glucose and xylose) obtained from the hydrolysis of lignocellulosic biomass for the production of second-generation ethanol (2G ethanol) has many economic and environmental advantages. The pretreatment step of this biomass is industrially performed mainly by steam explosion with diluted sulfuric acid and generates hydrolysates that contain inhibitory compounds for the metabolism of microorganisms, harming the next step of ethanol production. The main inhibitors are: organic acids, furan, and phenolics. Several strategies can be applied to decrease the action of these compounds in microorganisms, such as cell immobilization. Based on data published in the literature, this overview will address the relevant aspects of cell immobilization for the production of 2G ethanol, aiming to evaluate this method as a strategy for protecting microorganisms against inhibitors in different modes of operation for fermentation. This is the first overview to date that shows the relation between inhibitors, cells immobilization, and fermentation operation modes for 2G ethanol. In this sense, the state of the art regarding the main inhibitors in 2G ethanol and the most applied techniques for cell immobilization, besides batch, repeated batch and continuous fermentation using immobilized cells, in addition to co-culture immobilization and co-immobilization of enzymes, are presented in this work.

Bact-to-Batch: A Microbiota-Based Tool to Determine Optimal Animal Allocation in Experimental Designs.

The basis of any animal experimentation begins with the housing of animals that should take into account the need for splitting animals into similar groups. Even if it is generally recommended to use the minimum number of animals necessary to obtain reliable and statistically significant results (3Rs rule), the allocation of animals is currently mostly based on randomness. Since variability in gut microbiota is an important confounding factor in animal experiments, the main objective of this study was to develop a new approach based on 16S rRNA gene sequencing analysis of the gut microbiota of animals participating in an experiment, in order to correctly assign the animals across batches. For this purpose, a pilot study was performed on 20 mouse faecal samples with the aim of establishing two groups of 10 mice as similar as possible in terms of their faecal microbiota fingerprinting assuming that this approach limits future analytical bias and ensures reproducibility. The suggested approach was challenged with previously published data from a third-party study. This new method allows to embrace the unavoidable microbiota variability between animals in order to limit artefacts and to provide an additional assurance for the reproducibility of animal experiments.

Assessment of gas chromatography methodology approach for the trace evaluation of carcinogenic impurity, methyl chloride, in trimetazidine dihydrochloride.

OBJECTIVE: A simple and robust head space/gas chromatography with flame ionisation sensor (HS/GC/FIS) approach for the trace evaluation of carcinogenic impurity, methyl chloride, in trimetazidine dihydrochloride (TRD) drug ingredient and its formulation is described. METHOD: This HS/GC/FIS approach was based on separation and analysis of CH3Cl content on DB-624 [75.0m - length, 0.53mm - internal diameter, 3.0µm - film thickness] column using nitrogen as carrier gas flowing through the column at 3mL/min stream rate. Detection of eluted CH3Cl was accomplished with flame ionization sensor at a set temperature of 260̊C. RESULTS: The optimised HS/GC/FIS methodological approach was thoroughly validated, demonstrating that it was linear with range of 5.0ppm to 1508.4ppm, sensitive with detection limit of 1.65ppm and quantification limit of 5.01ppm, reproducible with RSD values of 2.10-2.35%, accurate with recoveries of 81.9-99.0%, robust with percent variation of 7.5-12.22% with respect to changes in oven temperature, injector temperature, detector temperature and practical for regular TRD quality control. CONCLUSION: The findings revealed that with this optimised HS/GC/FIS methodological approach, the trace amounts of carcinogenic impurity (methyl chloride) in TRD drug ingredient and formulation could be successfully measured.

Genomic evidence for stability of the Bacillus Calmette-Guérin (BCG) vaccine strain (Pasteur 1173P2) from different batches in Iran.

AIMS: Investigate the genetic stability of the BCG vaccine produced in Iran from different batches compared to the reference strain. METHODS AND RESULTS: We comparatively analyzed the whole genome sequences of the vaccine batches from different years. Eleven vials of different batches from 2010, 2018, and 2019 were included. Complete genome analyses revealed no difference between the old (2010) and new (2018 and 2019) vaccine batches. Additionally, minor genetic changes include five single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were observed compared to the BCG Pasteur 1173P2 reference strain, which were shared among all batches. Besides, the batches were identical to the reference strain in terms of antibiotic resistance genes, prophage sequences, and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems. CONCLUSIONS: High genetic stability of the BCG vaccine used in the national immunization program was confirmed, which indicates the optimal conditions in the vaccine production process. SIGNIFICANCE AND IMPACT OF THE STUDY: Genetic differences within and between vaccine strains have been declared as one of the main parameters related to the BCG vaccine variable protective efficacy. No study has been done to investigate the genetic variations of the vaccine batches at the single-base level.

[Dissolution test and evaluation of Guizhi Fuling Capsules].

Because of the complex components, simple content determination can hardly reflect the overall quality of Guizhi Fuling Capsules. Therefore, it is necessary to carry out a multi-component dissolution test. The variability of quality among different batches of products from different manufacturers is a common problem of Chinese medicine solid preparations. To comprehensively control the quality of Guizhi Fuling Capsules, we studied the dissolution behaviors of 7 index components in the capsules under different conditions, and investigated the consistency of dissolution behaviors among different batches of products from the same manufacturer. The basket method of general rule 0931 in Chinese Pharmacopoeia was adopted, and the rotating speeds were set at 50, 75, and 100 r·min~(-1), respectively. The hydrochloric acid solution(pH 1.2), acetate buffer solution(pH 4.0), pure water, and phosphate buffer solution(pH 6.8) were used as the dissolution media. Automatic sampling was carried out at the time points of 5, 10, 20, 30, 45, and 60 min, respectively. The cumulative dissolution of 7 index components was measured through ultra-performance liquid chromatography(UPLC). The difference factor f_1 and similarity factor f_2 were calculated to comprehensively evaluate the similarity of the dissolution curves among 8 batches of Guizhi Fuling Capsules, and a variety of dissolution and release equations were fitted. The results showed that multiple components had faster dissolution rates at higher rotating speed and in hydrochloric acid medium. The 8 batches of Guizhi Fuling capsules showed the average f_1 value lower than 15 and the average f_2 value higher than 50, which indicated that different batches of products had similar dissolution behaviors. Most components had synchronous dissolution behaviors and similar release cha-racteristics. This study provides a reference for the quality consistency evaluation among batches, processing optimization, and dosage form improvement of Guizhi Fuling Capsules.

Importance of Block Randomization When Designing Proteomics Experiments.

Randomization is used in experimental design to reduce the prevalence of unanticipated confounders. Complete randomization can however create imbalanced designs, for example, grouping all samples of the same condition in the same batch. Block randomization is an approach that can prevent severe imbalances in sample allocation with respect to both known and unknown confounders. This feature provides the reader with an introduction to blocking and randomization, and insights into how to effectively organize samples during experimental design, with special considerations with respect to proteomics.

A Glimpse into the Microbiota of Marketed Ready-to-Eat Crickets (Acheta domesticus).

The present study was aimed to get an insight into the bacterial biota of ready-to-eat small crickets (Acheta domesticus) already marketed in the European Union. 16S rRNA gene of the DNAs extracted from thirty-two samples of ready-to-eat crickets commercialized by 4 European Union producers located in Austria, Belgium, France and the Netherlands (2 batches per producer) was analyzed by Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE). The species belonging to the genera Hespellia, Ruminococcus and Clostridium were detected in samples from Austria, while those from genera Lysobacter, Staphylococcus and Clostridium were detected in samples from Belgium. Moreover, samples from France were characterized by Staphylococcus, Pseudomonas, and Hydrogenophilus genera. Finally, the genera Staphylococcus, Hydrogenophilus, Clostridium and Ruminococcus were identified in the samples produced in the Netherlands. When insects are intended for commercialization, rearing, processing and handling could affect the presence of the occurring microbial species. Hence, to assure a safe product, the need for a full standardization of production technologies, including feed supply as well as rearing and processing practices, is recommended.

Strategies for synchronizing chocolate conching batch process data using dynamic time warping.

In batch processing, process control is typically carried out comparing trajectories of process variables with those in an in-control set of batches that yielded products within specifications. However, one strong assumption of these schemes is that all batches have equal duration and are synchronized, which is often not satisfied in practice. To overcome that, dynamic time warping (DTW) methods may be used to synchronize stages and align the duration of batches. In this paper, three DTW methods are compared using supervised classification through the k-nearest neighbor technique to determine the in-control set in a milk chocolate conching process. Four variables were monitored over time and a set of 62 batches with durations between 495 and 1170 min was considered; 53% of the batches were known to be conforming based on lab test results and experts' evaluations. All three DTW methods were able to promote the alignment and synchronization of batches; however, the KMT method (Kassidas et al. in AIChE J 44(4):864-875, 1998) outperformed the others, presenting 93.7% accuracy, 97.2% sensitivity, and 90.3% specificity in batch classification as conforming and non-conforming. The drive current of the main motor was the most consistent variable from batch to batch, being deemed the most important to promote alignment and synchronization of the chocolate conching dataset.

Bayesian assurance and sample size determination in the process validation life-cycle.

Validation of pharmaceutical manufacturing processes is a regulatory requirement and plays a key role in the assurance of drug quality, safety, and efficacy. The FDA guidance on process validation recommends a life-cycle approach which involves process design, qualification, and verification. The European Medicines Agency makes similar recommendations. The main purpose of process validation is to establish scientific evidence that a process is capable of consistently delivering a quality product. A major challenge faced by manufacturers is the determination of the number of batches to be used for the qualification stage. In this article, we present a Bayesian assurance and sample size determination approach where prior process knowledge and data are used to determine the number of batches. An example is presented in which potency uniformity data is evaluated using a process capability metric. By using the posterior predictive distribution, we simulate qualification data and make a decision on the number of batches required for a desired level of assurance.

Harmonizing multisite data with the ComBat method for enhanced Parkinson's disease diagnosis via DAT-SPECT.

Background: Dopamine transporter single-photon emission computed tomography (DAT-SPECT) is a crucial tool for evaluating patients with Parkinson's disease (PD). However, its implication is limited by inter-site variability in large multisite clinical trials. To overcome the limitation, a conventional prospective correction method employs linear regression with phantom scanning, which is effective yet available only in a prospective manner. An alternative, although relatively underexplored, involves retrospective modeling using a statistical method known as "combatting batch effects when combining batches of gene expression microarray data" (ComBat). Methods: We analyzed DAT-SPECT-specific binding ratios (SBRs) derived from 72 healthy older adults and 81 patients with PD registered in four clinical sites. We applied both the prospective correction and the retrospective ComBat correction to the original SBRs. Next, we compared the performance of the original and two corrected SBRs to differentiate the PD patients from the healthy controls. Diagnostic accuracy was assessed using the area under the receiver operating characteristic curve (AUC-ROC). Results: The original SBRs were 6.13 ± 1.54 (mean ± standard deviation) and 2.03 ± 1.41 in the control and PD groups, respectively. After the prospective correction, the mean SBRs were 6.52 ± 1.06 and 2.40 ± 0.99 in the control and PD groups, respectively. After the retrospective ComBat correction, the SBRs were 5.25 ± 0.89 and 2.01 ± 0.73 in the control and PD groups, respectively, resulting in substantial changes in mean values with fewer variances. The original SBRs demonstrated fair performance in differentiating PD from controls (Hedges's g = 2.76; AUC-ROC = 0.936). Both correction methods improved discrimination performance. The ComBat-corrected SBR demonstrated comparable performance (g = 3.99 and AUC-ROC = 0.987) to the prospectively corrected SBR (g = 4.32 and AUC-ROC = 0.992) for discrimination. Conclusion: Although we confirmed that SBRs fairly discriminated PD from healthy older adults without any correction, the correction methods improved their discrimination performance in a multisite setting. Our results support the utility of harmonization methods with ComBat for consolidating SBR-based diagnosis or stratification of PD in multisite studies. Nonetheless, given the substantial changes in the mean values of ComBat-corrected SBRs, caution is advised when interpreting them.

Reducing manufacturing carbon emissions: Optimal low carbon production strategies respect to product structures and batches.

In the production process of industrial products, different product structures, batches, and the selection of different production methods directly affect the resource utilization, distribution, consumption, and carbon emission generation in the production process. In this study, a strategy to select low carbon production methods for product structure and batch is proposed to advance resource management and carbon emission reduction in manufacturing production processes. Specifically, taking a typical casting industry as an example, we analyze the two factors of product structures and batches on the resource consumption and environmental impact of the production process to establish a production process carbon emission model; using forty casting products as the research objects, the clustering algorithm and multiple linear regression analysis method are used to establish the influence relationship between product structure, batch, and production carbon emissions. Based on the characteristics of product structure and batch, a strategy is proposed for selecting a low carbon production method. The study shows that in sand casting production, the 3D printing method is more low carbon for small volumes, reducing 56.057 % of carbon emissions. However, traditional technology is more low carbon for large volumes, which can reduce at least 6.778 % of carbon emissions. In addition, as the number of casting batches increases, the advantage of low carbon in traditional casting technology will rise. The results of this study may provide a new way to help the manufacturing industry develop and optimize the environmental impact of the product manufacturing process.

Cell Immobilization for Erythritol Production.

Nowadays, commercial erythritol synthesis is performed by free-cell fermentation with fungi in liquid media containing high concentrations of pure carbon sources. Alternative fermentation techniques, such as cell immobilization, could imply an economic and energetic improvement for erythritol-producing factories. The present work describes, for the first time, the feasibility of achieving cell immobilization during erythritol production. Cells of the fungus Moniliella pollinis were successfully immobilized on a cotton cloth which was placed inside a 2-L bioreactor, where they were fed with red grape must supplemented with yeast extract. They produced 47.03 ± 6.16 g/L erythritol in 96 h (yield 0.18 ± 0.04 g/g) over four consecutive fermentation batches. The immobilized cells remained stable and operative during a 456 h period. The erythritol concentration attained was similar (p > 0.05; Tukey HSD test) to the reference value obtained with the use of free cells (41.88 ± 5.18 g/L erythritol) under the same fermentation conditions. The comparable results observed for free and immobilized cells evidences the efficiency of the immobilization system. Therefore, the proposed method for erythritol bioproduction eliminates the need for the continuous preparation of fungal inocula before each fermentation batch, thus reducing the costs of the reagents and energy.

Sustainable isomaltulose production in Corynebacterium glutamicum by engineering the thermostability of sucrose isomerase coupled with one-step simplified cell immobilization.

Sucrose isomerase (SI), catalyzing sucrose to isomaltulose, has been widely used in isomaltulose production, but its poor thermostability is still resisted in sustainable batches production. Here, protein engineering and one-step immobilized cell strategy were simultaneously coupled to maintain steady state for long-term operational stabilities. First, rational design of Pantoea dispersa SI (PdSI) for improving its thermostability by predicting and substituting the unstable amino acid residues was investigated using computational analysis. After screening mutagenesis library, two single mutants (PdSIV280L and PdSIS499F) displayed favorable characteristics on thermostability, and further study found that the double mutant PdSIV280L/S499F could stabilize PdSIWT better. Compared with PdSIWT, PdSIV280L/S499F displayed a 3.2°C-higher T m , and showed a ninefold prolonged half-life at 45°C. Subsequently, a one-step simplified immobilization method was developed for encapsulation of PdSIV280L/S499F in food-grade Corynebacterium glutamicum cells to further enhance the recyclability of isomaltulose production. Recombinant cells expressing combinatorial mutant (RCSI2) were successfully immobilized in 2.5% sodium alginate without prior permeabilization. The immobilized RCSI2 showed that the maximum yield of isomaltulose by batch conversion reached to 453.0 g/L isomaltulose with a productivity of 41.2 g/l/h from 500.0 g/L sucrose solution, and the conversion rate remained 83.2% after 26 repeated batches.

Action potential variability in human pluripotent stem cell-derived cardiomyocytes obtained from healthy donors.

Human pluripotent stem cells (PSC) have been used for disease modelling, after differentiation into the desired cell type. Electrophysiologic properties of cardiomyocytes derived from pluripotent stem cells are extensively used to model cardiac arrhythmias, in cardiomyopathies and channelopathies. This requires strict control of the multiple variables that can influence the electrical properties of these cells. In this article, we report the action potential variability of 780 cardiomyocytes derived from pluripotent stem cells obtained from six healthy donors. We analyze the overall distribution of action potential (AP) data, the distribution of action potential data per cell line, per differentiation protocol and batch. This analysis indicates that even using the same cell line and differentiation protocol, the differentiation batch still affects the results. This variability has important implications in modeling arrhythmias and imputing pathogenicity to variants encountered in patients with arrhythmic diseases. We conclude that even when using isogenic cell lines to ascertain pathogenicity to variants associated to arrythmias one should use cardiomyocytes derived from pluripotent stem cells using the same differentiation protocol and batch and pace the cells or use only cells that have very similar spontaneous beat rates. Otherwise, one may find phenotypic variability that is not attributable to pathogenic variants.

Discussion on several statistical problems in establishing quality standards of standard decoctions.

Since 2016, a number of studies have been published on standard decoctions used in Chinese medicine. However, there is little research on statistical issues related to establishing the quality standards for standard decoctions. In view of the currently established quality standard methods for standard decoctions, an improvement scheme is proposed from a statistical perspective. This review explores the requirements for dry matter yield rate data and index component transfer data for the application of two methods specified in "Technical Requirements for Quality Control and Standard Establishment of Chinese Medicine Formula Granules," which include the average value plus or minus three times the standard deviation ( X - ± 3 S D ) or 70% to 130% of the average value ( X - ± 30 % X - ). The square-root arcsine transformation method is used as an approach to solve the problem of unreasonable standard ranges of standard decoctions. This review also proposes the use of merged data to establish a standard. A method to judge whether multiple sets of standard decoction data can be merged is also provided. When multiple sets of data have a similar central tendency and a similar discrete tendency, they can be merged to establish a more reliable quality standard. Assuming that the dry matter yield rate and transfer rate conform to a binomial distribution, the number of batches of prepared slices that are needed to establish the standard decoction quality standard is estimated. It is recommended that no less than 30 batches of prepared slices should be used for the establishment of standard decoction quality standards.

A new strategy based on PCA for inter-batches quality consistency evaluation.

Due to cultivation position, climate, harvest times, storage conditions and processing method, the evaluation of intra- and inter- batches quality consistency of botanical drugs has always been a thorny problem since it concerns safety and efficacy. The combination of fingerprint based on instrumental analysis and chemometrics is a common evaluation method in recent years. The differences between groups can be judged intuitively and superficially through principal component analysis (PCA) multi-dimensional score plots, but there is a lack of scientific and quantitative index to quantify the differences between groups. How to quantify the difference between groups is basically a blank area of research. Based on traditional F-statistic, we proposed a new F*-statistic to quantify the difference between groups in PCA score plots from the perspective of statistics. As the results revealed, the calculated F*-statistic was 2.58, smaller than the critical value 3.17 (α = 0.05), which indicated that there was no significant difference between groups. Our study add another dimension for PCA application, which offers a new strategy to quantify differences between groups by a new perspective, namely, a combination of fingerprint, chemometrics and statistics to evaluate inter-batches quality consistency quantitatively and objectively. Therefore, this manuscript could provide new ideas and technical references for the quality consistency evaluation of natural drugs, thus better guarantee their clinical efficacy and safety, and better promote industrial development.

In Vitro Selection of Probiotics, Prebiotics, and Antioxidants to Develop an Innovative Synbiotic (NatuREN G) and Testing Its Effect in Reducing Uremic Toxins in Fecal Batches from CKD Patients.

We aimed to develop an innovative synbiotic formulation for use in reducing dysbiosis, uremic toxins (e.g., p-cresol and indoxyl sulfate), and, consequently, the pathognomonic features of patients with chronic kidney disease (CKD). Twenty-five probiotic strains, belonging to lactobacilli and Bifidobacterium, were tested for their ability to grow in co-culture with different vegetable (pomegranate, tomato, and grapes) sources of antioxidants and prebiotics (inulin, fructo-oligosaccharides, and ß-glucans). Probiotics were selected based on the acidification rates and viable cell counts. Inulin and fructo-oligosaccharides reported the best prebiotic activity, while a pomegranate seed extract was initially chosen as antioxidant source. The investigation was also conducted in fecal batches from healthy and CKD subjects, on which metabolomic analyses (profiling volatile organic compounds and total free amino acids) were conducted. Two out of twenty-five probiotics were finally selected. After the stability tests, the selective innovative synbiotic formulation (named NatuREN G) comprised Bifidobacterium animalis BLC1, Lacticaseibacillus casei LC4P1, fructo-oligosaccharides, inulin, quercetin, resveratrol, and proanthocyanidins. Finally, NatuREN G was evaluated on fecal batches collected from CKD in which modified the viable cell densities of some cultivable bacterial patterns, increased the concentration of acetic acid and decane, while reduced the concentration of nonanoic acid, dimethyl trisulfide, and indoxyl sulfate.

Fraud Detection in Batches of Sweet Almonds by Portable Near-Infrared Spectral Devices.

One of the key challenges for the almond industry is how to detect the presence of bitter almonds in commercial batches of sweet almonds. The main aim of this research is to assess the potential of near-infrared spectroscopy (NIRS) by means of using portable instruments in the industry to detect batches of sweet almonds which have been adulterated with bitter almonds. To achieve this, sweet almonds and non-sweet almonds (bitter almonds and mixtures of sweet almonds with different percentages (from 5% to 20%) of bitter almonds) were analysed using a new generation of portable spectrophotometers. Three strategies (only bitter almonds, bitter almonds and mixtures, and only mixtures) were used to optimise the construction of the non-sweet almond training set. Models developed using partial least squares-discriminant analysis (PLS-DA) correctly classified 86-100% of samples, depending on the instrument used and the strategy followed for constructing the non-sweet almond training set. These results confirm that NIR spectroscopy provides a reliable, accurate method for detecting the presence of bitter almonds in batches of sweet almonds, with up to 5% adulteration levels (lower levels should be tested in future studies), and that this technology can be readily used at the main steps of the production chain.

Batch-to-batch variation in domestic paints: Insights into the newly commercialized recycled paints.

A few recent studies attempted to evaluate the differentiation of paints at a production batch level and reported results depending largely on the paint type. The discrimination from production batches is much more random than brands and/or models levels and subject to many unknowns, which suggests that a particular production batch can suddenly present a substantially different composition than the one produced right before or right after it. To add to this existing complexity, most of the paint companies now propose a range of recycled paints among their products. These recycled paints are composed of wastes collected by recycling plants, sorted by their color and binder type (i.e. latex, alkyds), and mixed together in large tanks to form the basis material for future formulations. Quality controls on these recycled batches are voluntarily less precise, and a higher variation is expected in esthetic and chemical properties of the paint. In this project, we collaborated with a North American paint producer that gave us access to its samples, paint formulations recipes, and a summary of the quality controls and corrections they performed on each production batches. The whole study was conducted blindfold and a final verification was made with the manufacturer to evaluate the accuracy of the results. The data set comprised two models of regular paints and two models of recycled paint. Five different production batches were collected per model for a total of 20 samples analyzed by Microscopy, Infrared spectroscopy, Raman spectroscopy, and Pyrolysis GC/MS.