Fermentation Protocol Research Articles

Microbial Fermentation and Shelf Life of Potential Biotechnological Products Capable of Pesticide Degradation

The pesticide active ingredient azoxystrobin is widely used in agriculture and has negative effects for the environment and contained organisms. Bacterial strains have been reported to degrade azoxystrobin, but precise methodologies for producing and storing these strains as potential biotechnological products are lacking. The study focused on creating and optimising a non-sterile, small-scale microbial fermentation protocol to produce azoxystrobin-degrading products and to test their shelf life. By testing 14 variants and sampling at three production and two storage time points, the trial demonstrated the successful production and storage of microbial products capable of pesticide degradation. Various measurement parameters such as pH value and organic acids were used to monitor the quality of the microbial products during the production and storage. Further, we developed and validated qPCR assays to rapidly and specifically assess the concentration of the two azoxystrobin degrading strains, namely Bacillus subtilis strain MK101 and Rhodococcus fascians strain MK144. To ensure good specificity, the combination of two qPCR assays targeting two different genome regions was implemented for each strain. The study highlights the significant impact of media selection and bacterial inoculum quantity on the microbial product quality.

Simultaneous inoculation of non-Saccharomyces yeast and lactic acid bacteria for aromatic kiwifruit wine production

To further explore strain potential and develop an aromatic kiwifruit wine fermentation technique, the feasibility of simultaneous inoculation by non-Saccharomyces yeast and lactic acid bacteria was investigated. Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, and Limosilactobacillus fermentum, which have robust β-glucosidase activity as well as good acid and ethanol tolerance, were inoculated for simultaneous fermentation with Zygosaccharomyces rouxii and Meyerozyma guilliermondii, respectively. Subsequently, the chemical compositions and sensory characteristics of the wines were comprehensively evaluated. The results showed that the majority of the simultaneous protocols effectively improved the quality of kiwifruit wines, increasing the content of polyphenols and volatile compounds, thereby enhancing sensory acceptability compared to the fermentation protocols inoculated with non-Saccharomyces yeast individually. Particularly, the collaboration between Lacp. plantarum and Z. rouxii significantly increased the diversity and content of esters, alcohols, and ketones, intensifying floral and seeded fruit odors, and achieving the highest overall acceptability. This study highlights the potential significance of simultaneous inoculation in kiwifruit wine production.

Brewing Beer in Microgravity: The Effect on Rate, Yeast, and Volatile Compounds

The exploration of space is becoming more feasible, and with this comes the possibility of performing fermentations in microgravity. Our study explores the potential effects of microgravity on a standard brewing model fermentation. As the fermentation of barley wort has been studied for centuries, there is an established foundation of knowledge with which to compare any changes that occur under microgravity. A modified ASBC miniature fermentation protocol (Yeast-14) was conducted within a Yuri 2.0 microgravity chamber to examine the response of Saccharomyces pastorianus to simulated microgravity conditions. Our findings reveal that yeast exhibited accelerated growth rates under microgravity compared to standard conditions. Additionally, the microgravity environment led to significantly lower levels of volatile compounds such as isoamyl acetate and 2-phenethyl acetate in the final product. Further genetic analysis showed significant downregulation of the ATF2 gene in the control group, potentially the mechanism behind the observed decrease in volatile compounds. These results show that while fermentation under microgravity is likely to eventually be commonplace, there may be changes in rate and gene expression that are beneficial or detrimental to the final product depending upon the desired characteristics.

Statistical optimization for comparative hydrolysis and fermentation for hemicellulosic ethanolgenesis

The concept of ‘Energy from waste’ is one of the most focused areas of work to find a solution for controlling trash and combat energy crises. In Pakistan and other agricultural countries, because of their substantial use during the summer, watermelon peels as fruit waste are usually thrown out as a trash. This study supported the management of huge quantities of waste to value-added products at a commercial scale. The current study aims to select and subject xylanolytic and ethanologenic Bacillus cereus XG2 for water melon peels valorization appropriately with comparison of three hydrolysis techniques. The study will be helpful for selection of economical and environmentally beneficial valorization strategies. For ethanalogenesis, separate hydrolysis and fermentation (SHF) protocols with Saccharomyces cerevisiae K7 and Metchnikowia cibodasensis Y34 were used. For hydrolysis, three different saccharification approaches, viz. dilute sulfuric acid, enzymatic hydrolysis (using Bacillus cereus XG2 xylanases), and combined acidic and enzymatic hydrolysis, were adopted. Two statistical models, Placket-Burman (hydrolysis) and Central composite design (ethanologenesis) were used. In untreated watermelon waste (WW), reducing sugar, total lipids, total carbohydrates, and protein contents were calculated as 16.70±0.05 g/L, 3.20±0.02 g/L, 28.7±0.04 g/L, and 3.70±0.03 g/L, respectively. Similarly, the lignin (15.51±0.22%), hemicellulose (17.20±2.30%). and cellulose (52.26±0.33%) contents were also analyzed. Based on the significance of the Plackett–Burman model for enzymatic saccharification, the released reducing sugars as well as total sugars were 21.62±0.01 g/L and 43.30±1.55 g/L, respectively, and enzymatic hydrolyzate was adopted for further fermentation experiments. By CCD model, the highest ethanol yield calculated for yeast Metchnikowia cibodasensis Y34 was 0.4±0.04 g/g of fermentable sugars at 32.5oC with 50% enzymatic hydrolysate of WW by incubating for 8 days. It was suggested that SHF could be a beneficial approach to increase the conversion of hemicellulose to fermentable sugars to produce bioethanol on a large scale.

Advances in the Quality Improvement of Fruit Wines: A Review

Fruit wines have gained great interest in recent years due to the increasingly diverse demands of consumers for different fruit wines with different colors, flavors, and nutritional values. Some fruits such as blueberry and strawberry are perishable and have a short shelf life. The production of fruit wine reduces fruit losses after harvest and enhances fruit utilization. The production of fruit wine with premium quality is determined by both intrinsic (i.e., genetic background) and extrinsic factors (e.g., yeast and fermentation protocol). This article provides an updated overview on the strategies and technologies aiming to improve the quality of fruit wines. Recent progress in improving fruit wine quality by variety selection, post-harvest treatments, yeast selection, fermentation protocols, fermentation conditions, and aging technologies has been comprehensively reviewed.

New neuroprotective derivatives of cinnamic acid by biotransformation.

Microbial transformation is extensively utilized to generate new metabolites in bulk amounts with more specificity and improved activity. As cinnamic acid was reported to exhibit several important pharmacological properties, microbial transformation was used to obtain its new derivatives with enhanced biological activities. By manipulating the 2-stage fermentation protocol of biotransformation, five metabolites were produced from cinnamic acid. Two of them were new derivatives; N-propyl cinnamamide 2̲ and 2-methyl heptyl benzoate 3̲ produced by Alternaria alternata. The other 3 metabolites, p-hydroxy benzoic acid 4̲, cinnamyl alcohol 5̲ and methyl cinnamate 6̲, were produced by Rhodotorula rubra, Rhizopus species and Penicillium chrysogeneum, respectively. Cinnamic acid and its metabolites were evaluated for their cyclooxygenase (COX) and acetylcholinesterase (AChE) inhibitory activities. Protection against H2O2 and Aβ1-42 induced-neurotoxicity in human neuroblastoma (SH-SY5Y) cells was also monitored. Metabolite 4̲ was more potent as a COX-2 inhibitor than the parent compound with an IC50 value of 1.85 ± 0.07 μM. Out of the tested compounds, only metabolite 2̲ showed AChE inhibitory activity with an IC50 value of 8.27 μM. These results were further correlated with an in silico study of the binding interactions of the active metabolites with the active sites of the studied enzymes. Metabolite 3̲ was more potent as a neuroprotective agent against H2O2 and Aβ1-42 induced-neurotoxicity than catechin and epigallocatechin-3-gallate as positive controls. This study suggested the two new metabolites 2̲ and 3̲ along with metabolite 4̲ as potential leads for neurodegenerative diseases associated with cholinergic deficiency, neurotoxicity or neuroinflammation.

Systematic profiling of ale yeast protein dynamics across fermentation and repitching.

Studying the genetic and molecular characteristics of brewing yeast strains is crucial for understanding their domestication history and adaptations accumulated over time in fermentation environments, and for guiding optimizations to the brewing process itself. Saccharomyces cerevisiae (brewing yeast) is among the most profiled organisms on the planet, yet the temporal molecular changes that underlie industrial fermentation and beer brewing remain understudied. Here, we characterized the genomic makeup of a Saccharomyces cerevisiae ale yeast widely used in the production of Hefeweizen beers, and applied shotgun mass spectrometry to systematically measure the proteomic changes throughout 2 fermentation cycles which were separated by 14 rounds of serial repitching. The resulting brewing yeast proteomics resource includes 64,740 protein abundance measurements. We found that this strain possesses typical genetic characteristics of Saccharomyces cerevisiae ale strains and displayed progressive shifts in molecular processes during fermentation based on protein abundance changes. We observed protein abundance differences between early fermentation batches compared to those separated by 14 rounds of serial repitching. The observed abundance differences occurred mainly in proteins involved in the metabolism of ergosterol and isobutyraldehyde. Our systematic profiling serves as a starting point for deeper characterization of how the yeast proteome changes during commercial fermentations and additionally serves as a resource to guide fermentation protocols, strain handling, and engineering practices in commercial brewing and fermentation environments. Finally, we created a web interface (https://brewing-yeast-proteomics.ccbb.utexas.edu/) to serve as a valuable resource for yeast geneticists, brewers, and biochemists to provide insights into the global trends underlying commercial beer production.

Fermentation, Identification, and Antioxidant Activity of Saponins Produced by a Wild Ginseng Endophytic Fungus Umbelopsis dimorpha Strain NSJG

Background: Endophytes from ginseng plants may produce saponins through in vitro fermentation, and some saponins possess antioxidant activity. This study aimed to enhance the total saponin yield of an endophytic fungus isolated from wild ginseng, determine saponin types, and explore whether saponin extracts from optimized fermentation protocols have antioxidant activity. Methods: Umbelopsis dimorpha strain NSJG was fermented in liquid under different conditions. The types of saponins were analyzed by Liquid Chromatography/Mass Spectrometry (LC/MS). The antioxidant activity of saponin extracts was evaluated by measuring their hemolytic inhibition rate on erythrocytes. Results: The endophytic fungus strain NSJG produced 1.236 mg/mL total saponins through liquid potato dextrose (PD) fermentation. The total saponin concentrations of optimal protocols G-ED-8 (3.107 mg/mL) and G-DP-8 (2.045 mg/mL) were three times and twice that of G-PD. Saponin extracts from G-ED-8 and G-DP-8 contained seven and eight types of ginsenosides and had higher hemolysis inhibition rates on erythrocytes than Vitamin C (VC), which were 36.05% and 46.69%. Conclusions: The research highlights the potential implications of wild ginseng endophytic fungus strain NSJG for the in vitro production of ginsenosides and the development of novel antioxidants.

Optimization of Fermentation Conditions and Product Identification of a Saponin-Producing Endophytic Fungus.

Some fungal endophytes isolated from P. ginseng may present a new method of obtaining saponins. This experiment aimed to optimize the total saponin yield produced through in vitro fermentation by an endophytic fungus and analyze its saponin species in the fermented extract. Fermentation protocols were optimized with a uniform design and verified through regression analysis to maximize the total saponin yield. The saponin types under optimal fermentation conditions were then identified and analyzed using Liquid Chromatography-Mass Spectrometry. The Trametes versicolor strain NSJ105 (gene accession number: OR144428) isolated from wild ginseng could produce total saponins. The total saponin yield could be increased more than two-fold through the optimization of fermentation conditions. The concentration of the total saponins achieved by the verified protocol 105-DP was close to the predicted value. The fermentation conditions of the 105-DP protocol were as follows: potato concentration 97.3 mg/mL, glucose concentration 20.6 mg/mL, inoculum volume 2.1%, fermentation broth pH 2.1, fermentation temperature 29.2 °C, and fermentation time 6 d. It was detected and analyzed that the fermented extract of 105-DP contained the ginsenosides Rf and Rb3. The endophytic fungus Trametes versicolor strain NSJ105 has potential application value in saponin production.

Fungal Biotransformation of Chlordiazepoxide and Evaluation of Type and Kinetics of Fungal Enzyme

To produce an active metabolite of Chlordiazepoxide by fungal biotransformation in an easy and economic way and also to develop microbial models for drug metabolism studies. Chlordiazepoxide is metabolized in the liver by CYP3A4 and forms major active metabolite N-desmethyl chlordiazepoxide. The focus of the study was to explore the ability of six distinct fungi to biotransform the drug Chlordiazepoxide to its metabolites. Induction, Inhibition and kinetic studies were also conducted to find out the type and capability of enzyme involved in fungal biotransformation. The screening studies were performed and fermentation protocol was designed with two controls (culture control and drug control) and one sample. Extract metabolite samples were reconstituted and analysed using HPLC. Induction, Inhibition studies were conducted similarly by maintaining its respective controls using CYP3A4 inducer (Carbamazepine) and inhibitor (Fluoxetine), further kinetic studies were performed to find Km and Vmax of fungal biotransformation of Chlordiazepoxide. Among six organisms Aspergillus ochreus has shown an extra peak at 6.9 min. in HPLC when compared with its controls indicated the formation of metabolite. The metabolite thus formed was identified, isolated and structure was confirmed by mass spectrometry and NMR spectroscopy as Nor-chlordiazepoxide. During inhibition and induction studies, it was found that quantity of the metabolite was increased with inducer and decreased with inhibitor. The Km and Vmax of fungal metabolism of Chlordiazepoxide was 1.928 µg/ml and 0.1802 µg/ml/hr respectively. Aspergillus ochreus has the ability to biotransform the Chlordiazepoxide to its active metabolite by CYP3A4 like enzyme and it followed Michaelis-Menten kinetics.

Comparative Accuracy of In Vitro Rumen Fermentation and Enzymatic Methodologies for Determination of Undigested Neutral Detergent Fiber in Forages and Development of Predictive Equations Using NIRS

Undigested neutral detergent fiber (uNDF) is becoming more widely recognized as an important fiber fraction in forage quality assessment because it explains a portion of NDF that is inaccessible to digestion in the ruminant digestive system and is, thus, important in modeling the digestion kinetics of the potentially degradable component of NDF. In experiment 1, uNDF was determined in several forage species in order to compare the accuracy of two reference methods: (1) a long-term in vitro ruminal fermentation (240 h) using an Ankom DaisyII incubator and (2) a multi-step enzymatic method without ruminal fluid. The objective of experiment 2 was to construct predictive equations for uNDF estimation using acid detergent lignin (ADL) and near-infrared reflectance spectroscopy (NIRS) in a pool (n = 264) of alfalfa hay, timothy hay, and tall fescue straw, using the most accurate reference method selected in experiment 1. Partial least squares regression analysis was used to calibrate the reference values against NIRS spectra. Several indicators were used to assess the performance of validation results, including standard error of cross-validation (SECrV), coefficient of determination of cross-validation (R2CrV), and ratio percentage deviation (RPD). The findings of experiment 1 suggested that, relative to the in vitro ruminal methodology, the enzymatic approach overestimated uNDF concentration of forages. Repeatability coefficient was also greater when uNDF was determined using the in vitro versus enzymatic procedure, potentially disqualifying the enzymatic method for the uNDF analysis in forages. In experiment 2, a poor relationship was established between ADL and uNDF (R2 < 0.60), suggesting the inadequacy of ADL parameter to represent the uNDF pool size in these forages. The best predictive equation using NIRS was obtained for alfalfa hay (R2CrV = 0.92; SECrV = 1.16; RPD = 3.57), using the in vitro fermentation as a reference method. The predictive equations were moderately accurate for timothy hay (R2CrV = 0.80; SECrV = 1.31; RPD = 2.08) and tall fescue straw (R2CrV = 0.79; SECrV = 1.38; RPD = 2.18). Our findings suggested the inadequacy of the enzymatic procedure in accurately determining uNDF concentration of forages as compared with the in vitro rumen fermentation protocol. Although the NIRS equations developed using the alfalfa hay dataset were more accurate than that of timothy hay and tall fescue straw, the validation results verified applicability of the equations as a fast screening tool for qualitative prediction of uNDF in these forages, which is important in commercial settings.

Optimizing the production of docosahexaenoic fatty acid by Crypthecodinium cohnii and reduction in process cost by using a dark fermentation effluent

Recovery of omega-3 fatty acids (FAs) from microalgae constitutes a valuable alternative to fish oil, and offers the ability to isolate a single ω-3 FA, instead of a mixture. In the framework of the concept of circular economy and sustainability, in the current work, an alternative carbon source was used as feed for oleaginous microalga Crypthecodinium cohnii cultivation and, consequently, production of the omega-3 FA, docosahexaenoic acid (DHA). The conditions that improve DHA production in a fed-batch culture of C. cohnii, using an organic acid-rich liquid fraction of dark fermented biowaste as feed, were studied and optimized. The process was mathematically approached focusing on a proper Design of Experiments (DoE) method coupled with Response Surface Methodology (RSM). The temperature of 23 °C and the use of yeast extract (YE) as initial nitrogen source favored the amount of DHA recovered. In addition, the increase of the volatile FA (VFA) concentration of the feed enhanced the recovered cell biomass. In order to propose a final fermentation protocol with industrial potential and evaluate the annual total cost for the production of DHA, fermentation process was scaled-up and economically evaluated through cost analysis. The increase of bioreactor size from 1 to 20 m3 leads to a significant decrease in the cost of produced DHA while leveling at higher capacities.

Defatted durum wheat germ to produce type-II and III sourdoughs: Characterization and use as bread ingredient

A fermentation protocol including selected lactic acid bacteria has been applied to defatted durum wheat germ, resulting from the oil extraction, to produce a nutritionally valuable ingredient for bread production. An integrated approach was used to evaluate the microbiological, nutritional, technological, and sensory properties of the fermented ingredient and the corresponding fortified bread. The fermentation led to a significant increase of the concentration of free amino acids (3-times) and decrease of the phytic acid (50%) and raffinose (93%) contents. The bread fortified with the sourdough-fermented defatted wheat germ could be labelled as source of fiber (3.3 g/100 g of bread) and source of protein (15.4% of the energy value was provided by proteins), according to the Regulation EC No. 1924/2006. When the fermented ingredient was used, the free amino acids concentration was 80% higher and the glycemic index lower (84 vs 95) than the control bread. Although final volume, hardness and chewiness of bread fortified with the fermented ingredient were similar to those of the control bread, an easier fracturability was found probably due to the high content of dietary fibers and acidity. Sensory analysis showed that fermented defatted wheat germ conferred perceptible acidic odor and taste to the bread.

Oenological Characterization of Native Hanseniaspora uvarum Strains

The utilization of native yeast strains associated with a distinct terroir for autochthonous grape types represents a novel trend in winemaking, contributing to the production of unique wines with regional character. Hence, this study aimed to isolate native strains of the yeast H. uvarum from the surface of various fruits and to characterize its fermentation capability in Prokupac grape must. Out of 31 yeasts, 8 isolates were identified as H. uvarum. The isolates were able to grow at low (4 °C) temperatures, SO2 concentrations up to 300 ppm and ethanol concentrations up to 5%. Additionally, they provided a good profile of organic acids during the microvinification of sterile grape must. Although the content of acetic acid (0.54–0.63 g/L) was relatively high, the sniffing test proved that the yeast isolates developed a pleasant aroma characterized as fruity. All H. uvarum isolates produced twice the concentration of glycerol compared to commercial wine yeast Saccharomyces cerevisiae, contributing to the fullness and sweetness of the wine. The results for pure and sequential fermentation protocols confirmed that the selected S-2 isolate has good oenological characteristics, the capability to reduce the ethanol content (up to 1% v/v) and a potential to give a distinctive note to Prokupac-grape wines.

Exploring cocoa bean fermentation mechanisms by kinetic modelling.

Compared with other fermentation processes in food industry, cocoa bean fermentation is uncontrolled and not standardized. A detailed mechanistic understanding can therefore be relevant for cocoa bean quality control. Starting from an existing mathematical model of cocoa bean fermentation we analyse five additional biochemical mechanisms derived from the literature. These mechanisms, when added to the baseline model either in isolation or in combination, were evaluated in terms of their capacity to describe experimental data. In total, we evaluated 32 model variants on 23 fermentation datasets. We interpret the results from two perspectives: (1) success of the potential mechanism, (2) discrimination of fermentation protocols based on estimated parameters. The former provides insight in the fermentation process itself. The latter opens an avenue towards reverse-engineering empirical conditions from model parameters. We find support for two mechanisms debated in the literature: consumption of fructose by lactic acid bacteria and production of acetic acid by yeast. Furthermore, we provide evidence that model parameters are sensitive to differences in the cultivar, temperature control and usage of steel tanks compared with wooden boxes. Our results show that mathematical modelling can provide an alternative to standard chemical fingerprinting in the interpretation of fermentation data.

Simple Scalable Protein Expression and Extraction Using Two-stage Autoinducible Cell Autolysis and DNA/RNA Autohydrolysis in Escherichia coli.

Recombinant protein expression is extensively used in biological research. Despite this, current protein expression and extraction methods are not readily scalable or amenable for high-throughput applications. Optimization of protein expression conditions using traditional methods, reliant on growth-associated induction, is non-trivial. Similarly, protein extraction methods are predominantly restricted to chemical methods, and mechanical methods reliant on expensive specialized equipment more tuned for large-scale applications. In this article, we outline detailed protocols for the use of an engineered autolysis/autohydrolysis E. coli strain, in two-stage fermentations in shake-flasks. This two-stage fermentation protocol does not require optimization of expression conditions and results in high protein titers. Cell lysis in an engineered strain is tightly controlled and only triggered post-culture by addition of a 0.1% detergent solution. Upon cell lysis, a nuclease digests contaminating host oligonucleotides, which facilitates sample handling. This method has been validated for use at different scales, from microtiter plates to instrumented bioreactors. Graphic abstract: Two-stage protein expression, cell autolysis and DNA/RNA autohydrolysis. Reprinted with permission from Menacho-Melgar et al. (2020a). Copyright 2020 John Wiley and Sons.

Exploring the suitability of Saccharomyces cerevisiae strains for winemaking under aerobic conditions

Aerobic fermentation was previously proposed to reduce the ethanol content of wine. The main constraint found for Saccharomyces cerevisiae to be used under these conditions was the high levels of acetic acid produced by all S. cerevisiae strains previously tested. This work addressed the identification of S. cerevisiae wine yeast strains suitable for aerobic fermentation and the optimization of fermentation conditions to obtain a reduced ethanol yield with acceptable volatile acidity. This approach unveiled a great diversity in acetic acid yield for different S. cerevisiae strains under aerobic conditions, with some strains showing very low volatile acidity. Three strains were selected for further characterization in bioreactors, with natural grape must, under aerobic and anaerobic conditions. Ethanol yields were lower under aerobic than under anaerobic conditions for all strains, and acetic acid levels were low for two of them. Strain-dependent changes in volatile compounds were also observed between aerobic and anaerobic conditions. Finally, the process was optimized at laboratory scale for one strain. This is the first report of S. cerevisiae wine strains showing low acetic acid production under aerobic conditions and paves the way for simplified aerobic fermentation protocols aimed to reducing the alcohol content of wines.

Production of bioactive recombinant human fibroblast growth factor 12 using a new transient expression vector in E. coli and its neuroprotective effects.

In recent years, an increasing number of studies have shown that fibroblast growth factor 12 (FGF12) plays important roles in regulating neural development and function. Importantly, changes of FGF12 expression are thought to be related to the pathophysiology of many neurological diseases. However, little research has been performed to explore the protective effect of FGF12 on nerve damage. This study aims to explore its neuroprotective effects using our recombinant humanized FGF12 (rhFGF12). The hFGF12 gene was cloned and ligated into an expression vector to construct a recombinant plasmid pET-3a-hFGF12. Single colonies were screened to obtain high expression engineering strains, and fermentation and purification protocols for rhFGF12 were designed and optimized. The biological activities and related mechanisms of rhFGF12 were investigated by MTT assay using NIH3T3 and PC12 cell lines. The in vitro neurotoxicity model of H2O2-induced oxidative injury in PC12 cells was established to explore the protective effects of rhFGF12. The results indicate that the beneficial effects of rhFGF12 were most likely achieved by promoting cell proliferation and reducing apoptosis. Moreover, a transgenic zebrafish (islet) with strong GFP fluorescence in the motor neurons of the hindbrain was used to establish a central injury model caused by mycophenolate mofetil (MMF). The results suggested that rhFGF12 could ameliorate central injury induced by MMF in zebrafish. In conclusion, we have established an efficient method to express and purify active rhFGF12 using an Escherichia coli expression system. Besides, rhFGF12 plays a protective effect of on nerve damage, and it provides a promising therapeutic approach for nerve injury. KEY POINTS: • Effective expression and purification of bioactive rhFGF12 protein in E. coli. • ERK/MAPK pathway is involved in rhFGF12-stimulated proliferation on PC12 cells. • The rhFGF12 has the neuroprotective effects by inhibiting apoptosis.

An in vitro batch fermentation protocol for studying the contribution of food to gut microbiota composition and functionality.

Knowledge of the effect of foods on gut microbiota composition and functionality is expanding. To isolate the effect of single foods and/or single nutrients (i.e., fiber, polyphenols), this protocol describes an in vitro batch fermentation procedure to be carried out after an in vitro gastrointestinal digestion. Therefore, this is an extension of the previous protocol described by Brodkorb et al. (2019) for studying in vitro digestion. The current protocol uses an oligotrophic fermentation medium with peptone and a high concentration of fecal inoculum from human fecal samples both to provide the microbiota and as the main source of nutrients for the bacteria. This protocol is recommended for screening work to be performed when many food samples are to be studied. It has been used successfully to study gut microbiota fermentation of different foodstuffs, giving insights into their functionality, community structure or ability to degrade particular substances, which can contribute to the development of personalized nutrition strategies. The procedure does not require a specific level of expertise. The protocol takes 4-6 h for preparation of fermentation tubes and 20 h for incubation.

Enhanced antioxidant activity of Chenopodium formosanum Koidz. by lactic acid bacteria: Optimization of fermentation conditions.

In this study, different probiotics commonly used to produce fermented dairy products were inoculated independently for Chenopodium formosanum Koidz. fermentation. The strain with the highest level of antioxidant activity was selected and the fermentation process was further optimized via response surface methodology (RSM). Lactobacillus plantarum BCRC 11697 was chosen because, compared to other lactic acid bacteria, it exhibits increased free radical scavenging ability and can produce more phenolic compounds, DPPH (from 72.6% to 93.2%), and ABTS (from 64.2% to 76.9%). Using RSM, we further optimize the fermentation protocol of BCRC 11697 by adjusting the initial fermentation pH, agitation speed, and temperature to reach the highest level of antioxidant activity (73.5% of DPPH and 93.8% of ABTS). The optimal protocol (pH 5.55, 104 rpm, and 24.4°C) resulted in a significant increase in the amount of phenolic compounds as well as the DPPH and ABTS free radical scavenging ability of BCRC 11697 products. The IC50 of the DPPH and ABTS free radical scavenging ability were 0.33 and 2.35 mg/mL, respectively, and both protease and tannase activity increased after RSM. An increase in lower molecular weight (<24 kDa) protein hydrolysates was also observed. Results indicated that djulis fermented by L. plantarum can be a powerful source of natural antioxidants for preventing free radical-initiated diseases.