Comprehensive assessment of goat adipose tissue-derived mesenchymal stem cells cultured in different media.

Mesenchymal stem cells (MSCs) have demonstrated potential in treating livestock diseases that are unresponsive to conventional therapies. MSCs derived from goats, a valuable model for studying orthopaedic disorders in humans, offer insights into bone formation and regeneration. Adipose tissue-derived MSCs (ADSCs) are easily accessible and have a high capacity for expansion. Although the choice of culture media significantly influences the biological properties of MSCs, the optimal media for goat ADSCs (gADSCs) remains unclear. This study aimed to assess the effects of four commonly used culture media on gADSCs' culture characteristics, stem cell-specific immunophenotype, and differentiation. Results showed that MEM, DMEM/F12, and DMEM-LG were superior in maintaining cell morphology and culture parameters of gADSCs, such as cell adherence, metabolic activity, colony-forming potential, and population doubling. Conversely, DMEM-HG exhibited poor performance across all evaluated parameters. The gADSCs cultured in DMEM/F12 showed enhanced early proliferation and lower apoptosis. The cell surface marker distribution exhibited superior characteristics in gADSCs cultured in MEM and DMEM/F12. In contrast, the distribution was inferior in gADSCs cultured in DMEM-LG. DMEM/F12 and DMEM-LG culture media demonstrated a significantly higher potential for chondrogenic differentiation and DMEM-LG for osteogenic differentiation. In conclusion, DMEM/F12 is a suitable culture medium for propagating gADSCs as it effectively maintains cell morphology, growth parameters, proliferation and lower apoptosis while exhibiting desirable expression patterns of MSC-specific markers. These findings contribute to optimising culture conditions for gADSCs, enhancing their potential applications in disease treatment and regenerative medicine.

Organic Supplementation of Vaccinium corymbosum Micropropagation Media.

The best Vaccinium corymbosum plant growth under in vitro conditions can be achieved by using the right composition and pH of the medium. For the initial phase of in vitro culture, a combination of cytokinins-mostly zeatin-can usually be used. Organic supplementation of the medium enables the use of a replacement for the expensive natural cytokinin used in micropropagation of highbush blueberry. This chapter describes the experiments with silicon Hydroplus™ Actisil (Si), coconut water (CW), and different pH (5.0; 5.5, and 6.0) as a stress factor. The addition of 200 mg dm-3 silicon solution and 15% coconut water strongly stimulated highbush blueberry plant growth in vitro. Moreover, silicon solution benefits the negative effects of higher pH of the medium used for micropropagation of V. corymbosum. Maximum vegetative development of blueberry explants was obtained at pH 5.

Explainable AI for CHO cell culture media optimization and prediction of critical quality attribute.

Cell culture media play a critical role in cell growth and propagation by providing a substrate; media components can also modulate the critical quality attributes (CQAs). However, the inherent complexity of the cell culture media makes unraveling the impact of the various media components on cell growth and CQAs non-trivial. In this study, we demonstrate an end-to-end machine learning framework for media component selection and prediction of CQAs. The preliminary dataset for feature selection was generated by performing CHO-GS (-/-) cell culture in media formulations with varying metal ion concentrations. Acidic and basic charge variant composition of the innovator product (24.97 ± 0.54% acidic and 11.41 ± 1.44% basic) was chosen as the target variable to evaluate the media formulations. Pearson's correlation coefficient and random forest-based techniques were used for feature ranking and feature selection for the prediction of acidic and basic charge variants. Furthermore, a global interpretation analysis using SHapley Additive exPlanations was utilized to select optimal features by evaluating the contributions of each feature in the extracted vectors. Finally, the medium combinations were predicted by employing fifteen different regression models and utilizing a grid search and random search cross-validation for hyperparameter optimization. Experimental results demonstrate that Fe and Zn significantly impact the charge variant profile. This study aims to offer insights that are pertinent to both innovators seeking to establish a complete pipeline for media development and optimization and biosimilar-based manufacturers who strive to demonstrate the analytical and functional biosimilarity of their products to the innovator. KEY POINTS: • Developed a framework for optimizing media components and prediction of CQA. • SHAP enhances global interpretability, aiding informed decision-making. • Fifteen regression models were employed to predict medium combinations.

Enhanced high ß-carotene yeast cell production by Rhodotorula paludigena CM33 and in vitro digestibility in aquatic animals.

This study assessed Rhodotorula paludigena CM33's growth and ß-carotene production in a 22-L bioreactor for potential use as an aquatic animal feed supplement. Optimizing the feed medium's micronutrient concentration for high-cell-density fed-batch cultivation using glucose as the carbon source yielded biomass of 89.84 g/L and ß-carotene concentration of 251.64 mg/L. Notably, using sucrose as the carbon source in feed medium outperforms glucose feeds, resulting in a ß-carotene concentration of 285.00 mg/L with a similar biomass of 87.78 g/L. In the fed-batch fermentation using Sucrose Feed Medium, R. paludigena CM33 exhibited high biomass production rates (Qx) of 0.91 g/L.h and remarkable ß-carotene production rates (Qp) of 2.97 mg/L.h. In vitro digestibility assays showed that R. paludigena CM33, especially when cultivated using sucrose, enhances protein digestibility affirming its suitability as an aquatic feed supplement. Furthermore, R. paludigena CM33's nutrient-rich profile and probiotic potential make it an attractive option for aquatic nutrition. This research highlights the importance of cost-effective carbon sources in large-scale ß-carotene production for aquatic animal nutrition.

Improving Bioprocess Conditions for the Production of Prodigiosin Using a Marine Serratia rubidaea Strain.

The enormous potential attributed to prodigiosin regarding its applicability as a natural pigment and pharmaceutical agent justifies the development of sound bioprocesses for its production. Using a Serratia rubidaea strain isolated from a shallow-water hydrothermal vent, optimization of the growth medium composition was carried out. After medium development, the bacterium temperature, light and oxygen needs were studied, as was growth inhibition by product concentration. The implemented changes led to a 13-fold increase in prodigiosin production in a shake flask, reaching 19.7 mg/L. The conditions allowing the highest bacterial cell growth and prodigiosin production were also tested with another marine strain: S. marcescens isolated from a tide rock pool was able to produce 15.8 mg/L of prodigiosin. The bioprocess with S. rubidaea was scaled up from 0.1 L shake flasks to 2 L bioreactors using the maintenance of the oxygen mass transfer coefficient (kLa) as the scale-up criterion. The implemented parameters in the bioreactor led to an 8-fold increase in product per biomass yield and to a final concentration of 293.1 mg/L of prodigiosin in 24 h.

Survival of O157 and non-O157 shiga toxin-producing Escherichia coli in Korean style kimchi.

Korean style kimchi contaminated with Shiga toxin-producing Escherichia coli (STEC) O157:H7 was the cause of an outbreak in Canada from December 2021 to January 2022. To determine if this STEC O157:H7 has greater potential for survival in kimchi than other STEC, the outbreak strain and six other STEC strains (O26:H11, O91:H21, O103:H2, O121:H19, and two O157:H7) were inoculated individually at 6 to 6.5 log CFU/g into commercially sourced kimchi and incubation at 4 °C. At intervals of seven days inoculated and control kimchi was plated onto MacConkey agar to enumerate lactose utilising bacteria. The colony counts were interpreted as enumerating the inoculated STEC, since no colonies were observed on MacConkey agar plated with uninoculated kimchi. Over eight weeks of incubation the pH was stable at 4.10 to 4.05 and the STEC strains declined by 0.7-1.0 log, with a median reduction of 0.9 log. The linear rate of reduction of kimchi outbreak STEC O157:H7 was -0.4 log per 30 days (Slope Uncertainty 0.05), which was not significantly different from the other O157 and nonO157 STEC strains (P = 0.091). These results indicate that the outbreak was not due to the presence of strain better adapted to survival in kimchi than other STEC, and that STEC can persist in refrigerated Korean style kimchi with a minimal decline over the shelf-life of the product.

Microsclerotia from Metarhizium robertsii: Production, ultrastructural analysis, robustness, and insecticidal activity.

Microsclerotia (MS) are considered one of the most promising propagules for use as active ingredients in biopesticides due to their tolerance to abiotic factors and ability to produce infective conidia for the control of pests. Therefore, the objective of this research was to establish the conditions required to induce the formation of microsclerotia in Metarhizium robertsii Mt004 and to study its development process, tolerance to abiotic factors and insecticidal activity of MS-derived conidia. M. robertsii started to form hyphal aggregates after 2 days and looked more compact after 8 days. MS were mature and pigmented after 20 days. The final yield was 2.0 × 103 MS/mL and MS size varied between 356.9 and 1348.4 µm. Ultrastructure analysis revealed that mature MS contained only a few live cells embedded in an extracellular matrix. Mature MS were more tolerance to UV-B radiation, heat and storage trials than conidia from Solid State Fermentation. MS-derived conidia were as virulent as conidia against Diatraea saccharalis larvae. These results showed that MS are promising propagules for the development of more persistent and efficient biopesticides for harsh environmental conditions. Our findings provide a baseline for production and a better understanding of microsclerotia development in M. robertsii strains.

Physicochemical characterisation of casings in relation to mushroom (Agaricus bisporus) cropping performance.

Peat-based casings have been used for button mushroom (Agaricus bisporus) cultivation for decades but there is environmental pressure to find sustainable alternatives. This work aimed to characterise the physicochemical properties of peat and peat-substituted casings and to determine their influence on mushroom cropping to enable alternatives to be identified. British milled peat and German wet-dug peat casings produced smaller mushrooms than Irish wet-dug peat casing although yield was unaffected. Substitution of milled or wet-dug peat casings with 25% v/v bark, green waste compost or spent mushroom casing, except Irish wet-dug peat casing with spent peat mushroom casing, caused reductions in mushroom yield and/or size. These poorer results of casings compared with Irish wet-dug peat casing corresponded with lower water retention volumes at matric potential (Ψm) -15 kPa but not after drainage from saturation or at -1 kPa. Air-filled porosity (17-22% v/v), compacted bulk density after drainage (670-800 g L-1) and electrical conductivity (0.30-0.54 mS cm-1) of casings were unrelated to their mushroom cropping performance. In-situ casing measurements with electronic tensiometers confirmed laboratory casing physical analysis: at the same casing Ψm, Irish wet-dug peat casing had a higher water content than German wet-dug peat casing and produced larger mushrooms for the same yield. Solid-state foam-based tensiometers were more robust than water-filled tensiometers but they did not detect the full decrease in casing Ψm during a flush of mushrooms. The results indicate that if sustainable materials are to replace wet-dug peat casing with the same mushroom yield and size quality performance, they should have equivalent water retention volumes at Ψm -15 kPa. Measurement of casing Ψm with electronic tensiometers to control mushroom crop irrigation should assist in this transition.

Comparative Analysis of Effect of Culture Conditions on Growth and C-Phycocyanin Production in Helical and Linear Spirulina.

Spirulina (Arthrospira and Spirulina spp.) has always been characterized by the helical trichomes, despite the existence of linear forms. A great debate is now open on the morphological flexibility of Spirulina, but it seems that both trichome morphology and C-phycocyanin (C-PC) concentrations are influenced by the culture conditions.This work compared the effect of some key growth factors (medium pH as well as its carbon, potassium, and salt contents) on the growth and C-PC concentration of helical and linear Spirulina strains. Further, two-phase strategies, including light and nitrogen variation, were applied to increase the in vivo C-PC accumulation into the trichomes. Results showed that high pH induced trichomes elongation and improved growth but decreased C-PC content (+ 65 and + 43% vs. -83 and -49%, for helical and linear strains, respectively). Variations in carbon and salt concentrations negatively impacted growth and C-PC content, even if the linear strain was more robust against these fluctuations. It was also interesting to see that potassium increasing improved growth and C-PC content for both strains.The variation of light wavelength during the enrichment phase (in the two-phase strategy) improved by 50% C-PC accumulation in trichomes, especially after blue lighting for 96 h. Similar result was obtained after 48 h of nitrogen reduction, while its removal from the medium caused trichomes disintegration. The current work highlights the robustness of linear Spirulina strain and presents an efficient and scalable way to increase C-PC in vivo without affecting growth.

The responses of pepper plants to nitrogen form and dissolved oxygen concentration of nutrient solution in hydroponics.

BACKGROUND: The presence of oxygen in the growth medium is absolutely essential for root development and the overall metabolic processes of plants. When plants do not have an adequate oxygen supply for respiration, they can experience a condition known as hypoxia. In order to investigate the impact of different nitrogen forms and varying oxygen levels in nutrient solutions on the growth, photosynthesis, and chlorophyll fluorescence parameters of bell pepper plants, a comprehensive study was conducted. The experiment was designed as a factorial experiment, considering two main factors: nitrogen forms (calcium nitrate and ammonium sulfate) with a fixed nitrogen concentration of 5 mM, and the oxygen levels of the nutrient solutions (ranging from 1.8 ± 0.2 to 5.3 ± 0.2 mg. L-1). RESULTS: The study examined the effects of nitrogen (NH4+ and NO3-) application on various parameters of vegetative growth. The results demonstrated that the use of ammonium (NH4+) led to a reduction in the most measured parameters, including the fresh and dry mass of both the root and shoot, at low O2 concentrations of 1.8 ± 0.2; 2.6 ± 0.2 and 3.8 ± 0.2 mg. L-1. However, an interesting observation was made regarding the impact of oxygen levels on root growth in plants grown with nitrate (NO3-). Specifically, the highest levels of oxygen significantly increased root growth in NO3--fed plants. Additionally, the application of NH4+ resulted in an increase in chlorophyll concentration in the leaves, particularly when combined with high oxygen levels in the nutrient solution. On the other hand, leaves of plants fed with NO3- exhibited higher photosynthetic rate (A), intrinsic water use efficiency (iWUE), and instantaneous carboxylation efficiency (A/Ci) compared to those fed with NH4+. Furthermore, it was found that NO3--fed plants displayed the highest instantaneous carboxylation efficiency at oxygen levels of 3.8 and 5.3 mg. L-1, while the lowest efficiency was observed at oxygen levels of 1.8 and 2.6 mg. L-1. In contrast, NH4+-grown plants exhibited a higher maximal quantum yield of PSII photochemistry (Fv/Fm), as well as increased variable fluorescence (Fv) and maximum fluorescence (Fm), compared to NO3--grown plants. Interestingly, the NO3--fed plants showed an increase in Fv/Fm, Fv, and Fm with the elevation of oxygen concentration in the nutrient solution up to 5.3 mg. L-1. CONCLUSION: This study showed that, the growth and photosynthesis parameters in bell pepper plants are sensitive to oxygen stress in floating hydroponic culture. Therefore, the oxygen level in the nutrient solution must not be lower than 3.8 and 5.3 mg. L-1 in NH4+ and NO3- -supplied culture media or nutrient solutions, respectively.

Characterization of gelling agents in callus inducing media: Physical properties and their effect on callus growth.

In plant tissue culture, callus formation serves as a crucial mechanism for regenerating entire plants, enabling the differentiation of diverse tissues. Researchers have extensively studied the influence of media composition, particularly plant growth regulators, on callus behavior. However, the impact of the physical properties of the media, a well-established factor in mammalian cell studies, has received limited attention in the context of plant tissue culture. Previous research has highlighted the significance of gelling agents in affecting callus growth and differentiation, with Agar, Phytagel, and Gelrite being the most used options. Despite their widespread use, a comprehensive comparison of their physical properties and their subsequent effects on callus behavior remains lacking. Our study provides insights into optimizing plant tissue culture media by analyzing the physical properties of gelling agents and their impact on callus induction and differentiation. We compared the phenotypes of calli grown on media composed of these different gelling agents and correlated them to the physical properties of these media. We tested water retention, examined pore size using cryo-SEM, measured the media mechanical properties, and studied diffusion characteristics. We found that the mechanical properties of the media are the only quality correlated with callus phenotype.

Acidic Derivatization of Thiols Using Diethyl 2-Methylenemalonate: Thiol-Michael Addition Click Reaction for Simultaneous Analysis of Cysteine and Cystine in Culture Media Using LC-MS/MS.

Cysteine (Cys) and its oxidized form, cystine (Cys2), play crucial roles in biological systems and have considerable applications in cell culture. However, Cys in cell culture media is easily oxidized to Cys2, leading to solubility issues. Traditional analytical methods struggle to maintain the oxidation states of Cys and Cys2 during analysis, posing a significant challenge to accurately measuring and controlling these compounds. To effectively control the Cys and Cys2 levels, a rapid and accurate analytical method is required. Here, we screened derivatizing reagents that can react with Cys even under acidic conditions to realize a novel analytical method for simultaneously determining Cys and Cys2 levels. Diethyl 2-methylenemalonate (EMM) was found to possess the desired traits. EMM, characterized by its dual electron-withdrawing attributes, allowed for a rapid reaction with Cys under acidic conditions, preserving intact information for understanding the functions of target compounds. Combined with LC-MS/MS and an internal standard, this method provided high analytical accuracy in a short analytical time of 9 min. Using the developed method, the rapid oxidation of Cys in cell culture media was observed with the headspace of the storage container considerably influencing Cys oxidation and Cys2 precipitation rates. The developed method enabled the direct and simplified analysis of Cys behavior in practical media samples and could be used in formulating new media compositions, ensuring quality assurance, and real-time analysis of Cys and Cys2 in cell culture supernatants. This novel approach holds the potential to further enhance the media performance by enabling the timely optimal addition of Cys.

Identification and antimicrobial susceptibility profile of bacteria isolated from primary endodontic infections.

This study aimed to identify and characterize the antimicrobial susceptibility profile of bacteria found in primary endodontic infections in the teeth of patients treated at the Dental Clinic of the University of Ribeirão Preto, São Paulo, Brazil. From September to December 2019, samples were obtained from 21 patients with primary endodontic infections. The collections were carried out in triplicate using paper cones placed close to the total length of the root canal. Bacterial isolation was performed in Brain Heart Infusion agar, Blood agar, and other selective culture media cultured at 37°C for up to 48 h under aerobiosis and microaerophilic conditions. The bacterial species were identified using the Vitek 2 automated system. The disk diffusion method on agar Müeller-Hinton was used to assess antimicrobial susceptibility with the recommended antimicrobials for each identified bacterial species. A total of 49 antibiotics were evaluated. Fifteen of the 21 samples collected showed bacterial growth, and 17 bacterial isolates were found. There were 10 different bacterial species identified: Enterococcus faecalis (four isolates), Streptococcus mitis/oralis (three isolates), Streptococcus anginosus (three isolates) being the most common, followed by Staphylococcus epidermidis, Enterococcus faecium, Streptococcus constellatus, Streptococcus alactolyticus, Enterobacter cloacae, Klebsiella variicola, and Providencia rettgeri (one isolate of each species). The analysis demonstrated significant susceptibility to most of the tested antibiotics. However, some Enterococcus isolates resisted the antibiotic's erythromycin, ciprofloxacin, and tetracycline. A Staphylococcus epidermidis isolate was characterized as multidrug-resistant. Five Streptococcus isolates were non-susceptible to all antibiotics tested.

Using CHROMagar™ STEC medium exclusively does not recover all clinically relevant Shiga toxin-producing Escherichia coli in Aotearoa, New Zealand.

Diagnostic laboratories in Aotearoa, New Zealand (NZ) refer cultures from faecal samples positive for Shiga toxin genes to the national Enteric Reference Laboratory for isolation of Shiga toxin-producing Escherichia coli (STEC) for epidemiological typing. As there was variation in the culture media being referred, a panel of 75 clinical isolates of STEC, representing 28 different serotypes, was used to assess six commercially available media and provide guidance to clinical laboratories. Recommendations were subsequently tested for a 3-month period, where STEC isolations and confirmations were assessed by whole genome sequencing analysis against the culture media referred. CHROMagar™ STEC (CH-STEC; CHROMagar Microbiology, Paris, France) or CH-STEC plus cefixime-tellurite sorbitol MacConkey agar was confirmed inferior to CH-STEC plus blood agar with vancomycin, cefsulodin, and cefixime (BVCC). The former resulted in fewer STEC types (n = 18) being confirmed compared to those from a combination of CH-STEC and BVCC (n = 42). A significant (P < .05) association with an STEC's ability to grow on CH-STEC and the presence of the ter gene cluster, and eae was observed. Culturing screen positive STEC samples onto both CH-STEC and BVCC ensures a consistently higher recovery of STEC from all clinical samples in NZ than CH-STEC alone.

Mircrofabricating double-sided polydimethylsiloxane (PDMS) artificial phylloplane for microbial food safety research.

Leafy green surface microbiology studies often experience significant variations in results due to the heterogeneous nature of leaf surfaces. To provide a precise and controllable substitute, we microfabricated double-sided artificial leafy green phylloplanes using polydimethylsiloxane (PDMS) with a vinyl-terminated polyethylene glycol chain-based hydrophobicity modifier (PDMS-PEG) to modify PDMS hydrophobicity. We further tested the properties and applications of these artificial leaves, by examining the function of epicuticular wax, growth and survival of E. coli O157:H7 87-23 on the surface, and removal of attached E. coli cells via sanitation. The double-sided PDMS-PDMS-PEG leaves well-replicated their natural counterparts in macroscopic and microscopic structure, hydrophobicity, and E. coli O157:H7 87-23 attachment. After depositing natural epicuticular wax onto artificial leaves, the leaf surface wetting ability decreased, while E. coli O157:H7 87-23 surface retention increased. The artificial leaves supplied with lettuce lysate or bacterial growth media supported E. coli O157:H7 87-23 growth and survival similarly to those on natural leaves. In the sanitation test, the artificial lettuce leaves also displayed patterns similar to those of natural leaves regarding sanitizer efficiency. Overall, this study showcased the microfabrication and applications of double-sided PDMS-PDMS-PEG leaves as a replicable and controllable platform for future leafy green food safety studies.

Optimization of fermentation conditions and medium components for chrysomycin a production by Streptomyces sp. 891-B6.

BACKGROUND: Chrysomycin A (CA) is a promising antibiotic for treatment of Gram-positive bacterial infections and cancers. In order to enhance CA yield, optimization of fermentation conditions and medium components was carried out on strain Streptomyces sp. 891-B6, an UV-induced mutant with improved CA titer compared with its wide-type marine strain 891. RESULTS: Using one-way experiment, the optimal fermentation conditions for CA production in 1-L shake flask were obtained as follows: 12 days of fermentation time, 5 days of seed age, 5% of inoculum volume ratio, 200 mL of loading volume and 6.5 of initial pH. By response surface methodology, the optimal medium components determined as glucose (39.283 g/L), corn starch (20.662 g/L), soybean meal (15.480 g/L) and CaCO3 (2.000 g/L). CONCLUSION: Validation tests showed that the maximum yield of CA reached 1601.9 ± 56.7 mg/L, which was a 60% increase compared to the initial yield (952.3 ± 53.2 mg/L). These results provided an important basis for scale-up production of CA by strain 891-B6.

Identification and pathogenicity of Fusarium species associated with wilting and crown rot in almond (Prunus dulcis).

Severe Fusarium wilt and crown root symptoms were observed in almond orchards in Portugal. The present study elucidates the etiology of the disease through molecular, phenotypic, and pathogenic characterization. Three Fusarium isolates from Portugal were tested and 12 Fusarium isolates from almond from Spain were included for comparative purposes. Their identity was inferred by phylogenetic analysis combining tef1 and rpb2 sequences. The Portuguese isolates were identified as Fusarium oxysporum sensu stricto (s.s.), and the Spanish isolates as Fusarium nirenbergiae, F. oxysporum (s.s.), Fusarium proliferatum, Fusarium redolens (s.s.), Fusarium sambucinum (s.s.), and Fusarium sp. Fungal colonies and conidia were characterized on potato dextrose agar (PDA) and on Synthetischer Nährstoffarmer agar, respectively. The colonies had a variable morphology and their color ranged from white to pale violet. Typical Fusarium micro- and macroconidia were characterized. Temperature effect on mycelial growth was evaluated on PDA from 5 to 35 °C, with optimal growth temperature ranging between 16.8 and 26.4 °C. The pathogenicity of F. oxysporum was demonstrated by inoculating almond plants ('Lauranne') grafted on GF-677 or Rootpac 20 rootstocks. A significant reduction in plant growth, wilting, and xylem discoloration was observed, with Rootpac 20 being more susceptible than GF-677. Infections were also reproduced using naturally infested soils. Almond plants ('Lauranne') were inoculated with isolates of all Fusarium species, with F. redolens from Spain and F. oxysporum from Portugal being the most aggressive.

An alternative peptone preparation using Hermetia illucens (Black soldier fly) hydrolysis: process optimization and performance evaluation.

Background: Hermetia illucens (HI), commonly known as the black soldier fly, has been recognized for its prowess in resource utilization and environmental protection because of its ability to transform organic waste into animal feed for livestock, poultry, and aquaculture. However, the potential of the black soldier fly's high protein content for more than cheap feedstock is still largely unexplored. Methods: This study innovatively explores the potential of H. illucens larvae (HIL) protein as a peptone substitute for microbial culture media. Four commercial proteases (alkaline protease, trypsin, trypsase, and papain) were explored to hydrolyze the defatted HIL, and the experimental conditions were optimized via response surface methodology experimental design. The hydrolysate of the defatted HIL was subsequently vacuum freeze-dried and deployed as a growth medium for three bacterial strains (Staphylococcus aureus, Bacillus subtilis, and Escherichia coli) to determine the growth kinetics between the HIL peptone and commercial peptone. Results: The optimal conditions were 1.70% w/w complex enzyme (alkaline protease: trypsin at 1:1 ratio) at pH 7.0 and 54 °C for a duration of 4 h. Under these conditions, the hydrolysis of defatted HIL yielded 19.25% ±0.49%. A growth kinetic analysis showed no significant difference in growth parameters (µmax, Xmax, and λ) between the HIL peptone and commercial peptone, demonstrating that the HIL hydrolysate could serve as an effective, low-cost alternative to commercial peptone. This study introduces an innovative approach to HIL protein resource utilization, broadening its application beyond its current use in animal feed.

Biochemical and functional characterization of heat-inactivated coelomic fluid from earthworms as a potential alternative for fetal bovine serum in animal cell culture.

Fetal bovine serum (FBS) plays a pivotal role in animal cell culture. Due to ethical and scientific issues, searching for an alternative, comprising the three R's (Refinement, Reduction and Replacement) gained global attention. In this context, we have identified the heat inactivated coelomic fluid (HI-CF) of the earthworm, Perionyx excavatus as a potential alternative for FBS. Briefly, we formulated HI-CF (f-HICF) containing serum free medium which can aid the growth, attachment, and proliferation of adherent cells, similar to FBS. In this study, we investigated the biochemical characterization, sterility, stability, formulation, and functional analysis of HI-CF as a supplement in culturing animal cells. Notably, vitamins, micronutrients, proteins, lipids, and trace elements are identified and compared with FBS for effective normalization of the serum free media. HI-CF is tested to be devoid of endotoxin and mycoplasma contamination thus can qualify the cell culture grade. The f-HICF serum free media was prepared, optimised, and tested with A549, HeLa, 3T3, Vero and C2C12 cell lines. Our results conclude that f-HICF is a potential alternative to FBS, in accordance with ethical concern; compliance with 3R's; lack of unintended antibody interactions; presence of macro and micronutrients; simple extraction; cost-effectiveness and availability.

First description of adenosine production by Gnomoniopsis smithogilvyi, causal agent of chestnut brown rot.

Gnomoniopsis smithogilvyi (Gnomoniaceae, Diaporthales) is the main causal agent of chestnut brown rot on sweet chestnut worldwide. The rotting of nuts leads to alterations in the organoleptic qualities and decreased fruit production, resulting in significant economic losses. In 2021, there was an important outbreak of chestnut rot in southern Galicia (Spanish northwest). The profile of secondary metabolites from G. smithogilvyi was studied, especially to determine its capability for producing mycotoxins, as happens with other rotting fungi, due to the possible consequences on the safety of chestnut consumption. Secondary metabolites produced by isolates of G. smithogilvyi growing in potato dextrose agar (PDA) medium were identified using liquid chromatography coupled with high-resolution mass spectrometry. Three metabolites with interesting pharmacological and phyto-toxicological properties were identified based on their exact mass and fragmentation patterns, namely adenosine, oxasetin, and phytosphingosine. The capacity of G. smithogilvyi to produce adenosine in PDA cultures was assessed, finding concentrations ranging from 176 to 834 µg/kg. Similarly, the production of mycotoxins was ruled out, indicating that the consumption of chestnuts with necrotic lesions does not pose a health risk to the consumer in terms of mycotoxins.