Inoculation Concentration Research Articles

PSV-21 Pediococcus acidilactici survives an in vitro simulated digestion

Abstract In the swine industry, probiotics are recognized for optimizing growth performance and production efficiency by modulating host physiology, and the response across studies is not consistent. One area of focus is probiotic mitigation of post-weaning diarrhea, and further mechanistic understanding of probiotic and host physiology could help with consistent performance outcomes. An essential attribute of a probiotic is the ability to survive the harsh environments of the gastrointestinal tract (GIT). The aim of this study was to determine if Pediococcus Acidilactici PECh3A (PA) that has been previously genome sequenced for determining gut physiology mechanisms can survive an in vitro simulated digestion. In the in vitro digestion PA concentrations that were investigated included 108, 109, and 1010 colony forming units (CFU)/mL. The in vitro digestion (n = 4/concentration) consisted of 5 phases with incubation times simulating the GIT; initial inoculation (0 h), gastric (2 h), upper intestinal (4 h), lower intestinal (9 h) and extended lower intestinal phase (9 h). For the initial inoculation concentrations of PA were added into 25 mL of sterile milk replacer representing dietary requirements of a suckling pig. In the gastric phase pepsin was added and pH was adjusted to 2.5. In the upper intestinal phase bile and pancreatin were added, along with a pH adjustment to 6.8. In the lower intestinal phase, the multi-enzyme complex consisting of carbohydrase enzymes was added and pH adjusted to 4.8. There was no addition during the extended lower intestinal phase. At each sample collection at the end of a phase 1 mL was collected from each flask. For each sample, 100 µL were serially diluted, plated on deMan, Rogosa, and Sharpe (MRS) plates, and incubated at 37°C for 24h. Survivability was determined by counting CFUs and comparing the concentration with the initial CFU/mL at 0 h and reported as percent survival (%S). Data for the in vitro digestion were analyzed according to a randomized complete block design (RCBD) in a split plot using the Repeated Measures Glimmix Procedure of SAS. Additional orthogonal contrasts were analyzed across PA concentrations. Data showed survival for 108,109, and 1010CFU/mL at all sample phases (108 - 44.24, 60.14, 58.36, 55.69%S; 109 - 40.56, 55.60, 49.35, 50.7%S; 1010 - 32.98, 52.55, 46.72, 43.71%S ± 2.98 respectively; phase P < 0.01 and concentration P < 0.01). There is evidence of a phase and concentration effect on survivability, but no interaction. A linear concentration response was observed across concentrations (54.61, 49.05, 43.99%S ± 1.23; P < 0.05) indicating that as concentration increases the survivability decreases. These findings indicate that PA PECh3A can survive a simulated GIT and potentially impact swine intestinal physiology.

Bacillus velezensis YXDHD1-7 Prevents Early Blight Disease by Promoting Growth and Enhancing Defense Enzyme Activities in Tomato Plants.

Bacillus velezensis is well known as a plant growth-promoting rhizobacteria (PGPR) and biocontrol agent. Nevertheless, there are very few reports on the study of B. velezensis on tomato early blight, especially the biocontrol effects among different inoculation concentrations. In this study, an IAA-producing strain, Bacillus velezensis YXDHD1-7 was isolated from the tomato rhizosphere soil, which had the strongest inhibitory effect against Alternaria solani. Inoculation with bacterial suspensions of this strain promoted the growth of tomato seedlings effectively. Furthermore, inoculations at 106, 107, and 108 cfu/mL resulted in control efficacies of 100%, 83.15%, and 69.90%, respectively. Genome sequencing showed that it possesses 22 gene clusters associated with the synthesis of antimicrobial metabolites and genes that are involved in the production of IAA. Furthermore, it may be able to produce spermidine and volatile compounds that also enhance plant growth and defense responses. Our results suggest that strain YXDHD1-7 prevents early blight disease by promoting growth and enhancing the defense enzyme activities in tomato plants. This strain is a promising candidate for an excellent microbial inoculant that can be used to enhance tomato production.

Optimization of the fermentation media and growth conditions of Bacillus velezensis BHZ-29 using a Plackett-Burman design experiment combined with response surface methodology.

Bacillus velezensis occurs extensively in the soil environment. It produces a range of antimicrobial compounds that play an important role in the field of biological control. However, during the actual application process it is often affected by factors such as the medium formulation and fermentation conditions, and therefore biocontrol measures often do not achieve their expected outcomes. In this study, the B. velezensis BHZ-29 strain was used as the research object. The carbon and nitrogen sources, and inorganic salts that affect the number of viable bacteria and antibacterial potency of B. velezensis BHZ-29, were screened by a single factor test. A Plackett-Burman design experiment was conducted to determine the significant factors affecting the number of viable bacteria and antibacterial potency, and a Box-Behnken design experiment was used to obtain the optimal growth of B. velezensis BHZ-29. The medium formula that produced the highest number of viable bacteria and most antibacterial substances was determined. The initial pH, temperature, amount of inoculant, liquid volume, shaking speed, and culture time were determined by a single factor test. The factors that had a significant influence on the number of viable bacteria of B. velezensis BHZ-29 were selected by an orthogonal test. A Box-Behnken design experiment was conducted to obtain the optimal fermentation conditions, and highest number of viable bacteria and antibacterial titer. Molasses, peptone, and magnesium sulfate had significant effects on the viable count and antibacterial titer of B. velezensis BHZ-29. The viable count of B. velezensis BHZ-29 increased from 7.83 × 109 to 2.17 × 1010 CFU/mL, and the antibacterial titer increased from 111.67 to 153.13 mm/mL when the optimal media were used. The optimal fermentation conditions for B. velezensis BHZ-29 were as follows: temperature 25.57°C, pH 7.23, culture time 95.90 h, rotation speed 160 rpm, amount of inoculant 2%, and liquid volume 100 ml. After the optimization of fermentation conditions, the number of viable bacteria increased to 3.39 × 1010 CFU/mL, and the bacteriostatic titer increased to 158.85 mm/ml.The plant height and leaf number of cotton plants treated with BHZ-29 fermentation broth were higher than those of cotton inoculated with Verticillium dahliae. The number of bacteria was 1.15 × 107 CFU/g, and the number of fungi was 1.60 × 105 spores/g. The disease index of the cotton seedlings treated with the optimized fermentation broth was 2.2, and a control effect of 93.8% was achieved. B. velezensis BHZ-29 could reduce the disease index of cotton Verticillium wilt and had a controlling effect on the disease. The best effect was achieved in the treatment group with an inoculation concentration of 2 × 108 CFU/ml, the disease index was 14.50, and a control effect of 84.18% was achieved. The fermentation process parameters of the number of viable bacteria and antibacterial titer by strain B. velezensis BHZ-29 were optimized to lay a foundation for the practical production and application of strain B. velezensis BHZ-29 in agriculture.

Exploration on the Mildew Conditions of Cladosporium asperulatum CY-H1, a Pathogenic Fungus on Tobacco Leaves

Aims: Mildew poses a significant threat to tobacco industry. This study focuses on a strain of Cladosporium asperulatum isolated from moldy tobacco leaves to investigate its growth under different temperature and humidity conditions, aiming to provide a theoretical basis for local prevention and control strategies against this fungus.
 Methodology: Potential fungal strains were obtained from moldy tobacco leaves utilizing isolation techniques and individual colonies were cultured on PDA medium subsequently. Microscopic examination and molecular identification were employed to determine the species of the isolated strains. Isolated strain was initially inoculated with varying spore powder concentrations at water activities of 0.99, 0.95, 0.90, 0.87, 0.83, and 0.77, combined with diverse culture temperatures of 15°C, 20°C, 25°C, 30°C, and 35°C on PDA. The growth of colony area was daily measured by ImageJ software, and spore powder yield was observed after a 7-day incubation period under these specified culture conditions.
 Results: (1) colony area of isolated C. asperulatum CY-H1 has a rising trend with increasing culture time at consistent temperatures. Sporulation exhibited an initial increase followed by a decrease, yet overall demonstrated an upward trajectory. The maximum value was attained at a water activity of 0.95. Under identical water activity, escalating temperatures initially augmented colony area until reaching a maximum at 25°C. Similarly, spore production displayed an initial increase followed by a decrease, with the highest production occurring at 25°C. (2) Varied concentrations of initial spore suspension influenced colony growth differently. A high concentration (initially 106 spores/mL) exhibited an earlier growth trend compared to medium (105 spores/mL) and low concentrations (104 spores/mL). (3) C. asperulatum CY-H1 did not thrive under the culture conditions of 35°C, irrespective of the initial inoculation concentration and water activity.
 Conclusion: C. asperulatum assumes a crucial role as a predominant mold in tobacco storage environments. Effective prevention of fungal mildew in stored tobacco leaves is achieved by maintaining temperatures exceeding 35°C and sustaining water activity below 0.77. These findings highlight the significance of environmental conditions in controlling mold growth during tobacco storage.

Steinernema yirgalemense (Steinernematidae) to control Phlyctinus callosus and Phlyctinus xerophilus (Curculionidae)

Summary Considerable progress has been made in the surveying, taxonomy, screening, mass production and formulation of entomopathogenic nematodes (EPN) and their associated symbiotic bacteria in South Africa. Steinernema yirgalemense isolate 157-C is one of the most promising native EPN candidates with regards to virulence, its broad insect-host spectrum, and can be readily mass-produced and formulated into a commercial product. The banded fruit weevils, Phlyctinus callosus sensu stricto and Phlyctinus xerophilus, previously grouped together under the Phlyctinus callosus sensu lato species concept, are native entimine weevils of economic importance to deciduous fruit, grapevine and berries in the Western Cape province of South Africa. This study investigated potential differences in baseline susceptibility of larvae and pupae of the two weevil species to S. yirgalemense in laboratory screenings. The test arena used was 24-well bioassay plates, with an inoculation concentration of 200 infective juveniles (IJ) insect−1 for larvae and 100 IJ insect−1 for pupae. Infection was determined 48 h and 96 h after inoculation. Field-efficacy of S. yirgalemense, applied at a concentration of 60 IJ cm−2, against larvae of the two weevil species was determined in an ecologically relevant semi-field trial. In all cases in vitro mass-produced IJ of S. yirgalemense were used. No baseline differential susceptibility between P. callosus and P. xerophilus larvae was obtained in laboratory screenings. Phlyctinus pupae were approximately twice as susceptible compared to larvae, with significant differences between bioassay batches. Approximately 45% control of P. xerophilus larvae was obtained after 96 h of exposure to S. yirgalemense in the field, differing significantly from the control and P. callosus treatment. Low levels of Phlyctinus larval infection by native EPN (confirmed as Heterorhabditis bacteriophora from one P. xerophilus cadaver) occurred in both control and EPN treatment groups under field conditions.

Unraveling the pathogenic potential of the Pentatrichomonas hominis PHGD strain: impact on IPEC-J2 cell growth, adhesion, and gene expression.

Pentatrichomonas hominis, a flagellated parasitic protozoan, predominantly infects the mammalian digestive tract, often causing symptoms such as abdominal pain and diarrhea. However, studies investigating its pathogenicity are limited, and the mechanisms underlying P. hominis-induced diarrhea remain unclear. Establishing an in vitro cell model for P. hominis infection is imperative. This study investigated the interaction between P. hominis and IPEC-J2 cells and its impact on parasite growth, adhesion, morphology, and cell viability. Co-cultivation of P. hominis with IPEC-J2 cells resulted in exponential growth of the parasite, with peak densities reaching approximately 4.8×105 cells/mL and 1.2×106 cells/mL at 48h for initial inoculation concentrations of 104 cells/mL and 105 cells/mL, respectively. The adhesion rate of P. hominis to IPEC-J2 cells reached a maximum of 93.82% and 86.57% at 24h for initial inoculation concentrations of 104 cells/mL and 105 cells/mL, respectively. Morphological changes in IPEC-J2 cells co-cultivated with P. hominis were observed, manifesting as elongated and irregular shapes. The viability of IPEC-J2 cells exhibited a decreasing trend with increasing P. hominis concentration and co-cultivation time. Additionally, the mRNA expression levels of IL-6, IL-8, and TNF-α were upregulated, whereas those of CAT and CuZn-SOD were downregulated. These findings provide quantitative evidence that P. hominis can promote its growth by adhering to IPEC-J2 cells, inducing morphological changes, reducing cell viability, and triggering inflammatory responses. Further in vivo studies are warranted to confirm these results and enhance our understanding of P. hominis infection.

DEVELOPMENT OF THE TECHNOLOGY OF DEEP CULTIVATION OF A TRANSFORMED B. SUBTILIS STRAIN

Deep cultivation of recombinant cultures in bioreactors makes it possible to accumulate a large number of specific antigens used in the production of immunobiological preparations and diagnostic test systems in a short period of time, taking into account stimulating and limiting growth factors. The main purpose of the research was to determine the influence of the factors of seed concentration, pH level and the level of dissolved oxygen on deep cultivation, with the control of accumulation kinetics by turbidimetry. During the research, it was found that the most optimal inoculation concentration is 1 million cells/cm3 with a maximum final concentration of B. subtilis 4x106 cells/cm3, the most optimal pH level is a dynamic mode from 8.0 to 6.0 with a maximum final concentration of 5x106 cells/cm3, the most optimal level of dissolved oxygen is stationary mode with a minimum oxygen level of 25%, with a maximum final concentration of 4x106 cells/cm3.Bacteriological, technological and biochemical methods were used in the work to develop the modes of deep cultivation of the transformed B. subtilis strain with control of pH parameters and the level of dissolved oxygen.The scientific significance of the research is based on the development of deep cultivation, with an assessment of external growth factors and their influence on the kinetics of accumulation of the transformed B. subtilis strain, which will provide effective conditions for the growth and production of microorganisms. The practical significance of the research is based on the possibility of optimizing the biotechnological stages of the cultivation of the transformed B. subtilis strain, which will positively affect the efficiency of submerged cultivation as the main method for the accumulation of specific antigens.

Transfer of generic Escherichia coli and attenuated Salmonella enterica Typhimurium from the soil to the surface of in-shell pecans during harvest

During harvest pecan nuts are at risk of contamination with foodborne pathogens from extended contact with the ground. The objective of this study was to determine the potential transfer of Escherichia coli and Salmonella from the ground to in-shell pecans during the harvesting process. Plots (2 m2) were sprayed with 1 L of a rifampicin (rif) resistant strain of either E. coli TVS 353 or an attenuated Salmonella Typhimurium inoculum at a low (∼4 log CFU/ml), mid (∼6 log CFU/ml) or high (∼8 log CFU/ml) concentrations. The following day, nuts were mechanically harvested and samples from each plot were collected at 1 min, 4 h, and 24 h. Samples were enumerated for Salmonella and E. coli on tryptic soy agar supplemented with rif. The Salmonella levels in the soil from the inoculated plots were 2.0 ± 0.3, 4.1 ± 0.1, and 6.4 ± 0.2 log CFU/g for the low, mid, and high inocula, respectively. The E. coli levels in the soil from the inoculated plots were 1.5 ± 0.4, 3.7 ± 0.3, and 5.8 ± 0.1 log CFU/g for the low, mid, and high inocula, respectively. There was a significant difference in the average daily rainfall among the three trials. Trial 3 received 23.8 ± 9.2 cm, while trials 1 and 2 received much less (0.1 ± 0.1 0.0 ± 0.0 cm, respectively). Inoculation concentration and trial were significant (P<0.05) factors that influenced the transfer of E. coli and Salmonella to pecans. For the high inoculum treatment, bacterial transfer to pecans ranged from 0.7 ± 0.3 to 4.1 ± 0.2 for E. coli and 1.3 ± 0.7 to 4.3 ± 0.4 log CFU/g for Salmonella. For the medium inoculum treatment, transfer ranged from <0.3 to 1.5 ± 0.1 for E. coli and <0.3 to 1.9 ± 0.2 log CFU/g for Salmonella. For the low treatment, transfer ranged from <0.3 to 0.4 ± 0.2 and <0.3 to 0.5 ± 0.1 log CFU/g for E. coli and Salmonella, respectively. These results show the need for implementing agricultural practices that prevent potential transfer of foodborne pathogens onto the surface of in-shell pecans during harvest.

Fermentation and Quality Characteristics of Yogurt Treated with Bifidobacterium longum.

The fermentation and quality characteristics of yogurt were investigated according to the inoculation concentration of Bifidobacterium longum. The total sugar content of yogurt decreased as the fermentation time increased, and with an increased concentration of B. longum treatment the fermentation time decreased rapidly. As fermentation progressed, the lactose content decreased rapidly at the beginning and gradually decreased as the pH decreased. Depending on the B. longum treatment concentration, the lactose content varied from 0.29 ± 0.01 to 0.47 ± 0.01% and was 0.5% or less in all experimental groups. The experimental group inoculated with 0.0015% of B. longum displayed the best results in all categories, including pH, total acidity, lactic acid content, solid non-fat content, and total lactic acid bacteria count, which are factors that determine the quality of yogurt. In summary, the experimental group inoculated with 0.0015% of B. longum was determined to be the highest quality yogurt.

Remediation of Acid Mine Drainage in the Haizhou Open-Pit Mine through Coal-Gangue-Loaded SRB Experiments

To address the pollution problem of acid mine drainage (AMD) characterized by high concentrations of Fe2+, Mn2+, and SO42−, a combination of coal gangue (CG) and sulfate-reducing bacteria (SRB) was employed. The effects of coal-gangue dosage, SRB inoculation concentration, and temperature on AMD treatment with coal-gangue-loaded SRB were determined through single-factor experiments and response surface methodology (RSM) experiments. By considering the principles of adsorption isotherms, adsorption kinetics, and reduction kinetics, the removal mechanisms of SO42−, Fe2+, and Mn2+ in AMD using coal gangue-loaded SRB in the the Haizhou open-pit mine was revealed. The results showed that the overall effectiveness of the four types of coal-gangue-loaded SRB in repairing AMD was as follows: 3# CG-loaded SRB &gt; 2# CG-loaded SRB &gt; 1# CG-loaded SRB &gt; 4# CG-loaded SRB, with coal-gangue-loaded SRB in the the Haizhou open-pit mine showing the best performance. According to the RSM test, the optimum conditions for repairing AMD with coal-gangue-loaded SRB in the open-pit mine were a coal-gangue dosage of 52 g, SRB inoculation concentration of 11.7%, and temperature of 33.4 °C. The order of factors affecting the removal of SO42− and Fe2+ from AMD by SRB loaded on coal gangue was SRB inoculation concentration &gt; temperature &gt; coal-gangue dosage. For Mn2+, the order of influence was temperature &gt; SRB inoculation concentration &gt; coal-gangue dosage. In the process of repairing Fe2+ with coal-gangue-loaded SRB in the the Haizhou open-pit mine, the biological activity metabolism of SRB played a leading role, while the adsorption isotherm of Mn2+ followed the Freundlich model. The adsorption kinetics of coal-gangue-loaded SRB in the the Haizhou open-pit mine for Fe2+ and Mn2+ in AMD conformed to Lagergren’s second-order kinetic model, while the reduction kinetics of SO42− conformed to a first-order reaction model.

Applications-oriented algicidal efficacy research and in-depth mechanism of a novel strain Brevibacillus sp. on Microcystis aeruginosa

The utilization of algicidal bacteria for the control of harmful algal blooms (HABs) is a promising technology for ecological remediation. In our most recent publication, a novel strain of Brevibacillus sp. was isolated and proved to have significant algicidal activity and stability against Microcystis aeruginosa. In order to verify the algicidal effect of the strain in the practical application scenario, the algicidal efficacy of Brevibacillus sp. under conditions close to water in the environment was investigated. Results indicated that the algicidal threshold of Brevibacillus sp. culture was 3‰ inoculation concentration, and the removal rate of M. aeruginosa reached 100%. The process of Chl-a degradation followed a first-order kinetic model, which could be used to predict the degradation effect of M. aeruginosa in practical applications. Additionally, the inoculation of Brevibacillus sp. culture introduced additional nutrients, some of which remained in the water. Furthermore, the algicidal substances demonstrated good sustainability, with a removal rate of up to 78.53% at 144 h after three repeated uses. At 12 h, the algicidal substances caused a 78.65% increase in malondialdehyde (MDA) content in M. aeruginosa compared to the control group, thereby triggering the antioxidant system of M. aeruginosa. Moreover, algal cell fragments were observed to aggregate. This study provides a promising direction for treating cyanobacterial blooms using algicidal bacteria in practical applications.

Strain Screening and Conditions Optimization in Microalgae-Based Monosodium Glutamate Wastewater (MSGW) Treatment

The wastewater generated from monosodium glutamate production displays distinctive features of elevated salinity, organic content, as well as nitrogen and phosphorus concentrations, and its indiscriminate disposal poses a significant threat to water quality and can cause detrimental impacts on aquatic ecosystems. The application of microalgae for monosodium glutamate wastewater (MSGW) treatment can result in simultaneous wastewater purification and biomass recovery. In this study, the algae species capable of thriving in diluted MSGW were screened, and the wastewater composition and growth conditions were optimized to obtain high algal biomass and nutrient removal rate. Among the tested species, Chlorella sp. FACHB-30 demonstrated superior potential for MSGW treatment and achieved a maximum specific growth rate of 0.28 d−1 and the highest COD removal rate of 61.50% over a 20-day cultivation period with trace metals supplementation in the wastewater. Moreover, the cultivation of Chlorella sp. FACHB-30 yielded considerable reductions in total phosphate (69.09%), total nitrogen (26.93%), and NH4+-N (51.91%) levels in the wastewater. The optimum conditions for achieving maximum algal density and highest nutrient removal were determined as light intensity of 150 μmol m−2s−1, inoculation concentration of 1 × 105 cells mL−1, and an iron concentration of 10−5 mol L−1. Finally, under the optimized conditions, the removal rates of total phosphate, total nitrogen, NH4+-N, and COD were determined to be 87.60%, 68.05%, 75.89%, and 77.96%, respectively. The findings of this study highlight the potential for enhancing the nutrient removal efficiency of microalgae-based MSGW treatment through the implementation of a combined approach that involves the selection of tolerant strains, optimization of cultivation conditions, and refinement of wastewater composition.

Biomass production and astaxanthin accumulation of Haematococcus pluvialis in large-scale outdoor culture based on year-round survey: Influencing factors and physiological response

Astaxanthin is an economically valuable carotenoid pigment, and most natural astaxanthin is produced via the large-scale cultivation of the green alga Haematococcus pluvialis in outdoor culture systems. The effects of various environmental factors on Haematococcus and the physiological response of algal cells have rarely been investigated at commercial production scale, which are important for maximizing the yield and efficiency of algae production. This study found that the light intensity and cumulative light intensity are the most important factors affecting the biomass production and astaxanthin accumulation in outdoor large-scale cultivation of Haematococcus based on a year-round survey, and the cumulative light intensity between 550 and 850 mol photons·m−2 was an optimal range for a reasonable productive harvest. The temperature, diurnal temperature difference, culture period and initial inoculation concentration were found to be associated with production performance. Light requirements for green cell growth and for astaxanthin accumulation of immotile cells were quite different, the late showed a very high tolerance to light intensity. Under high light intensity or low temperature, non-photochemical quenching seemed to play a role in quenching excess energy in red immotile spore cells. This study provided detailed information on Haematococcus biomass production and astaxanthin accumulation at big commercial scale via tubular photobioreactors, which is valuable for improvement of commercial production of Haematococcus.

Subculturing and Gram staining of blood cultures flagged negative by the BACTEC™ FX system: Optimizing the workflow for detection of Cryptococcus neoformans in clinical specimens.

To investigate whether an incubation time of 5 days (Aerobic/F, Anaerobic/F) and 14 days (Myco/F) blood culture bottles is sufficient to prevent false-negative results. We evaluated 1,244 blood bottles (344 patients) defined as negative by the BACTEC™ FX system. We also reviewed published cases and our own cases of bloodstream infection caused by Cryptococcus neoformans and simulated different scenarios, including different inoculation concentrations, bottle types, and clinical isolates. Two bottles (0.16%) were found to contain C. neoformans when subcultured and Gram stained. A 5-day protocol with Aerobic/F bottles was insufficient for the growth of C. neoformans in some cases, and C. neoformans grew better in Myco/F bottles than in Aerobic/F bottles. Subculturing and Gram staining after a 5-day protocol were important for the detection of C. neoformans, and Myco/F bottles should be collected for the blood culture of C. neoformans.

Optimizing the Fermentation Conditions of Cudrania tricuspidata Fruit Using Bacillus amyloliquefaciens for Anti-Inflammatory Activity and GC-MS-Based Volatile Component Characteristics.

The aim of this study is to optimize the performance conditions used for maximum anti-inflammatory activity and to clarify in vitroanti-inflammatory properties of fermented C. tricuspidata fruit. Based on the single-factor experiment and Box-Behnken design, the optimized fermentation conditions of C. tricuspidata fruit for maximum anti-inflammatory activity were 3.8 d fermentation period, 8.4% (v/w) inoculation concentration, and 29.2°C fermentation temperature. Under optimal conditions, anti-inflammatory activity-based nitric oxide of fermented C. tricuspidata fruit reached 93.9%. Moreover, this study provides a theoretical basis and experimental data containing β-hexosaminidase and reactive oxygen species for the medical use and industrialization of C. tricuspidata fruit fermentation. Interestingly, the results of GC-MS analysis confirmed that fermented C. tricuspidata fruits detect volatile components different from unfermented C. tricuspidata fruits. We suggested that this volatile component may have been involved in the anti-inflammatory reaction, but scientific verification of this is needed later. Therefore, an in-depth study of volatile components detected from fermented C. tricuspidata fruits will need to be conducted later.

Phytoremediation Potential of Arbuscular Mycorrhizal Fungi and Berseem in Soil Irrigated with Industrial Wastewater

This article describes a new idea about industrial pollution abatement through berseem ( Trifolium alexandrinum L.) using arbuscular mycorrhizal fungi (AMF) Glomus Spp. as an accelerating agent. For this purpose, berseem plant was grown on soil irrigated with industrial wastewater in pots. Three cuttings of plants were obtained at different intervals. At each cut, plant biomass, plant height, and rate of mycorrhizal infection were recorded and plant tissues (roots, stems and leaves) were analyzed to determine the concentrations of Cd, Cr, Pb, and Zn as indicators. The results showed that the biomass of the roots, stems, and leaves of the inoculated plants was higher than that of the un-inoculated plants. The height of inoculated plants was higher compared with that of uninoculated plants. The mycorrhizal infection rate was recorded highest at the third cut. Heavy metals in plants were within permissible limits for crops irrigated with wastewater) at each cutting. With inoculation, Zn and Cr concentrations in roots and leaves increased and Pb and Cd enhanced in roots but reduced in leaves as compared to un-inoculated plants, indicating that metals accumulated more in roots due to inoculation. The effectiveness can be confirmed by calculating the difference between the inoculated and non-inoculated plants. The new results showed that AMF inoculation promoted plant biomass and height, reduced metal transfer in leaves and stems, and to roots. This research confirmed that the procedure can be used for protecting plants from toxicity. Keywords: arbuscular mycorrhizal fungi, heavy metals, biomass, mycorrhizal infection rate, berseem. https://doi.org/10.55463/issn.1674-2974.50.4.6

Development and application of a virus-induced gene silencing system for functional genomics in pecan (Carya illinoinensis)

Pecan is a high-value nut tree that cultivated globally. Nowadays, in-depth studies of this species are often hampered by lacking of efficient molecular genetic tools. To facilitate functional genomics in pecan, a virus-induced gene silencing (VIGS) system based on tobacco rattle virus (TRV) was explored in this study. We used pecan magnesium chelatase subunit H (CiChlH) gene as a visual indicator to evaluate the effect of several factors including infiltration method, insert size, insert orientation, inoculation concentration, host genotype, and culture temperature on VIGS. Our results showed that the optimized conditions for pecan VIGS system were as followed: construction of TRV-based vector harboring ∼300 bp target sequence in sense or antisense orientation, adjustment of inoculation solution to a final OD600 of 0.7, use of syringe infiltration method, inoculation of ‘Pawnee’ or ‘Shaoxing’ seedlings at 2∼4 true-leaf stage with obvious shoot apical growth, and subjecting plants to a temperature cycle of 24 °C (day, 12 h)/16 °C (night, 12 h). Using the optimized system, we obtained an expected photobleaching at 4 weeks post infiltration. To further verify the stability of the system, a pecan phenylalanine ammonialyase (CiPAL) gene was used for silencing and we detected a significant reduction in total phenolics in silenced plants with respect to control seedlings, as anticipated. Altogether, the TRV-based VIGS system has been successfully established in pecan and could be used for future reverse genetic studies.

Optimization of environmental factors in a dual invitro biofilm model of Candida albicans-Streptococcus mutans.

The biofilm formation of Streptococcus mutans-Candida albicans is an important virulence factor for dental caries. The purpose of this study was to determine the effect of some environmental conditions on the biofilm formation like inoculation concentration, temperature, sugar, amino acid, metal ions and saliva, and then establish a persistent invitro biofilm model for further research. Based on the single factor experiment, the factors participating in the biofilm formation including sugar, inoculation concentration, and saliva increased the biofilm mass, while amino acid, metal ions, temperatures reduced biofilm mass. Optimal conditions for biofilm formation were the inoculation dosage of S. mutans and C. albicans of 108 and 107 , respectively, the addition of 0·3 g l-1 sucrose and sterile saliva. These results contribute to a deep understanding of the factors involved in oral biofilm formation of the important cariogenic pathogen S. mutans and the opportunistic pathogen C. albicans to study better for biofilm and promote the design of new therapeutic approaches. The present research also provides a model for evaluating the therapeutic potential for drugs in the future.

Effect of Metschnikowia pulcherrima on Saccharomyces cerevisiae PDH By-Pass in MixedFermentation with Varied Sugar Concentrations of Synthetic Grape Juice and Inoculation Ratios

The effects of Metschnikowia pulcherrima and high glucose osmolality on S. cerevisiae pyruvate dehydrogenase pathway (PDH) by-pass were examined by varying the starting sugar concentration of synthetic grape juice and the inoculation ratio of S. cerevisiae to M. pulcherrima. The findings revealed that M. pulcherrima and osmolarity impacted S. cerevisiae’s PDH by-pass. The inoculation concentration of M. pulcherrima significantly affected pyruvate decarboxylase (PDC) activity and acs2 expression when the initial sugar concentration was 200 g L−1 and 290 g L−1. The osmolarity caused by the initial sugar (380 g L−1) significantly influenced the enzymatic activity of S. cerevisiae, which decreased PDC and acetaldehyde dehydrogenase (ALD) activities while increasing Acetyl-CoA synthetase (ACS) activity. The reduction in acetic acid in the wine was caused by M. pulcherrima altering the initial sugar concentration faced by S. cerevisiae, which in turn affected enzymatic activity. The alteration of enzyme activity and accumulation of primary metabolites revealed why mixed fermentation could reduce the acetic acid content in wine by altering the enzymatic activity and affecting the expression of several key genes. The M. pulcherrima inoculation levels had no significant effect on the acetic acid and glycerol concentration in the same fermentation medium.

Red Sea Bream Iridovirus (RSIV) Kinetics in Rock Bream (Oplegnathus fasciatus) at Various Fish-Rearing Seawater Temperatures.

Simple SummaryRed sea bream iridoviral disease (RSIVD) generates serious economic losses by causing mass mortality events of rock bream during the season with high water temperature in the Republic of Korea and other Asian countries. However, very few studies have investigated RSIV kinetics in rock bream under various rearing water temperatures. In this paper, we investigated the viral load shedding of RSIV into seawater after artificially infecting rock bream (Oplegnathus fasciatus) with the virus. Overall, our data suggest that the viral load shedding of RSIV into seawater varies depending on water temperature and virus inoculation concentration. Our results reveal the potential of non-invasive virus detection approaches, such as the utilization of environmental DNA in fish farms. In addition, we showed that the quantitative analysis of seawater viruses can indirectly improve our understanding of disease progression in fish, potentially contributing to enhanced disease control.Red sea bream iridoviral disease (RSIVD) causes serious economic losses in the aquaculture industry. In this paper, we evaluated RSIV kinetics in rock bream under various rearing water temperatures and different RSIV inoculation concentrations. High viral copy numbers (approximately 103.7–106.7 RSIV genome copies/L/g) were observed during the period of active fish mortality after RSIV infection at all concentrations in the tanks (25 °C and 20 °C). In the group injected with 104 RSIV genome copies/fish, RSIV was not detected at 21–30 days post-infection (dpi) in the rearing seawater. In rock bream infected at 15 °C and subjected to increasing water temperature (1 °C/d until 25 °C) 3 days later, the virus replication rate and number of viral copies shed into the rearing seawater increased. With the decrease in temperature (1 °C/d) from 25 to 15 °C after the infection, the virus replicated rapidly and was released at high loads on the initial 3–5 dpi, whereas the number of viral copies in the fish and seawater decreased after 14 dpi. These results indicate that the number of viral copies shed into the rearing seawater varies depending on the RSIV infection level in rock bream.