Incubation Temperature Research Articles

Spectrophotometric and cloud point extraction methods to detect Quercetin Dihydrate in supplement formulations and urine samples

The accurate detection and quantification of quercetin dihydrate (QRC) are vital for quality control, pharmacokinetic studies, and bioavailability assessments in pharmaceutical and biological samples. This study aimed to develop and validate a cloud point extraction (CPE) method combined with spectrophotometry for the sensitive and environmentally friendly detection and quantification of QRC in pharmaceutical formulations and spiked urine samples. Methods. The CPE method employed Triton X-114 as a non-ionic surfactant to extract QRC from samples. The extraction process was optimized by evaluating key parameters, including surfactant concentration, incubation temperature, extraction time, and centrifugation speed. Spectrophotometric analysis was conducted before and after extraction to assess the sensitivity and linearity of the method. The method was validated using spiked urine samples and pharmaceutical formulations of QRC, with recovery rates, limits of detection (LOD), and linearity evaluated to ensure accuracy and precision. Results. The optimized CPE conditions included an incubation temperature of 65°C, a 5-minute extraction time, and centrifugation at 3500 rpm. The CPE method significantly improved the sensitivity of QRC detection, reducing the LOD from 0.0351 μg/mL (without CPE) to 0.0234 μg/mL (with CPE). The method exhibited excellent linearity (r² > 0.998) over a wide concentration range (1–12 μg/mL). High recovery rates (98.88% to 101.6%) and low relative standard deviations (RSD < 2%) were observed in pharmaceutical formulations and spiked urine samples, demonstrating the method’s accuracy and precision. The enrichment factor was 1.75, and the preconcentration factor was 4.6. Conclusions. The proposed CPE method combined with spectrophotometry provides a simple, sensitive, and environmentally friendly approach for QRC analysis. It offers significant advantages over conventional methods, including reduced organic solvent use and waste generation, making it suitable for routine analysis in pharmaceutical quality control and pharmacokinetic studies. The method’s adaptability to complex matrices, such as urine, and its potential for broader applications, including the analysis of other polyphenolic compounds, were also demonstrated.

Mechanical Microvibration Device Enhancing Immunohistochemistry Efficiency.

Immunohistochemistry (IHC) is a widely used technique in diagnostic pathology and biomedical research, but there is still a need to shorten the operation process and reduce the cost of antibodies. This study aims to assess a novel IHC technique that incorporates mechanical microvibration (MMV) to expedite the process, reduce antibody consumption, and enhance staining quality. MMV was generated using coin vibration motors attached to glass slides mounted with consecutive tissue sections. Multiple antibodies targeting various antigens were used to stain cancerous and normal tissues, with and without microvibration. Various parameters were tested, including incubation durations, temperatures, and antibody dilutions. The novel method showed the potential to achieve comparable or superior outcomes in significantly less time, utilizing over 10 times less antibody than controls. MMV improved specific staining quality, yielding stronger, and better-defined positive reactions. This was validated through a multicenter double-blind assessment and quantitative image analysis. The possible mechanisms were also investigated. MMV shortens immunohistochemical staining duration, reduces antibody usage, and enhances staining specificity, likely by accelerating antibody movement and diffusion. These improvements translate to time and cost savings, offering clinical and financial value for diagnostic pathology and biomedical research.

Optimized biosynthesis of lytic enzymes by special Trichoderma citrinoviride

The use of Trichoderma filamentous fungi in the wide concept of biocontrol is still a highly relevant topic. The multifaceted nature of their impact on phytopathogenic microorganisms results from the species diversity and complexity of their antagonistic action. The presented research aimed to determine optimal cultivation conditions of two T. citrinoviride strains for the biosynthesis of major enzymes especially those involved in the biocontrol process. Culture conditions were optimized using a three-factor Box-Behnken design to maximize the yield of chitinase and lichenase. The following independent variables were included in the model: incubation temperature, initial pH, and supplementation with fungal biomass. As a result of statistical optimization, unprecedented activities of extracellular lytic enzyme were achieved. For the B1 and B3 strains, the optimal pH was 3.5 or 7.5, respectively, in the determination of chitinase biosynthesis. It was similar for the biosynthesis of β-1.3 and β-1.4 glucanases, but at higher cultivation temperature. The exception was the B3 strain, for which the optimal pH in glucanase biosynthesis was 5.5. The most stimulating culture temperature in the process of chitinase biosynthesis and β-1.3 and β-1.4 glucanases was above 25 °C. In that, the levels of enzyme biosynthesis and corresponding composition culture environment were confirmed to be strain-dependent.

Insights into the methodological perspectives for screening polyunsaturated fatty acids-containing bacteria.

Polyunsaturated fatty acids (PUFA) are vital molecules in the pharmaceutical, medical, and nutritional industries. Exploration of bacterial strains capable of producing significant amounts of PUFAs offers a promising avenue for biotechnological applications and industrial-scale production. However, an extensive screening of several samples from diverse sources is highly needed to identify a potential strain. The present study provides the results of the evaluation of 15 different screening methodologies (including changes in existing protocols in terms of reagent concentration, incubation temperature and time) for identifying PUFA-producing bacteria in comparison to the gold standard method (Gas chromatography-mass spectrometry), for the first time. The results determined the most effective techniques for each critical PUFA, leading to an optimized screening process that saves time and resources. The H2O2 plate assay using 0.5% or 1% H2O2 for 72 & 96h of incubation at 15 °C consistently outperformed others for finding bacteria containing total nutritionally important long chain-PUFA (LC-PUFA), linoleic acid, and arachidonic acid. Whereas the 2,3,5-triphenyl tetrazolium chloride broth assay at 10-15 °C was the most effective and semiquantitative screening methodology for eicosapentaenoic acid (EPA) and alpha-linolenic acid-containing bacteria. Apart from the methodological perspectives, the study also revealed certain potential strains to be targeted in the ongoing research on PUFA-containing bacteria. Further, the manuscript forms the first report on the presence of docosahexaenoic acid (DHA) in Shewanella decolorationis, EPA in Psychrobacter maritimus and Micrococcus aloeverae, and both EPA and DHA in Arthrobacter rhombi. Altogether, the paper generates several thought-provoking insights on the methodological perspectives and identifies potential PUFA-containing bacteria with practical applications in future bacteria-based PUFA research.

Microbial Degradation of Polyester Microfibers Using Indigenously Isolated Bacterial Strain Exiguobacterium Sp.

ABSTRACTSynthetic microfibers are emerging environmental microplastic pollutants released from different industrial and domestic sources. The present investigation describes the isolation of potential bacterial strains from microplastic‐contaminated sites of Bhubaneswar city of Odisha, India. Four morphologically distinct bacterial strains were isolated using 2% polyethylene glycol (PEG) supplemented nutrient agar (NA) medium and were screened for their polymer tolerance ability by growing them on 2%–8% PEG. A single microorganism capable of growing on 8% PEG was selected for biodegradation experiment. Through 16S rRNA sequencing, the selected bacterial strain was identified as Exiguobacterium sp. with gene bank accession number ON318396. The microbial strain's microfiber biodegradation ability was assessed in a laboratory setting over a period of 28 ± 2 days, utilizing optimized conditions with an initial pH of 7, 2 mL inoculum volume, an incubation temperature of 30°C ± 2°C, and 150 rpm, using 2 g of polyester microfiber. In optimum conditions, the weight loss of the treated sample with the selected microbial strain was 19.2%. The polyester degradation was confirmed through scanning electron microscopic images viewing the degradation of the polyester microfiber surfaces. Variation in functional groups confirmed through Fourier transform infrared spectrophotometry. Detection of carbonyl (C═O) group stretching band at 1711 cm−1 through ATR‐FTIR analysis in the treated sample confirmed the polymer biodegradation. The potential isolate can efficiently degrade polyester and, in the future, can be employed as a promising solution for the sustainable treatment of synthetic microfiber pollution.

Prodigiosin, a promising biocontrol agent against Thaumetopoea wilkinsoni (Tams, 1926) (Lepidoptera: Notodontidae)

Serratia marcescens Bizio (Enterobacteriaceae: Serratia) is an entomopathogenic bacterium that produces hydrolytic enzymes and toxins. It also produces a pigment with various biological properties called prodigiosin. The study was conducted at Bilecik Seyh Edebali University in 2023. In this study, the effects of medium, incubation temperature and time on the process of prodigiosin production by S. marcescens strain Se9 and the extraction efficiency of different solvents were optimized for the first time using the orthogonal Taguchi array design. The optimal yield of pigment was achieved by methanol extraction from bacteria grown in tyriptic soy broth medium at 30ºC for 96 hours. The yield of prodigiosin pigment was 83.4±1.7 mg/L in the validation experiment conducted under the optimum conditions determined. The insecticidal potential of pigment against the larvae of Thaumetopoea wilkinsoni (Tams, 1926) (Lepidoptera, Notodontidae) was demonstrated for the first time. While the mortality rate in larvae exposed to 1000 ppm of the pigment was only 40%, it was observed that doubling the applied concentration led to a significant increase in larval mortality, reaching 91%. The LC50 value of the pigment for the fourth larval stage of T. wilkinsoni was determined to be 1192 ppm. The study showed that the pigment prodigiosin may be a promising biocontrol agent for the control of T. wilkinsoni.

Influence of Asparaginase on Acrylamide Content, Color, and Texture in Oat, Corn, and Rice Cookies.

The safety of cereal-based baked goods can be compromised by acrylamide, a processing contaminant and class 2A carcinogen. One method to prevent acrylamide formation is by converting asparagine to aspartic acid using asparaginases. Four different asparaginases were tested using two dough incubation temperatures and dosages for oat, corn, and rice cookies. To evaluate the impact of asparaginases on product quality, color and texture were measured. Acrylamide was reduced by up to 97, 95, and 92% for oat, corn, and rice cookies, respectively, compared to the control. Asparaginase treatment resulted in minor changes in color and texture. There was a strong correlation between acrylamide concentrations in cookies and the free asparagine content of the flour. By minimizing the formation of acrylamide while maintaining product quality, the use of asparaginases offers a promising approach to enhancing food safety standards and protecting public health, potentially influencing regulatory guidelines and consumer preferences.

Investigation of optimal sperm storage conditions for short-term storage

Objective The quality of sperm cells is important role in the success rates of assisted reproduction technology (ART) treatments. The quality of the sperm cells shows variations depending on the temperature of short-term semen storage as well as the methods of semen preparation. Thus, this study aimed to investigate the sperm viability, motility and DNA fragmentation following different sperm preparation methods and short-term storage conditions, respectively. Materials and Methods A total of 25 semen samples were evaluated. In the first part of this study, different incubation temperatures were investigated in two groups, in such the first group involved the semen samples and the second group involved the sperm cells separated by density gradient centrifugation method, respectively. The samples in each group were incubated at 4°C, room temperature (21°C) and 37°C for 24 hours, respectively. The sperm cell qualities were evaluated by mobility analysis, DNA fragmentation by acridine orange staining and sperm cell viability by propidium iodide staining. Results and Discussion The analysis outcome demonstrated that the mobility, DNA fragmentation and viability of the sperm cells were statistically different when incubated at RT (21°C) in both groups. Furthermore, samples prepared by the density gradient centrifugation method were shown to have better quality. The optimum short-term storage temperature was detected to be the room temperature. Conclusion The conclusion of this investigation is crucial to assess storage conditions in ART clinics. This study provides essential data for short-term sperm storage and preparation methods to improve the success rates of ART clinics. Bangladesh Journal of Medical Science Vol. 23 No. 04 October’24 Page : 1137-1141

B-021 Impact of Assay Conditions on FT4 Measurement Accuracy of Equilibrium Dialysis LC/MS/MS Clinical Assay

Abstract Background Reliable FT4 measurements are vital for accurate diagnosis and effective management of thyroid disorders. However, currently used FT4 immunoassays (IAs) lack standardization, leading to significant variations between platforms. Equilibrium dialysis (ED) coupled with LC-MS/MS analysis, considered the gold standard for FT4 measurement, has been adopted as a reference measurement procedure (RMP) for IA standardization. This study aimed to establish a high-throughput version of the routine clinical FT4 assay based on ED-LC/MS/MS technology and assess the impact of altered assay conditions on measurement accuracy. Methods A commercially available micro-ED plate was employed for ED-LC/MS/MS analysis. FT4 extraction from the dialysate was performed by utilizing a 96-well C18 SPE plate, followed by FT4 quantitation via LC-MS/MS in positive ion mode. Certified primary reference material was used to calibrate the assay. To evaluate assay robustness, the effects of minor method modifications were studied, including incubation time of ED, serum/buffer ratios in ED inserts, ED incubation temperature, shaker speed during ED, serum sample dilution ratio, and dialysate sample dilution ratio. Additionally, short-term and long-term stability of serum FT4 were assessed. Results The developed ED-LC/MS/MS assay successfully resolved T4, T3, reverse triiodothyronine (rT3), and other interferences within 4 minutes using C18 chromatography. The assay's linear range (0.5-100 pg/mL) encompassed clinically relevant FT4 concentrations across hypo- and hyperthyroid patient samples. Excellent agreement was observed between the routine assay based on ED-LC/MS/MS and CDC RMP with a Deming regression slope near 1 (95%CI: 0.87-1.06). Furthermore, serum/buffer ratios from 1:1 to 1:2.5 in ED inserts, shaker speeds from 75 to 150 rpm, and ED incubation times from 16 to 20 hours did not significantly affect FT4 measurement. However, elevated ED temperature (38°C) led to a more than 10% increase in FT4 values. Serum FT4 remained stable when exposed to room temperature for 12 hours, five freeze-thaw cycles, and -70°C storage for 1.5 years. Additionally, dilution of sera by up to 3-fold before ED did not alter FT4 measurement results. Diluting dialysate samples at least 3 times prior to SPE extraction proved accurate for samples exceeding the calibration curve range. Conclusions In conclusion, we successfully developed a routine clinical FT4 assay based on ED-LC/MS/MS that generates data comparable to the RMP. The assay exhibits robustness, with several altered conditions not significantly impacting measurement accuracy. This high-throughput and accurate assay demonstrates suitability for both clinical laboratories and large-scale epidemiological studies. Disclaimer: The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry. Use of trade names is for identification only and does not imply endorsement by the Centers for Disease Control and Prevention, the Public Health Service, and the US Department of Health and Human Services.

Random mutagenesis and disulfide bond formation improved thermostability in microbial transglutaminase

Microbial transglutaminase (MTG) from Streptomyces mobaraensis is widely used in the food and pharmaceutical industries for cross-linking and post-translational modification of proteins. It is believed that its industrial applications could be further broadened by improving its thermostability. In our previous study, we showed that the introduction of structure-based disulfide bonds improved the thermostability of MTG, and we succeeded in obtaining a thermostable mutant, D3C/G283C, with a T50 (incubation temperature at which 50% of the initial activity remains) 9 °C higher than that of wild-type MTG. In this study, we performed random mutations using D3C/G283C as a template and found several amino acid substitutions that contributed to the improvement of thermostability, and investigated a thermostable mutant (D3C/S101P/G157S/G250R/G283C) with three amino acid mutations in addition to the disulfide bond. The T50 of this mutant was 10 °C higher than that of the wild type, the optimal temperature for enzymatic reaction was increased to 65 °C compared to 50 °C for the wild type, and the catalytic efficiency (kcat/Km) at 37.0 °C was increased from 3.3 × 102 M−1 s−1 for the wild type to 5.9 × 102 M−1 s−1. X-ray crystallography of the D3C/G283C MTG showed no major structural differences against wild-type MTG. Structural differences were found that may contribute to thermostabilization and improve catalytic efficiency.Key points• Improved heat resistance is essential to broaden the application of MTG.• The MTG mutant D3C/S101P/G157S/G250R/G283C showed improved thermostability.• X-ray crystallography of the disulfide bridge mutant D3C/G283C MTG was elucidated.

Prolonged incubation of frozen-thawed equine spermatozoa for invitro fertilization: A preliminary study using low temperature and INRA96 medium.

A protocol for conventional invitro fertilization (IVF) in horses using fresh semen has been described, using a prolonged incubation in FERT-TALP medium (22 h) at 38.2°C in the presence of penicillamine, hypotaurine and epinephrine (PHE). Our work aimed to develop a protocol that maintains quality parameters in frozen-thawed equine spermatozoa incubated for 22 h in the presence of PHE using different media (FERT-TALP and INRA96) and incubation temperatures (30 and 38.2°C). Twelve frozen ejaculates from four stallions were thawed and then incubated in either FERT-TALP or INRA96 with PHE at 30 or 38.2°C for 22 h. Following incubation, total motility (TM), progressive motility (PM), viability and acrosome integrity were evaluated. The results showed that TM was significantly higher (p < .001) at 30°C in both media, while PM was higher for INRA96 at 30°C compared to 38°C (p < .05). Moreover, INRA96 at 30°C exhibited higher sperm viability and acrosome integrity (p < .001) compared to the other experimental groups. These preliminary results suggest that incubating thawed equine spermatozoa at 30°C with PHE in INRA96 successfully maintains motility, viability and acrosome integrity in equine spermatozoa, indicating its potential use for conventional equine IVF.

A Fluorescent Probe for Hydrazine Based on 4-hydroxycoumarin with High Selectivity and Sensitivity

Background: Therefore, the development of reliable analytical techniques with high selectivity and sensitivity to detect hydrazine is required for the protection of human health and safety. Objectives: Traditional methods for detecting N2H4 are frequently time-consuming, less accurate, and unsuitable for the analysis of living systems. Numerous fluorescent probes for hydrazine have been produced and gained some valuable results recently. The creation of a simple fluorescent probe for hydrazine detection is the goal of this project Method: In this study, 300 µL of probe 3-methyl-2-oxo-2H-chromen-7-yl propionate (MOCP) was mixed with an equivalent amount of the solution of each analyte to obtain the measurement solution. Following a 10-minute room temperature incubation period, the fluorescence spectra of the resultant solution were recorded. Results: The fluorescence intensity of the probe was noticeably enhanced when N2H4 was added to the probe, but almost no fluorescence enhancement was observed when other competitive ions were added. Conclusion: A hydrazine fluorescent probe based on 4-hydroxycoumarin fluorophore was developed. The probe MOCP displayed high sensitivity and selectivity for hydrazine, with a color change from colourless to blue for detection by the naked eye. Moreover, it demonstrated a low detection limit of 20 nM and a fast reaction time of 30 s.

Effect of The Temperature on The Size of Inhibition Zone of the Clindamycin, Levofloxacin, Tetracycline, and Trimethoprim Activity Against Staphylococcus aureus ATCC 25923

Antibiotic sensitivity should be tested. In the sensitivity test, there are technical factors that influence the formation of the inhibition zone diameter. Based on several research one of the technical factors that affect the diameter of the inhibition in the disc diffusion method is the temperature incubation of the media, this must be examined so that it can be controlled to ensure the validity of the sensitivity test results. This study aims to determine the mean, difference, and analyze the diameter of the inhibition zone of the antibiotics namely Clindamycin, Levofloxacin, Tetracycline, and Trimethoprim against Staphylococcus aureus on Mueller-Hinton agar media with incubation temperatures of 33°C, 34°C, 35°C, 36°C and 37°C for 18 hours. This research is observational, with a cross-sectional design. The data used are primary data with 100 data on the diameter of the antibiotic inhibition zone, obtained by measuring the diameter of the inhibition zone with different incubation temperatures. The selection of antibiotics is based on the mechanism of action of antibiotics inhibiting bacteria namely, the cell wall or membranes that surrounds the bacterial cell; the machineries that make the nucleic acids DNA and RNA and the machinery that produce proteins (the ribosome and associated proteins) with a range of inhibition zones based on Internal Quality Control CLSI. The data will be processed univariately and the Repeated Measure statistical test to determine the significance of the difference in the diameter of the formed inhibition zone using the ANOVA test. The results of the measurement of the inhibition zone diameter on the incubation temperature variation showed a significant difference with p-value 0.000 for Levofloxacin, Tetracycline and Trimethoprim, while for p-value Clindamycin is 0.010. Levofloxacin, Tetracycline, and Trimethoprim antibiotics, the higher the incubation temperature, the average diameter of the inhibition zone is smaller, while for Clindamycin the higher the incubation temperature, the higher the average diameter of the inhibition zone is the same. There is an effect of incubation temperature volume on the diameter of the antibiotic inhibition zone in the disc diffusion method antibiotic sensitivity test. The research indicates that incubation temperature affects the diameter of the antibiotic inhibition zone in disc diffusion tests, underscoring the need for standardized and precise testing conditions to ensure accurate and reliable antibiotic sensitivity results.

Establishment and characterization of novel spontaneously immortalized larval cell lines from sablefish Anoplopoma fimbria.

Sablefish Anoplopoma fimbria is a groundfish of the North PacificOceantypically found in sea floor habitat at depths to 2700m. Prized as a food fish with exceptionally high market value, sablefish aquaculture has been sought to provide a sustainable source of this fish to meet market demands. While commercial culture has successfully produced market-sized fish in Pacific coastal environments, production has been hampered by disease and the overall lack of information on sablefish health and immunology. To begin to address these knowledge gaps, herein we describe the isolation and characterization of spontaneously immortalized sablefish larval cell lines (AFL). Six sublines were established from pools of early yolk-sac larvae, while attempts to develop tissue-specific-derived cell lines were unsuccessful. The six yolk-sac larval cell lines each display two morphologies in culture, an elongated fibroblast-like cell type, and a rounded squamous or epithelial-like cell type. Cytogenetic characterization suggests that both cell types are diploid (2n = 48) with 24 pairs of chromosomes, 23 pairs of autosomes, and 1 pair of sex chromosomes. A small proportion (11%) of AFL cells display tetraploidy. Incubation temperature and medium composition experiments revealed HEPES buffered L-15 media containing 10-20% FBS at temperatures between 15 and 18° C yielded optimal cell growth. These growth characteristics suggest that sablefish larval cells display a robustness for varying growth conditions. The establishment of AFL cell lines provides a foundational tool to study the physiology, health, immunology, and cell and molecular biology of sablefish.

Establishment of an Efficient Screening Methods for Resistance of Chinese Cabbage to Clubroot Disease

Clubroot caused by &lt;i&gt;Plasmodiophora brassicae&lt;/i&gt; is an important disease of crucifer crops worldwide. This study aimed to establish an efficient screening method to determine resistant cultivars of Chinese cabbage against &lt;i&gt;P. brassicae&lt;/i&gt;. To do this, we investigated the virulence of seven &lt;i&gt;P. brassicae&lt;/i&gt; isolates using seedlings of susceptible Chinese cabbage cultivar. The isolates exhibited different virulence in the plants and were divided into three groups based on their virulence. When we explored the disease occurrence in Chinese cabbage seedlings according to photoperiod after inoculation of &lt;i&gt;P. brassicae&lt;/i&gt; and incubation temperature, the plants with all-day light showed higher disease severity than seedlings cultivated under 14 hr of light a day. The occurrence of clubroot disease was most severe at 25&lt;sup&gt;o&lt;/sup&gt;C, followed by 20&lt;sup&gt;o&lt;/sup&gt;C and 18&lt;sup&gt;o&lt;/sup&gt;C, but the fresh weight of clubroot of the seedlings cultivated at 20&lt;sup&gt;o&lt;/sup&gt;C was the highest, followed by plants grown at 25&lt;sup&gt;o&lt;/sup&gt;C and 18&lt;sup&gt;o&lt;/sup&gt;C. When the seedlings of two commercial resistant cultivars were inoculated with the mixed spore suspensions of two different pathotype isolates of &lt;i&gt;P. brassicae&lt;/i&gt;, disease severity increased as the spore concentration of the susceptible &lt;i&gt;P. brassicae&lt;/i&gt; isolate among the two strains increased, suggesting that the clubroot development by different pathotype isolates was independent and not influenced by each other. Taken together, our results provide a faster and more accurate screening methods to determine the resistance of Chinese cabbage against &lt;i&gt;P. brassicae&lt;/i&gt;.

Effect of egg incubator temperature on sex differentiation in Korat chickens

The effect of incubator temperature on sex differentiation in Korat chickens was investigated. The experiments were divided into two sets: temperature applied throughout the entire incubation period and temperature applied during certain periods (days 3–6 of incubation) by either increasing above the standard or decreasing below the standard temperature. In each experiment, 300 Korat chicken eggs were separated into three groups of 5 repetitions, with 20 eggs in each group. This was done using a completely randomized design for each experiment: a group using a temperature below the standard for incubation (36.0 °C), a group using the standard incubation temperature (37.7 °C), and a group using a temperature above the standard for incubation (38.0 °C). W chromosomes were detected at hatch; histology examined reproductive structures after 35 days. Increasing the temperature to 38.0 °C throughout the entire incubation period resulted in no significant difference in hatching rates compared to the standard temperature (P > 0.05). Raising the temperature to 38.0 °C throughout the entire incubation and during certain periods resulted in changes in the reproductive structure of chickens, leading to a mismatch between chromosomal and gonadal sex, observed at 9.7% and 5.9% of individuals with W chromosomes possessed testes, indicating a mismatch between chromosomal and gonadal sex. However, decreasing the temperature to 36.0 °C throughout the incubation period resulted in lower hatching rates compared to the standard temperature (P < 0.05). Incubating eggs at 36.0 °C for specific periods resulted in 19.4% of genetic males developing ovaries instead of testes. The presence of ovaries in individuals without W chromosomes indicated this mismatch. The results of this study provide evidence that temperature plays a role in sex differentiation in Korat chickens, as demonstrated by the detection of W chromosomes and histological studies of testes and ovaries. Moreover, this study presents the first evidence in broilers that temperature can affect sex differentiation.

Designing the Properties of Probiotic Kefir with Increased Whey Protein Content

This research unveiled new insights on the impact of incorporating whey proteins into kefir produced using three different methods. This aims to improve its quality and health benefits, primarily as a result of optimal proliferation of probiotic bacteria. In the initial part of the experiment, samples were prepared using three different methods (methods 1, 2, and 3) to examine the impact of introducing whey protein on bacterial count, the content of L(+)-lactic acid, lactase activity, and the lactic acid and ethanol levels. The methods differed primarily in the sequence of the inoculation milk with probiotic bacteria stage in the production cycle, as well as incubation time and temperature. No significant differences were found in the number of yeasts and bacteria between samples with and without whey proteins. However, it was revealed that the 5% addition of whey proteins enhanced the number of probiotic bacteria in kefir produced with method 2 (from 4.86 to 5.52 log cfu/mL) and method 3 (from 3.68 to 4.01 log cfu/mL). The second part of the research investigated the impact of whey proteins on firmness, consistency, cohesiveness, viscosity, color, and water activity of kefir. This part focused on testing samples with lower whey protein contents (1 and 3%, w/v). We found that the addition of 1% and 3% whey proteins resulted in decreased firmness, consistency, cohesiveness, and viscosity compared to the control kefir. On the other hand, the addition of 5% whey proteins resulted in increased firmness and consistency compared to the addition of 1% and 3% whey proteins. The addition of whey protein decreased the white index WI of the kefir samples. Overall, our results revealed that incorporating whey protein concentrate (WPC) in the production of probiotic kefir can enhance its health benefits while maintaining its rheological properties and overall quality.

Potato peel as substrate for Single Cell Protein in animal feed by submerged fermentation

Single-cell protein (SCP) has gained attention in scientific research as a vital source of protein in animal feed and human food. Taking into account the importance of SCP, an attempt was made in the current research to grow Rhizopus oligosporus and Saccharomyces cerevisiae biomass by selecting potato peel as a substrate. The potato peel was treated with sulfuric acid to form potato peel hydrolysate following acid hydrolysis. In 1.0-L potato peel hydrolysate, 2.5-g/L KH2PO4, 0.5-g/L MgSO4, 0.5-g/L NaCl, and 10 g/L yeast extract were taken to prepare growth media. The moisture content of potato peel was examined by following standard procedure of the Association of Official Agricultural Chemists (AOAC). The Kjeldahl method was used to determine the protein content, and glucose was examined by using the dinitrosalicylic acid (DNS) method. Optimization of SCP yield was accomplished by following the ‘one factor at a time’ method. The effect of each parameter, such as incubation period, temperature and pH on SCP production was analyzed. Bioreactor scale-up production of SCP was performed in a jar fermenter. To enhance SCP, yield co-culture of Saccharomyces cerevisiae and Rhizopus oligosporus was used in a bioreactor that yielded 19 g/L of SCP. Thus, as a nutritional supplement for living organisms, SCP can effectively fulfill protein scarcity globally.

Biological characteristics of marine Streptomyces SK3 and optimization of cultivation conditions for production of compounds against Vibiriosis pathogen isolated from cultured white shrimp (Litopenaeus vannamei).

Antibiotic resistance in shrimp farms has emerged as an extremely serious situation worldwide. The main aim of this study was to optimize the cultural conditions for producing new antibiotic agents from marine Streptomyces species. Streptomyces SK3 was isolated from marine sediment and was identified by its 16S rDNA as well as biochemical characteristics. This microbe produced the highest concentration of bioactive secondary metabolites (BSMs) when cultured in YM medium (YM/2). It produced the maximum total protein (41.8 ± 6.36 mg/ml) during the late lag phase period. The optimum incubation temperature was recorded at 30 °C; BSMs were not produced at ≤10 °C within an incubation period of 3-4 days. The suitable agitation speed was found to be 200 rpm with pH 7.00. The proper carbon, nitrogen, and trace elements supplementation consisted of starch, malt extract, calcium carbonate (CaCO3), and magnesium sulfate (MgSO4). The ethyl acetate extract was found to act strongly against three vibriosis pathogens, Vibrio harveyi, Vibrio parahaemolyticus, and Vibrio vunificus, as indicated by the inhibition zones at 34.5, 35.4, and 34.3 mm, respectively. The extract showed the strongest anti-V. harveyi activity, as indicated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of 0.101 ± 0.02 and 0.610 ± 0.04 mg/ml, respectively. Basic chemical investigation of the crude extract using thin layer chromatography (TLC), bioautography, liquid chromatography tandem mass spectrometry (LC‒MS/MS), Fourier transform infrared spectroscopy (FTIR), and proton nuclear magnetic resonance (1H-NMR) revealed that the active components were the terpenoid and steroid groups of compounds. They showed carboxylic acid and ester functions in their molecules.

Lactic acid production by fermentation of hydrolysate of the macroalga Gracilaria corticata by Lactobacillus acidophilus

Macroalgae (Ulva fasciata, Gracilaria corticata, and Sargassum wightii) were collected from the marine environment and used as the substrate for lactic acid production. These macroalgae were pretreated with hydrochloric acid (0.2 to 0.4 N) for various times (20 to 60 min). Additionally, the algal hydrolysate was incubated with cellulase for 24 h at 30 ± 1 °C to achieve enzymatic saccharification. Proximate analysis of these macroalgae was performed, and the yield was high in G. corticata. The G. corticata hydrolysate was composed of 10.01 ± 0.12% ash content, 1.25 ± 0.2% total fat, 10.2 ± 0.1% crude protein, 9.2 ± 0.2% moisture content, and a higher level of total carbohydrate (69.33 ± 1.5%) than the other two macroalgae. In G. corticata, the enzymatic treatment showed the maximum reducing sugar (33.5 ± 2.3%) relative to the other macroalgal hydrolysates and was considered for optimization of lactic acid production. Lactobacillus acidophilus (MTCC447) utilized pretreated G. corticata hydrolysate (enriched with 5% yeast extract), and maximum lactic acid yield was achieved after 72 h, 30 °C incubation temperature, and 6% inoculum (1×108 CFU/mL) in static culture condition. Batch fermentation was performed in the 1-L bioreactor at room temperature (30 °C) for 96 h. Lactic acid production was maximum within 72 h and the pH value was depleted. The present finding indicates that G. corticata could be used as a substrate for lactic acid production.