Selection of Optimal Conditions for Cultivation of Bacillus megaterium UCM B-5710 – Producer of Keratinase

Every year the volume of production of poultry products all over the world is growing steadily. This contributes to a constant increase in the amount of by-products of poultry processing in the form of down and feather waste, which are dangerous for the environment due to the hard-to-degrade keratin protein and a large number of microbial pathogens. Therefore, the use of environmentally friendly methods for the destruction of keratin substrates due to keratinases of microorganisms is an urgent area of research. The aim of this work was to select the optimal cultivation conditions for the Bacillus megaterium strain UCM B-5710 to increase the activity of the keratinase synthesized by it. Methods. The culture was grown at 28°C, 201 rpm for 7 days on a basic nutrient medium containing defatted chicken feathers as the only source of carbon and nitrogen. The selection of optimal cultivation conditions was carried out according to the following parameters: temperature (21°C, 28°C, 42°C), stirring speed (201 rpm, 212 rpm), amount of inoculum (5%, 10%, 15% , 20%, 25%), the initial pH value of the nutrient medium (4.0–11.0), concentration of keratin-containing substrate (0.1%, 0.2%, 0.5%, 1.0%, 1.5%, 2.0%), additional carbon source (glucose, galactose, lactose, maltose, sucrose, mannitol, potato and corn starch, soluble starch, soybean meal) and nitrogen (NH4Cl, NH4NO3, (NH4)2SO4, NaNO3, urea, peptone, tryptone, yeast extract and soybean meal) at a concentration of 1%. Keratinase activity was assessed by the UV absorption at 280 nm of the hydrolysis products of keratin-containing raw materials. Protein was determined by the Lowry method. Results. The dynamics of the enzyme synthesis showed that the culture of B. megaterium UCM B-5710 exhibited the highest keratinase activity on the 3rd day, and complete splitting of feathers was observed on the 4–5th days. The selection of the concentration of the keratin-containing substrate showed that 0.5% is the optimal concentration. The study of the influence of the initial pH value of the nutrient medium indicates that the culture grew well at pH 6.0–7.0 and pH 9.0–11.0, but at pH 8.0 its growth was very weak. The culture exhibited the maximum keratinase activity at pH 10.0. In addition, at this pH value, complete splitting of feathers was visually observed. The influence of such a key factor as temperature on the growth and synthesis of the enzyme by B. megaterium UCM B-5710 culture demonstrated complete splitting of feathers already on the 2nd day of cultivation at 42°C, at 21°C the culture split feathers very poorly. The introduction of the inoculum into the composition of the nutrient medium in an amount of 15% of the volume of the medium and the mixing intensity of 212 rpm turned out to be optimal. Besides, it was shown that the introduction of an additional source of carbon or nitrogen had an ambiguous effect on the level of keratinase activity of B. megaterium UCM B-5710. Complete inhibition of enzyme synthesis was observed when ammonium sulfate was added to the nutrient medium, and partial inhibition was observed in the case of glucose, lactose, and maltose. Potato, corn, and soluble starch stimulated keratinase synthesis. The majority of inorganic nitrogen sources (ammonium chloride and nitrate) did not affect the synthesis of B. megaterium UCM B-5710 keratinase, while organic sources (urea, peptone, tryptone, yeast extract) increased the level of keratinase activity by 20–50%. However, the most effective result was obtained using soybean meal, the addition of which to the nutrient medium increased the keratinase activity by 2.5 times. Conclusions. As a result of the studies, the optimal conditions for cultivation of the B. megaterium UCM B-5710 strain were selected: the optimum temperature for the growth and development of the culture is 42°C, the initial pH value is 10.0, the stirring speed is 212 rpm and the amount of inoculum introduced is 15%, an additional source of carbon and nitrogen in the form of soybean meal at a concentration of 0.5%. This made it possible to increase the activity of keratinase by 4 times.

Growth of Pancreas and Intestinal Enzyme Activities in Growing Goats: Influence of a Low-Protein Diet

A dependence between dietary protein and starch levels flowing to the duodenum has been characterized in monogastric animals for optimal enzymatic secretions of the pancreas, but those in ruminants remain unclarified. The present experiment was conveyed to assess the pancreas growth and mRNA expression of the small intestine enzymes in growing goats fed a low-protein diet. Twenty-four Liuyang goats (19.55 ± 3.55 of body weight (BW)) and aged approximately 8 months were randomly assigned to either a control protein diet (NP: 10.77% CP) or a low-protein diet (LP: 5.52% CP) for 70 days. The results show that no statistical differences (p > 0.05) were observed in the pancreas growth indices between the groups. Pancreas and small intestine α-amylase and lipase activities were unaffected (p > 0.05) by the LP diet, while activities of trypsin and chymotrypsin were decreased (p < 0.05). The LP diet reduced (p < 0.05) the mRNA expressions of trypsin and chymotrypsin in the duodenum and jejunum, and had no effects (p > 0.05) on the mRNA expressions of α-amylase and lipase. Goats fed with the LP diet had higher (p < 0.05) concentrations of cholecystokinin and insulin than those fed with the NP diet. In conclusion, feeding an LP diet (5.52% CP) had no profound influence on pancreas growth and digestive enzyme synthesis in goats.

The effect of probiotics on digestive enzyme activity during larvae and juvenile stage of Yellow Fin Tuna (Thunnus albacares)

Yellow fin tuna fry production technology is not successful yet. This was assumed related to the biological and physiological characteristics of the fish. The physiological approach to regulating digestion through probiotics is needs to be studied. The aim of this study was to examine the effect of probiotics bacterial strains of the enzymatic activity in digestion system for the larvae to juvenile stages of yellow fin tuna, Thunnus albacares. The experiment was initiated by culturing 3 probiotic isolates (Bacillus subtilis strain TA-1, Bacillus amyloliquefaciens strain TN-2, Bacillus subtilis strain TO-4) and apply to the larval rearing of yellow fin tuna. In the experiment the treatments applied were supplemented probiotics and without probiotic, each treatment was held with 2 replications. Fish samples were collected every day and analyze the gene expression profile associated with digestion enzyme synthesis by total RNA isolation and cDNA synthesis followed analysis of its by using the RT –qPCR method. The results showed that the growth response of T. albacares tuna larvae reared with probiotics tended to be faster (P<0.05) than the control (without probiotics). The supplementation of probiotic could improved the expressed digestibility of the target genes associated with enzymatic synthesized. Amylase enzymatic synthesized increase 200-1200 times in larvae 10 –17 Day After Hatch (DAH), while in the lipase enzymatic activity started with 13 DAH ( 25 time) and 16 – 21 DAH by 150-300 times. In trypsin the highest enzymatic activity was at 16 DAH, which was 200 times. While in control, the expression of enzymatic synthesis of amylase, lipase, and trypsin was relatively low.

Influence of Bacterial Inoculation on Growth and Plant Nutrition of Peach Grafted in Different Rootstocks in Calcareous Soil

The highly calcareous soil limits plant growth parameters due to inadequate uptake of plant nutrients. Calcareous soil conditions affect plant growth through impaired chlorophyll synthesis, root growth, enzyme synthesis, and nutrient uptake. To overcome the negative effect of calcareous soil, six bacterial strains namely Alcaligenes 637Ca, Agrobacterium A18, Staphylococcus MFDCa1, Staphylococcus MFDCa2, Bacillus M3, and Pantoea FF1 were inoculated in one-year-old plants of peach cultivar ‘Elegant Lady’ grafted onto GF677 and Nemaguard rootstocks. The bacterial treatments were observed to improve plant growth and nutrient content compared to the control. Moreover, the GF677 rootstock was observed to be more tolerant to high calcareous soil conditions than Nemaguard, showing better plant growth and nutrient content. At the Nemaguard rootstocks, the largest leaf area was observed to be upon inoculation with MFDCa2 (29.1 cm2), FF1 (28.8 cm2), and M3 (28.1 cm2), whereas at the GF677 rootstock, the highest leaf area was observed upon inoculation with MFDCa1 (34.7 cm2), FF1 (32.6 cm2), and 637Ca (31.5 cm2). The leaf iron content was higher in bacterial treatments than the control. In the Nemaguard rootstock, the highest iron content was measured in plants inoculated with 637Ca (133.49 mg kg–1) and M3 (127.64 mg kg–1), whereas in the GF677 rootstock, the treatments MFDCa1 (131.51 mg kg–1), 637Ca (131.21 mg kg–1), FF1 (127.72 mg kg–1), and M3 (127.68 mg kg–1) resulted in high iron content. The results indicate that bacterial inoculations have a significant potential to improve plant growth and can be used as biofertilizers for peach grafted onto Nemaguard and GF677 in high calcareous soil conditions.

Selenium Nanoparticles as a Natural Antioxidant and Metabolic Regulator in Aquaculture: A Review

Balanced aquafeed is the key factor for enhancing the productivity of aquatic animals. In this context, aquatic animals require optimal amounts of lipids, proteins, carbohydrates, vitamins, and minerals. The original plant and animals’ ingredients in the basal diets are insufficient to provide aquafeed with suitable amounts of minerals. Concurrently, elements should be incorporated in aquafeed in optimal doses, which differ based on the basal diets’ species, age, size, and composition. Selenium is one of the essential trace elements involved in various metabolic, biological, and physiological functions. Se acts as a precursor for antioxidative enzyme synthesis leading to high total antioxidative capacity. Further, Se can enhance the immune response and the tolerance of aquatic animals to infectious diseases. Several metabolic mechanisms, such as thyroid hormone production, cytokine formation, fecundity, and DNA synthesis, require sufficient Se addition. The recent progress in the nanotechnology industry is also applied in the production of Se nanoparticles. Indeed, Se nanoparticles are elaborated as more soluble and bioavailable than the organic and non-organic forms. In aquaculture, multiple investigations have elaborated the role of Se nanoparticles on the performances and wellbeing of aquatic animals. In this review, the outputs of recent studies associated with the role of Se nanoparticles on aquatic animals’ performances were simplified and presented for more research and development.

Low Light Alters the Photosynthesis Process in Cigar Tobacco via Modulation of the Chlorophyll Content, Chlorophyll Fluorescence, and Gene Expression

Shading or low light (LL) conditions are a key and necessary cultivation technique in cigar wrapper tobacco production. However, the effect of low light on the photosynthesis in cigar tobacco is not clear. Therefore, this study is designed to know the photosynthesis of cigar tobacco under different light intensities (T200, T100, and T50 μmol m−2 s−1). The results reveal that under low light, T50 especially improved the light interception and increased carbon utilization, as witnessed by a higher specific leaf area and lower specific leaf weight. Low light intensity caused better light interception and carbon utilization in cigar tobacco leaves, and thus thinner leaves are more able to use low light efficiently. The chlorophyll content is related to the photosynthesis process; thus, LL affected the photosynthesis process by lowering the chlorophyll content. Similarly, LL also altered the photosynthetic efficiency by lowering the QY_Lss, qP_Lss, and Rfd_Lss. Additionally, higher expression of Lhcb4.2, Lhcb6, PsbA, PsbB, and PsbD under low light, especially T50, shows that the PSII and antenna proteins complex efficiently utilized the absorbed energy for photosynthesis. Finally, the lower photosynthesis, particularly in T50, is attributed to the downregulation of genes related to NADPH production (petH) and the rubisco enzyme synthesis-related gene (rbcs) for CO2 fixation in the Calvin cycle. Overall, the results show that the photosynthesis is decreased under LL intensities which might be related to lower chlorophyll content and downregulation of petH and rbcs genes.

Dual effects of 9-cis retinoic acid on ACTH-dependent hyperplastic adrenal tissues

Retinoids play a pivotal role in adrenal development and differentiation. Recent clinical trials revealed therapeutic potential of both all-trans and 9-cis retinoic acid in patients with cortisol excess due to a pituitary ACTH-secreting adenoma and indicated that retinoids might act also on the adrenal. Aim of the present study was to evaluate the effect of 9-cis retinoic acid on adrenals from patients with ACTH-dependent Cushing’s syndrome. Adrenal specimens from six patients with Cushing’s disease were incubated with 10 nM–1 µM 9-cis retinoic acid with and without 10 nM ACTH. Cortisol secretion was measured by immunoassay and expression of genes involved in steroidogenesis as well as retinoic acid action were evaluated by real-time RT-PCR. Incubation with 10–100 nM 9-cis retinoic acid increased spontaneous cortisol secretion and expression of STAR and CYP17A. On the other hand, in wells treated with ACTH, 9-cis retinoic acid markedly diminished ACTH receptor upregulation and no stimulatory effect on cortisol secretion or steroidogenic enzyme synthesis was observed. ACTH itself increased ligand-induced retinoic acid receptor expression, possibly enhancing sensitivity to retinoic acid. Our findings indicate that the effect of 9-cis retinoic acid in presence of ACTH is distinct from unchallenged wells and support the hypothesis of a direct adrenal action in patients with Cushing’s disease.