Growth and Phytase Activities of Bacillus subtilis IMV B-7023 During Cultivation with Sodium Phytate

Bacteria of the genus Bacillus are known for their ability to mineralize organic phosphorus compounds. Phytates constitute up to 60–80% of the total plant phosphorus and almost 50% of soil organic phosphorus. Phytates phosphorus is unavailable for plants. Bacillus can synthesize phosphatases both wide spectrum of action, and highly specific phytases that catalyze the hydrolysis of phytates. Therefore, the aim of this work was to study the growth and phytase activity of Bacillus subtilis IMV B-7023, which is the component of the ”Azogran” complex bacterial preparation for plant growing. Methods. The growth activity of bacteria was studied by cultivation methods, the phytase activity – by measuring the amount of phosphate released from sodium phytate during the enzymatic reaction. Results. It was shown that B. subtilis IMV B-7023 assimilated phytate as the source of phosphorus nutrition during cultivation in media with 0.5, 1.0 and 2.0 g/L of sodium phytate. The highest growth activity of these bacteria was observed after two days of cultivation in medium with 1.0 g/L of phytate. The number of bacteria was (3.91±0.32)×109 CFU/mL under these conditions. At the same time, B. subtilis IMV B-7023 demonstrated a low level of phytate assimilation as a source of carbon nutrition. Thus, after two days of cultivation the number of bacteria increased from (4.12±0.09)×106 CFU/mL to (1.07±0.07–3.11±0.51)×107 CFU/mL in the presence of 0.5–2.0 g/L phytate in the medium and the absence of another carbon source. It was determined that strain B. subtilis IMV B-7023 had phytase activity, the highest activity (221.85±0.12 U/g) was on the first day of their cultivation in medium with inorganic phosphates. It should be noted that B. subtilis IMV B-7023 phytase activity was lower during cultivating in medium with sodium phytate as a source of phosphorus nutrition, than in medium with inorganic phosphates. The obtained fact may be due to phytate hydrolysis by widespecific phosphatases. Higher rates of phytase activity obtained on the first and third days compared to the second and fourth days of bacterial cultivation may indicate the expression of phosphatases genes only in the period required for maximum bacterial development, in the absence of these proteins in the media. At the same time, the phytase activity of B. subtilis IMV B-7023 after 2 days cultivation in a media with 0.5 and 1.0 g/L of sodium phytate (194.80±0.15 U/g and 160.90±0.13 U/g, respectively) as the source of carbon and phosphorus was higher compared to the activity of bacteria on medium with inorganic phosphates (137.79±0.10 U/g). This may be caused by the synthesis of a larger number of highly specific phosphatases (phytases) in bacterial cells at the presence of only phytate in the medium as a substrate. Conclusions. B. subtilis IMV B-7023 strain is characterized by growth on nutrient medium with sodium phytate and phytase activity. Because they are soil microorganisms used as the component of the ”Azogran” complex bacterial preparation for plant growing, the ability to hydrolyze and assimilate phytate is important for functioning of this strain in the rhizosphere. The obtained results extend the understanding of B. subtilis IMV B-7023 influence on phosphorus nutrition and development of plants.

Effect of Carbon Sources on Different Strains of Phytase-Producing Bacteria Isolated from Malaysia’s Hot Spring

Animal feed from cereal grains and oilseed meals mainly containing phytic acid which has adverse effects on animal nutrition and its environment. Ruminants can easily digest the phytic acid as they have fungi and bacteria in their guts which can produce phytase to degrade the phytic acid. Meanwhile, phytic acid in non-ruminant animals is poorly digested due to the lack of sufficient phytase in their guts. Thus, the feed must be supplemented with inorganic phosphate to ensure it can absorb adequate nutrients. This study aimed to determine the effects of using different carbon sources to the growth of different strains of phytase producing bacteria based on optical density (OD), colony forming unit (CFU), and their phytase production. All four strains of potentially producing-phytase bacteria have been isolated from several hot springs in Malaysia. The bacteria were grown in modified Phytase Screening Medium (PSM) with glucose and lactose as a carbon source and under optimum culture conditions (pH 5.5, 37˚C, 200 rpm) for 72 hours. For quantitative screening of phytase production, the bacterial cultures were harvested to obtain the supernatants that were used to measure the amount of inorganic phosphorus released by the bacterial strains. Among these carbon sources, glucose has shown consistency between their CFU counts and the observed ODs whereas lactose shown inconsistency. Meanwhile, the maximum phytase activity was recorded for all strains in the presence of glucose in which bacteria strain L3 (0.0404 U/mL), RT (0.0359 U/mL), B9 (0.0262 U/mL), and A (0.0263 U/mL). As for the overall, strain L3 (Labis, Johor) gave a promising rate of inorganic phosphate released with optimum phytase activity value of 0.0404 U/mL in presence of glucose and lactose. The optimisation of the fermentation medium can contribute to more economical production of industrial enzyme as phytase has the potential to produce feed additives for poultry feeding.

Isolation and identification of phytate-degrading yeast from traditional fermented food

Suryani AE, Anggraeni AS, Istiqomah L, Damayanti E, Karimy MF. 2021. Isolation and identification of phytate-degrading yeast from traditional fermented food. Biodiversitas 22: 866-873. Application of phytase (myo-inositol hexakisphosphate phosphohydrolase) to catalyze the release of phosphate from phytates contained on grain-based feed has been used widely in poultry feed industry. In this study, yeast as phytase producer from traditional fermented food was isolated, screened and identified their morphological, biochemical, and molecular characteristics. Production of extracellular phytase from yeast was quantified using spectrophotometer. The results showed that among 8 yeast isolates that had phytase activity, there were two isolates with the highest phytase activity and specific activity which were TKd3 isolate (6.57 U/mL and 54.230 U/mg) and GF1 (6.07 U/mL and 53.68 U/mg). Morphological identification using Scanning Electron Microscope revealed that TKd3 cells isolated from soybean tempeh had an elongated oval cell structure, whereas the GF1 isolated from fresh gatot had a rounder cell structure. TKd3 isolate with accession number MW131530 had homology with Candida tropicalis ATCC 750 28S rRNA with 99.83% similarity and GF1 isolate with accession number MW131531 had homology with Candida tropicalis ATCC 750 28S rRNA with 100% similarity. It could be concluded that C. tropicalis yeast from traditional fermented food produced the extracellular phytase for further use of phytase in poultry feed additive.

Phytic Acid in Brown Rice Can Be Reduced by Increasing Soaking Temperature

Phytic acid (PA) is a storage form of phosphorus in seeds. Phytase enzyme is activated at germination and hydrolyses PA into myo-inositol and inorganic phosphate. PA inhibits the absorption of minerals in the human intestine by chelation. Its degradation, therefore, is a key factor to improve mineral bioavailability in rice. Germinated brown rice (GBR) is favoured because it improves the availability of nutrients, and thus have a positive effect on health. In this study, we show the effects of soaking temperature on phytase activity and PA content in GBR. Rice phytase showed thermostability and its activity peaked at 50 °C. After 36 h of soaking, phytase activity was significantly increased at 50 °C and PA content was significantly decreased, compared to that at 30 °C. Zinc (Zn) analysis revealed that there was no significant difference in Zn content among different temperature treatments. Calculated total daily absorbed Zn (TAZ) was significantly higher in GBR compared with non-soaked seeds. Moreover, brown rice grains germinated at 50 °C showed a higher TAZ value than that at 30 °C. Seed germination and seed water soaking at high temperatures reduce PA content in brown rice showing a potentially effective way to improve mineral bioavailability in brown rice.

Effect of pH on Different Strains of Phytase Producing Bacteria from Malaysia’s Hot Spring

In the recent research, the optimisation of culture condition for phytase production rarely done for Acetinobacter baumanii. The optimisation of the phytase production from the bacterial strains largely contributed by Bacillus sp. The study on the phytase originated from hot spring are limited and the species that identified from the hot spring samples are not in the same species from the previous study and mainly the species isolated from Bacillus sp. In this study, four potential strains of bacteria producing phytase isolated from hot spring in several regions in Malaysia. For enrichment of the bacterial, Nutrient Agar was used, meanwhile for batch culture optimisation, the bacteria producing phytase grown in modified liquid Phytase Screening Media with soy extract as agro residual substrate as a replacement for sodium phytate, the chemical substrate. The bacteria were screened for their ability to produce clear zone in solid PSM with sodium phytate as substrate. Optimisation of media through its physical factor that is pH of the media carried out using shake flask scale in laboratory. The growth of the bacterial strains and phytase activity measured quantitatively through the two different pH of media at pH 5.5 and pH 7. The analysis of colony-forming unit and pH determination after fermentation was carried out in this study. From the study, bacterial strain L3 from Labis, Johor has the highest phytase activity in the two parameters studied where the inorganic phosphate released at pH 5.5 (0.21953 U/mL) and pH 7 (0.2047 U/mL). Optimisation carried out through manipulating the culture condition that is pH of the media to determine at which condition has the highest phytase production. Several effects on enzyme activity caused by culture conditions identified. The optimisation of the fermentation medium able to contribute to the less cost production of the industrial enzyme as phytase has potential production for feed additives for poultry feeding. In the future research, molecular identification of the bacterial strains from the better-quality bacteria producing phytase grown in optimised culture media to investigate the molecular identity of the bacterial.