Studies on phytase producing yeasts and its application in food and agriculture

Abstract

Introduction: Phosphorus (P) is one of the major plant nutrients which affects physiology and morphology of the plants. Plants mainly utilize inorganic P, which is supplied through fertilizers. The fertilizer demand and consumption in India has been tremendously increased nearly 100 times in last few decades while P source is getting diminished. Sustainable agriculture and finding of alternative phosphorus sources are the only solutions for the above problem (Gujar et al. 2013). Phytic acid (myo-inositol - hexakisphosphate) is an abundantly available form of organic phosphorus (P) present in soil (50-60%) and plants products like edible legumes, cereals, nuts, oil seeds and pollens (1-5%) (Vohra et al. 2011). It is the primary storage form of both phosphorus and inositol in plants (Savita et al. 2017), phytate content of different cereals and roots are mentioned in Haefner et al. (2005). Phytate considered as an anti nutrient factor, because of its high chelating capability towards the important metal ions and binding to proteins. Because of that, the bioavailability of important proteins and minerals get diminished (Vohra et al. 2011, Mikulski and Kłosowski 2017). Monogastric animals like poultry, pigs, fish and humans are unable to utilize this form of phosphorous due to lack of inadequate level of phytate hydrolyzing enzymes in their gastrointestinal tracts. This undigested phytate leads to the growth of algal blooms in water bodies which cause the environmental pollution and detrimental effects on the food chain (He et al. 2017). To remove such anti-nutrients factors (Phytic acid) from food obtained from plant sources, phytases were found very efficient and cheap enzymatic sources.Some of the studies have been reported showing the utilization of phytic acid very efficiently from different microbial sources (Bhavsar and Khire 2014). Yeasts are known for producing industrially important enzymes like Invertase, lactase, lipase, raffinase and pectinases (Moharib et al. 2000). Due to the nonpathogenic status of most yeast, they are commonly used in brewing, baking, winemaking and are most preferable enzyme sources for their application in feed and foods. The potential applications of yeast phytases are enormous, majorly in animal feeds and human nutrition. Yeasts like Candida parapsilosis (Ranjan and Sahay, 2013), Pichia anomala were for known their extracellular and cell-bound phytases. Therefore, further research efforts are called for discovering new phytases from yeast sources as very few are presently known, and to develop an economical process for their large scale production (Kaur and Satyanarayana 2009). Our objective was to screen and isolate a potential phytase producing yeast strains and its identification. Positive phytase producing strains further optimized for production and media optimization using different statistical methods. Scale up the process upto 10 L fermenter to increase the productivity of the enzyme. The resulted yeast phytases applications were assessed in the field of plant growth promotion and as probiotics in the health sector, where the yeast phytases are unexplored. The thesis is divided into four chapters. Chapter 1 gives a detailed introduction to the importance of phosphate and its scarcity effects on future generations, phytases and its bioavailability, types of phytases, mechanism of action of different phytases and its applications in different sectors. Chapter 2 deals with initial screening, characterization and production of phytase producing yeasts and to find out the enzyme characteristics. Chapter 3 gives insights into optimization of media by using a statistical approach for higher phytase production by Candida tropicalis NCIM 3321 and scale up for commercialization perspective to improve phytase productivity and the application of its phytases in the agricultural sector by improving the plant growth. Chapter 4 includes the optimization of media components for enhanced phytase production by Saccharomyces cerevisiae NCIM 3662 and scale up studies for better productivity and evaluation of probiotic properties of NCIM 3662 and dephytinizing potential of the strain. Chapter 1: Introduction: Yeast phytases The first chapter provides a general introduction about yeast phytases and its reaction mechanisms on the substrate. It also discusses the phytase impotence in terms of increasing food demand and population, phosphate security. It also outlined the alternative sources of phosphates, contribution of phytases in food supplementation, unexplored yeast phytases for plant growth promotion, role of phytases in food fortification, unexplored role of phytase producing organisms as a probiotics and various applications of phytases. At the end, it described the basic work done in our lab and the objectives of the presented work. Chapter 2: Screening, characterization and production of phytase producing yeasts This chapter mainly focused on screening the yeast strains available in NCIM Resource Center and soil isolated yeasts for phytase production. The phytase screening consist of mainly two steps one is plate method in phytase screening media agar (PSM Agar) with calcium phytate as a substrate and the second one is confirmative quantification phytase enzyme activity assay. The strains were selected based on the ability to create a zone of clearance or hydrolysis around the colony on PSM containing calcium phytate. To avoid the false positive results provide by the strains from plate method was checked by quantifying the phytase production of the positive strains in submerged fermentation in MGYP media. Candida tropicalis NCIM 3321 was selected from NCIM cultures and Saccharomyces cerevisiae NCIM 3662 was selected from soil isolates in the screening process for studying the phytase production. The strains were identified and characterized by using morphological (SEM), biochemical (VITEK) and genetic level identification was done by sequencing the ITS region of the yeasts. Both the yeasts were submitted in NCIM and MCC culture collections center as a safe deposit. After identification, we find out the enzyme characteristics of both the strains Candida tropicalis NCIM 3321 and Saccharomyces cerevisiae NCIM 3662 Chapter 3: Optimization, Scale-up and evaluation of Candida tropicalis (NCIM 3321) phytases having plant growth promotion potential Phytase is known to provide a solution for depletion of phosphorus (P). It helps it by hydrolyzing the insoluble P source in the soil, which is phytate. In this chapter, we provide insight on yeast Candida tropicalis (NCIM 3321) which produces cell-bound and extracellular thermostable phytase. The media components were optimized to enhance the enzyme production and checked.

for plant growth promoting activity. On optimization, the isolate exhibited enhanced cell-bound and extracellular phytase activity by four folds (from 236 to 1024 IU DCG −1 ) and by five folds (from 0.46 to 1.95 IU ml −1 ) respectively in 36 h. The production time decreased to 24 h compared to shake flask on Up-scaling the production process upto 10 L scale, thus increasing the productivity of cell-bound (1810 IU DCG −1 day −1 ) and extracellular phytase (6.08 IU ml

day −1 ). The crude phytase (12 IU) from NCIM 3321 strain was studied for plant growth promotion activity in lab scale and field level experiments with maize crop. Findings of the study revealed that the extracellular phytase derived from nonpathogenic C. tropicalis (NCIM 3321) was found to be plant growth stimulating by increasing the available P in soil. Our findings of phytase isolated from non-pathogenic yeast C. tropicalis NCIM 3321 exhibited dephytinization potential. Therefore, the current study may have profound application in sustainable agriculture. Chapter 4: Evaluation of phytase producing Saccharomyces cerevisiae (NCIM 3662) probiotic properties and its application in food fortification The increase of undigested complexes of phytic acid in food is gaining serious attention to overcome nutritional challenges due to chelation effects. In this chapter, we investigated soil borne yeast phytase from Saccharomyces cerevisiae (NCIM 3662) for dephytinization of foods, probiotic properties and process development. The strain produced 45 IU/DCG of cell-bound phytase in an un-optimized medium, which was increased by four folds (164 IU/DCG) in 12 h using statistical media optimization. The process was scale-up upto 10L fermenter scale with increased phytase productivity of 6.4 IU/DCG/h as compared to the lab scale. The strain displayed probiotic characteristics like tolerance to artificial gastric acid conditions, hydrophobicity, autoaggregation, coaggregation and bile salt hydrolase (BSH) activity. Further, it could dephytinize (removal of phytic acid; an anti-nutritional factor) functional foods like Ragi (Finger millet) flour, Soya flour, Chickpea flour and poultry animal feed. Results less phytic acid content in food and it will make food more nutritive by releasing the important minerals in the process. It was confirmed by analyzing the important mineral content by using atomic emission spectroscopy. A combination of cell bound dephytinizing phytase, nutrition ameliorating and probiotic traits of S. cerevisiae (NCIM 3662) present profound applications in food technology sector.