Review Article
Microbial phytase in animal nutrition: Unlocking phytate for
sustainable feed utilization
M. Nitheesh Gopan, K. Madhavan Nampoothiri
Biosciences and Bioengineering Division, CSIR-National Institute for Interdisciplinary
Science and Technology (NIIST), Thiruvananthapuram, Kerala, India
Corresponding author: K. Madhavan Nampoothiri, Email: madhavannampoothiri.niist@csir.res.in
Journal of Experimental Biology and Zoological Studies. 2(1): p 4-19, Jan-Jun 2026.
Received: 20/10/2025; Revised: 31/10/2025; Accepted: 12/11/2025; Published: 01/01/2026
___________________________________________________________________________
Abstract
Phytase enzyme have earned significant importance in recent years, due to their critical role as
biocatalysts in the hydrolysis of phytic acid, thereby enhancing the bioavailability of
phosphorus in plant-based diets. In plants, phosphorus is predominantly stored in the form of
phytic acid, which exists mainly as mineral salts such as phytate and phytin. Phytic acid is
considered an anti-nutritional factor due to its strong chelating ability, which binds essential
minerals such as phosphorus, calcium, iron, and zinc, reducing their bioavailability, especially
for monogastric animals. To overcome this limitation, supplementation of the phytase enzyme
along with raw feed or incorporating phytase into feed formulations has become a common
strategy to enhance nutrient utilization efficiency of animal diets, by reducing the reliance on
expensive inorganic phosphate supplements. In this review, we focus on the application of
phytase enzyme in sustainable animal feed for monogastric animals. Numerous studies are
ongoing in this field to develop advanced variants of phytase with superior catalytic
performance, elevated thermal stability, broad pH activity range across various substrates,
coupled with greater enzyme productivity. Moreover, recombinant phytases developed through
various microbial expression systems have demonstrated superior performance in animal feed
processing, to achieve maximal nutrient utilization while ensuring feed stability throughout
processing. These advancements highlight the global potential of phytase in food processing,
agriculture, human nutrition and health, development of transgenic plants, environmental
protection, and various other industrial applications.
Keywords: Animal feed application, anti-nutrient, phosphorus recycling, phytase, phytate
(phytic acid), recombinant phytase.
___________________________________________________________________________
Introduction
Global demand for animal-derived food products is steadily increasing due to population
growth and urbanization creating pressure on the livestock sector to enhance productivity while
ensuring cost efficiency and environmental sustainability. Phytase has become one of the most
important feed enzymes in modern livestock nutrition, offering a sustainable solution to unlock
the nutritional potential of plant-based feed formulations. Phytase, an enzyme of the
phosphatase family, is widely recognized for its ability to hydrolyse phytic acid, thereby
releasing bound nutrients in the feed. Most feed ingredients used in livestock diets are of plant
origin, primarily consisting of cereals, oilseeds, and their by-products derived from agro-
industrial residues, such as wheat bran, soybean meal, and various types of oil cakes. These
collectively serve as the major sources of energy and protein for animal nutrition. However,
phytic acid, the natural storage form of phosphorus in plants, is deposited in substantial
amounts, particularly in cereals, legumes, nuts, and oilseeds.[1]
Phytic acid is poorly digested by monogastric animals such as poultry, swine, and fish because
they lack sufficient endogenous phytase to hydrolyse phytate. As a result, the phosphorus
bound to phytate remains unavailable, reducing overall nutrient utilization, while the excretion
of undigested phytic acid in manure contributes to environmental pollution. Excessive
phosphorus runoff from animal farming is a significant contributor to water eutrophication,
resulting in ecosystem imbalance and posing risks to both the environment and human health.[2]
Moreover, to compensate for the poor availability of phytate-bound phosphorus, farmers often
add inorganic phosphate supplements to animal diets to meet the nutritional requirements,
which in turn raises feed costs.
Supplementation with exogenous microbial phytase helps overcome these challenges by
hydrolyzing phytate, thereby enhancing phosphorus and mineral bioavailability, in addition to
reducing feed costs and minimizing phosphorus excretion into the environment. To achieve
sustainable livestock production, it is essential to maximize nutrient utilization from plant-
derived feed ingredients, thereby reducing reliance on non-renewable phosphate supplements
and lowering nutrient waste.
The development of sustainable practices in animal nutrition through enzyme technology has
been significantly advanced by supplementing diets with microbial phytase. This strategy has
proven to be a practical and effective method for improving phytate digestibility in monogastric
animal diets. The global feed industry spends billions of dollars annually on improving feed
formulations by adding nutrient supplements, with inorganic phosphate being a major
contributor. Recent advancements in enzyme technology and ongoing research have focused
on engineering phytases with enhanced catalytic efficiency, broader substrate specificity,
higher thermal stability, and improved resistance to proteolysis during feed processing. A key
area of current development is the production of phytases with high thermal stability, as
significant heat is generated during the feed pelleting process. This ensures consistent nutrient
release under commercial processing conditions, which would otherwise inactivate
conventional enzymes. The development of next-generation phytases with enhanced catalytic
activity and the ability to function across a broad pH range in the gastrointestinal tract has been
achieved through site-directed mutagenesis and protein engineering.[3] These innovations have
given a new dimension to the use of phytase in animal feed applications, as it not only improves
phosphorus bioavailability and optimizes feed efficiency but also reduces reliance on inorganic
phosphate supplementation, thus lowering feed costs and mitigating environmental impacts
associated with phosphorus excretion. Collectively, these advancements place phytase as a
cornerstone of modern feed enzyme technology, aligning economic, nutritional, and
environmental objectives in sustainable livestock production.
Phytic acid- the anti-nutritional factor
Phytic acid, also known as D-myo-inositol (1,2,3,4,5,6)-hexakisphosphate (inositol
hexaphosphate/IP6), consists of six phosphate groups connected to an inositol ring by ester
bonds. It is the principal storage form of phosphorus in many plant-derived foods such as
cereals, legumes, and oilseeds (Figure 1).[4] Phytic acid predominantly occurs as a complex by
binding to divalent and monovalent metal cations such as calcium (Ca²⁺), iron (Fe²⁺), zinc
(Zn²⁺), magnesium (Mg²⁺), potassium (K⁺), and manganese (Mn²⁺), forming stable mineral–
phytate complexes.[4] These salts of phytic acid are collectively referred to as phytate, and in
plants they predominantly exist as mixed salts with calcium and magnesium, commonly termed
phytin. In association with other nutrients, phytate can also form complexes known as
“lipophytins” with lipids and their derivatives.[5] However, phytate phosphorus is poorly
available to monogastric animals, reducing the bioavailability of essential nutrients such as
calcium, iron, and zinc. Furthermore, it inhibits the activity of key digestive enzymes, including
amylase, trypsin, and pepsin, in the gut. This inhibition arises from the nonspecific binding of
phytate to proteins and the chelation of calcium ions, which are essential cofactors for the
proper functioning of these enzymes.[6]
Phytic acid has traditionally been regarded as an antinutrient, as numerous studies have shown
that its reactive phosphate groups, attached to the inositol ring, form insoluble complexes with
cations, thereby reducing their intestinal absorption in humans and other monogastric animals.
The primary reason for the antinutritional effects of phytic acid is its strong chelating effect,
which arises from its six reactive, negatively charged phosphate groups.[7] These groups readily
bind to positively charged minerals, such as calcium, iron, zinc, and magnesium, forming
insoluble complexes that significantly reduce the bioavailability of these minerals. When
phytate-containing plant-based food products, mainly derived from cereals, oilseeds and
legumes, are consumed by themselves without being processed or cooked in large quantities
can reduce the absorption of phosphorus and other important minerals in monogastric animals,
Figure 1: Different sources of phytic acid in feed ingredients[4,6]
which lack sufficient endogenous phytase activity to digest phytate. This results in poor
phosphorus bioavailability. When the stomach pH rises above the isoelectric point of
proteins,phytate readily binds to protein–mineral complexes, generating insoluble aggregates
that are resistant to enzymatic hydrolysis. This reduced solubility impairs the efficiency of
protein digestion and limits the availability of nutrients. Moreover, these complexes can hinder
the activity of endogenous proteases such as pepsin, which depend on substrate accessibility
for optimal function, thereby decreasing the release and absorption of amino acids. Such
interference not only reduces dietary protein utilization but also causes deficiency of amino
acids, representing a hidden nutritional cost. The calcium–phytate complexes have been shown
to adversely affect lipid digestion, as calcium bound to phytate promotes the precipitation of
fatty acids into indigestible metallic soaps within the gut lumen. Collectively, these interactions
highlight the multifaceted anti-nutritional role of phytate, impairing both protein and fat
utilization in monogastric animals. [7,8]
To overcome this challenge, microbial phytases have been widely explored and utilised in both
the animal feed and food processing industries. These enzymes, primarily produced by fungi
but also by certain bacterial strains, play an important role in enhancing phosphorus
bioavailability, reducing dependence on inorganic phosphate supplements, and minimizing
environmental phosphate discharge. Beyond their established use in feed, phytases are
increasingly recognized for their potential applications in food processing, agriculture, and
human nutrition, making them a valuable tool in sustainable biotechnological practices.
Phytase
Phytase (myo-inositol hexakisphosphate phosphohydrolase; EC 3.1.3.26) is an enzyme
belonging to the phosphatase family that catalyses the hydrolysis of phytic acid (myo-inositol
hexakisphosphate), primarily releasing inorganic phosphate, lower inositol phosphates, and
essential minerals such as zinc, iron, and calcium that are bound within the phytic acid complex
(Figure 2). It is a naturally occurring enzyme widely distributed across various biological
systems, including microorganisms, plants, and certain animal tissues. Suzuki et al. (1907) first
identified phytase in rice bran.[9] The pioneering research on phytase application in poultry feed
was conducted by Nelson et al. (1968), in which soybean meal was treated with fermented
Aspergillus ficuum for inclusion in chicken feed. Their findings showed improved utilization
of phosphorus made available by the degradation of phytic acid in the substrate. This was
SOURCE OF PHYTIC ACID
LEGUMES
Soya beans
Kidney
beans
Chickpea
Lentils
Mung
beans
Black
gram
OIL SEEDS
Sesame
seeds
Sunflower
seeds
Mustard
seeds
Flax seeds
Cotton seed
Rape seed
NUTS
Almonds
Walnuts
Cashew
Peanuts
Pistachios
hazelnuts
CEREALS
Wheat
Rice
Maize
Barley
Oats
Sorghum
indicated by a significant increase in bone ash content, which outperformed the results
observed with inorganic phosphate supplementation.[10]
Figure 2: Breakdown of phytic acid by phytase[11]
The breakdown of phytic acid or phytate is referred to as dephytinization, a process that plays
a crucial role in enhancing the nutritional quality of plant-based foods and animal feeds by
mitigating the anti-nutritional effects of phytate. Several strategies can be employed to achieve
dephytinization, including milling, soaking, germination, fermentation, thermal processing and
application of exogenous microbial phytase. Soaking and germination are traditional
approaches widely used to reduce phytate levels in plant-based foods. Extended soaking for
12–16 hours initiate hydration and activates endogenous enzymes, while germination induces
metabolic changes that enhance phytase and associated phosphatase activity, leading to the
stepwise hydrolysis of phytic acid into lower inositol phosphates and free phosphate groups.
Enzymatic hydrolysis of phytate during soaking is strongly influenced by temperature and pH,
with intrinsic plant phytases capable of degrading 26–100% of phytate when conditions are
close to optimal (45–65 °C, pH 5.0–6.0).[12] According to Azeke et al. (2010), germination for
10 days led to an 81–88% reduction in phytate content across cereals, with maximum decreases
observed between the 7th (Maize, Millet and Wheat) and 10th day (Sorghum) depending on the
grain. This reduction corresponded with a significant (P < 0.05) increase in phytase activity,
whereas minimal changes occurred within the first 24 hours.[13]
Fermentation and thermal processing are widely recognized as effective strategies to lower
phytate levels. Fermentation promotes the production of microbial phytases and related
phosphatases, which, in turn, cause hydrolysis of phytate, releasing bound minerals and
improving nutrient absorption and protein digestibility. Thermal processes, such as boiling,
cooking, and autoclaving, reduce phytate levels, although the extent of reduction depends on
the temperature and duration, making them fast and scalable methods. Elhardallou and Walker
(1994) reported that boiling reduced the phytic acid content of lentils by 60%, butter beans by
50%, and broad beans by only 11%.[14] Prolonged thermal treatments are not always optimal,
as they can degrade heat-sensitive nutrients and lead to substantial nutrient losses. In
comparison, fermentation offers a gentler means of reducing phytic acid while preserving
nutritional quality, although it requires longer processing times, which can increase costs.
Germination is also effective, as 6–10 days of sprouting significantly lowers phytate levels;
however, since the process is time- and space-intensive, it can increase processing costs. While
these methods are suitable for home-based food preparation, they are generally not cost-
effective for large-scale industrial food production due to the extended time, labour, and
resources required. Among the various dephytinization methods, the application of exogenous
microbial phytase is considered one of the most effective approaches, as it directly catalyzes
phytate hydrolysis and ensures a more efficient release of bound nutrients. Phytase is found
naturally in many living organisms, including animals, plants, fungi, and bacteria (Table 1).[15]
Phytases are a structurally diverse group of enzymes classified according to their catalytic
mechanism, sequence homology, and pH optima for enzymatic activity. Notably, phytase
enzymes are classified into four types based on their structural and catalytic mechanism.
Histidine acid phosphatases (HAPhy) are a common type of phytase found in filamentous fungi
(Aspergillus niger, Aspergillus fumigatus) and yeast. They are optimally active at acidic pH
(around 2.5–5.5) and have a wide range of applications in the feed industry for developing
nutrient-rich feed for monogastric animals.[15] Purple acid phosphatases (PAPs), members of
the calcineurin-type metallophosphoesterase superfamily, are named for their distinctive purple
colour caused by a charge-transfer interaction between ferric iron (Fe³⁺) and a tyrosine residue
Table 1: Phytase enzyme sources
PHYTASE SOURCE
Animal Source
Plant Source
Bacterial Source
Fungal Source
in the active site.[16,17] PAPs are mostly found in plants (soybean) and exhibit optimal activity
at acidic pH. β-Propeller phytases (BPPs) are alkaline phytases primarily discovered in Bacillus
species, characterized by a unique three-dimensional structure resembling a six-bladed
propeller. BPPs hydrolyze phytic acid by specifically recognizing a Ca²⁺-mediated bidentate
chelation (P–Ca²⁺–P) between two adjacent phosphate groups and play a crucial role in
maintaining the phosphorus cycle in soil. This mechanism directs sequential cleavage,
producing myo-Ins (2,4,6) P₃ (confirmed by ¹H- and ¹³C NMR with the aid of 2D NMR) along
with free phosphates as the final products.[18] Cysteine phosphatases represent a new class of
phytase characterized by a cysteine-based active site, mainly found in anaerobic bacteria from
the gut of ruminant animals. The active site cysteine is located within a highly conserved motif,
HCxxGxxR, and these enzymes are optimally active at a pH range of approximately 4.5 to
6.0.[19]
In addition to structural classifications, phytases are also categorized based on their site of
initial hydrolysis on the phytic acid molecule. This classification includes three main types: 3-
phytase (EC 3.1.3.8), 5-phytase (EC 3.1.3.72), and 6-phytase (EC 3.1.3.26), which differ with
respect to the specific position on the inositol ring where they first cleave the phosphate
group.[20] Notably, based on their structure and functional diversity, these enzymes ensure
efficient degradation of phytic acid under various physiological conditions and highlights their
significance in animal nutrition and biotechnological applications.
Application of phytase in animal feed
The primary application of phytase is in animal nutrition, where it plays a vital role in
enhancing phosphorus utilization from plant-based feed ingredients in monogastric animals
such as poultry, swine, and fish. In such feeds, phytic acid (phytate) constitutes around 60–80%
of the total phosphorus content; however, monogastric animals lack sufficient endogenous
phytase to break it down, leading to poor mineral absorption. Microbial phytase
supplementation facilitates the enzymatic hydrolysis of phytate, releasing bound essential
minerals such as phosphorus, zinc, iron, and calcium, thereby significantly enhancing nutrient
bioavailability.[15] This not only reduces the dependence on expensive inorganic phosphate
supplements but also promotes more cost-effective and sustainable livestock production.
Notably, several commercial phytase products are available in the market, as shown in Table
2.[21] Although many more commercial phytase enzymes are produced locally in various
countries, the market is dominated by enzymes from developed nations, with European
countries being key producers. The widespread use of these enzymes in feed for poultry, pigs,
and fish underscores their importance as a microbial feed supplement.
Phytase enzymes can be incorporated into probiotic supplements for livestock to support gut
microbiota and enhance nutrient utilization. Bandari et al. (2024) developed a biologically
engineered probiotic by cloning the phy gene from Bacillus subtilis into Lactococcus lactis,
Table 2: Commercially available Phytase enzyme
enabling it to produce phytase. The engineered strain produced phytase in a granular form,
enhancing phosphorus availability from plant-based feed ingredients, particularly benefiting
monogastric animals.[22] Multi-enzyme formulations have become a prominent strategy in
amylase to target a broader range of anti-nutritional factors commonly present in plant-based
feed ingredients, including phytate, non-starch polysaccharides (NSPs), and complex proteins.
Multi-enzyme supplementation enhances nutrient availability, improves feed efficiency, and
promotes gut health by breaking down complex feed components, thereby reducing the need
for costly dietary nutrients without compromising animal growth and productivity.
The synergistic effect of exogenous multi-enzyme and phytase supplement was assessed by
Kim et al. (2021) in corn, wheat and soybean-based diets for broiler chickens. It was found that
muti-enzyme phytase combination significantly improved body weight gain and feed
conversion ratio during the finisher period. Birds receiving this combination also showed
markedly higher digestibility of dry matter, crude protein, gross energy, calcium, and
phosphorus, along with elevated serum Ca and P levels. Moreover, intestinal morphology was
enhanced, with increased villus height, crypt depth, and villus height-to-crypt depth ratio.
These improvements were superior to those achieved with phytase alone, while gut microflora
remained unaffected.[23]
Further investigations are essential to optimize feed formulation strategies by examining the
interactions between phytase and various feed components, including other enzymes, minerals,
dietary fibres, and nutrients, as well as the influence of feed processing methods. The effect of
phytase on the gut microbiota is also an important factor that collectively impacts nutrient
absorption and overall animal health. Assessing both synergistic and antagonistic interactions
will aid in optimizing phytase efficiency across diverse and unconventional feed formulations.
These studies are vital not only for maximizing phytase utilization in animal feeds but also for
promoting environmental sustainability by reducing phosphorus footprints resulting from
animal waste, thereby improving the bioavailability of minerals and other nutrients, and
enhancing overall feed efficiency.
Impact of phytase on animal performance and productivity
Animal feeds are primarily composed of cereals, legumes, and oilseeds, which are
predominantly plant-based. In addition to breaking down phytate, phytase improves bone
mineralization, enhances nutrient utilization, and promotes better digestion of proteins and fatty
Trade name
Protein origin
pH optima
Thermal
stability
Company
Country
Natuphos® E
Aspergillus niger
2.0-5.0
(5.5)
90°C
BASF Corporation
Germany
Ronozyme® P
Peniophora lycii
4.0-4.5
70-90°C
DSM-firmenich &
Novozyme
Denmark
Ronozyme®
Hiphos
Citrobacter
braakii
-
80°C
DSM-firmenich &
Novozyme
Denmark
Phyzyme® XP
Escherichia coli
5.5
-
DuPont Nutrition
USA
Finase® EC
Escherichia coli
5
70- 85°C
AB Vista
UK
Quantum®
Blue
Escherichia coli
5.5
80–85°C
AB Vista
UK
Quantum®
Escherichia coli
4.5
85°C
AB Vista
UK
OptiPhos®
Escherichia coli
3.4,5.0
75- 90°C
Huvepharma
USA
Axtra® PHY
15 000 L
Buttiauxeralla sp.
3.5-4.5
-
Danisco Animal
Nutrition (Dupont)
Denmark
Allzyme®SSF
Aspergillus niger
6.0
-
Alltech
USA
acids. Phytate forms electrostatic interactions with the terminal amino groups of proteins,
resulting in phytate–mineral–protein complexes that lower the availability of essential amino
acids (lysine and arginine) and reduce overall protein digestibility. Moreover, phytate inhibits
key gastrointestinal enzymes (e.g., trypsin, pepsin, and α-amylase), thereby reducing protein
and starch digestibility and diminishing overall nutrient utilization.[4,6,24,25] Phytate also reduces
α-amylase activity through two mechanisms: direct noncompetitive inhibition of the enzyme
and sequestration or interaction with essential divalent cations required for enzyme
stabilization and activation.
Experimental studies by Deshpande and Cheryan (1984) demonstrated that in the presence of
6–30 mM phytate, calcium ions (Ca²⁺) lowered α-amylase activity by 9–34%, whereas
magnesium ions (Mg²⁺) caused a much stronger inhibition, ranging from 24% to 49%. This
indicates that Mg²⁺ amplifies the negative effects of phytate more significantly than Ca²⁺.[25]
The anti-nutritional effects of phytate are particularly pronounced in legumes such as soybean
and chickpea, as well as in cereals. In soybeans and chickpeas, protein digestibility is reduced
due to strong phytate-protein binding. [26] Collectively, these interactions emphasize the anti-
nutritional role of phytate, posing major nutritional challenges in plant-based diets dominated
by legumes and cereals. To overcome these effects, the application of phytase has been
extensively studied in animal nutrition, especially in monogastric species such as poultry and
swine.[27,28]
Adeshakin (2023) investigated the impact of varying phytase (PHY) levels, with or without the
inclusion of multi-carbohydrase (MC), on growth performance, nutrient digestibility, and bone
characteristics in nursery pigs fed phosphorus-deficient diets. The findings revealed that PHY
supplementation, whether used alone or in combination with MC, significantly enhanced
average daily gain (ADG), feed efficiency, and the apparent total tract digestibility (ATTD) of
ash and phosphorus release (p < 0.05). Phosphorus release refers to the enzymatic hydrolysis
of phytate by phytase, resulting in the liberation of inorganic phosphorus (Pi) from phytate
complexes. Phytase activity is defined as the amount of enzyme that liberates 1 µmol of
inorganic phosphorus per minute from 0.0015 mol/L sodium phytate at pH 5.5 and 37 °C,
indicating the enzyme’s efficiency in converting unavailable phytate-bound phosphorus into
an absorbable form. Additionally, PHY improved bone mineralization, as indicated by
increased ash and phosphorus content. While MC alone had limited influence, its combined
use with PHY demonstrated a synergistic effect, further improving nutrient digestibility and
bone quality.[27]
The study by Leyva-Jimenez et al. (2018) evaluated four commercially available phytase
sources across different parameters by supplementing broilers at regular and super-dose levels
to assess their effects on performance, bone mineralization, and apparent ileal digestible energy.
Results show that broilers fed super-dose levels of phytase had significantly higher body
weight, weight gain, and bone mineralization compared to birds on the negative control.
Additionally, broilers fed super-dose phytase showed a 17% increase in apparent ileal energy
digestibility at day 24, indicating improved nutrient utilization. Overall, high levels of phytase
supplementation more effectively enhanced growth performance, bone characteristics, and
energy utilization while compensating for phosphorus deficiency compared to broilers fed low
levels of phytase.[28] Supplementation of Schizosaccharomyces pombe expressed phytase
(500–1,500 FTU/kg) in broiler diets from day 1 to 45 linearly improved body weight gain (p =
0.001–0.018) and feed conversion ratio (p = 0.001–0.012) throughout the trial. Additionally,
ileal phosphorus digestibility (p = 0.049), toe ash content (p < 0.001), and carcass weight (p =
0.035) were increased, while footpad lesions decreased (p = 0.040), with no significant effects
on organ indices, calcium content, crude protein digestibility, or meat quality.[29]
Supplementation of microbial phytase in low-phosphorus pig diets improved digestibility and
growth performance, including feed intake, while enhancing overall mineral utilization. It also
increased the retention of phosphorus, calcium, magnesium, and copper, reducing
environmental excretion of P and Cu by 39% and 33%, respectively.[30] Babatunde and Adeola
(2022) conducted two trials to investigate the impact of phytase supplementation on
phosphorus utilization in growing and finishing pigs, assessing parameters such as growth
performance, apparent total tract digestibility (ATTD) of nutrients, phosphorus excretion, and
plasma mineral levels. In both experiments, pigs receiving the positive control diet exhibited
greater body weight compared to those on the negative control diet. Inclusion of phytase in the
diet significantly enhanced growth performance, with increases in body weight, average daily
gain, and both linear and quadratic improvements in gain-to-feed ratio (G:F) (P < 0.01 for
growing pigs; P < 0.05 for finishing pigs) relative to negative control. Phytase supplementation
also improved nutrient digestibility, particularly ATTD of phosphorus and calcium, and
markedly reduced water-soluble phosphorus excretion by 45% during the growing phase, 32%
during finishing, and 35% across the full grow-finish period. Additionally, plasma phosphorus
concentrations were elevated in pigs fed phytase, indicating its effectiveness in enhancing
mineral utilization and overall growth performance.[31] Overall, phytase supplementation has
been shown to improve growth performance in monogastric animals while enhancing nutrient
digestibility, mineral absorption, bone mineralization, and energy utilization.
Optimisation of fermentation strategies for industrial scale phytase production
Optimisation of fermentation strategies is a key focus area for the industrial-scale production
of phytase enzymes. Numerous ongoing studies aim to optimize phytase fermentation
processes to achieve maximum yield at minimal cost. Submerged fermentation is commonly
utilized for producing phytase from both bacterial and fungal sources, whereas solid-state
fermentation is more suitable for filamentous fungi due to its reliance on low-moisture solid
substrates.[32] Semi-solid fermentation offers an intermediate moisture environment, supporting
specific microbial growth for specialized applications. Low cost agricultural and industrial
residues such as wheat bran, soybean meal, molasses, corn steep liquor, various oil cakes, and
other agro-industrial wastes are commonly used as effective substrates in fermentation
processes to reduce production costs.[33] Moreover, each fermentation strategy must be
carefully optimized for industrial-scale applications to ensure higher enzyme yields and reduce
downstream processing costs. This includes fine-tuning media composition, pH, temperature
and bioreactor design for efficient, scalable production.
Enzyme immobilization is a key industrial strategy used to reduce production costs and
enhance enzyme efficiency, stability, and reusability. Immobilization of phytase enzyme from
Chenopodium album onto chitosan-coated iron oxide (Fe₃O₄) nanoparticles was effective in
reducing phytic acid in soymilk. The immobilized phytase exhibited broad substrate specificity
towards various natural polymers and retained 70% of its catalytic activity over a 40-day
storage period. Additionally, it demonstrated good operational stability with successful reuse
over seven batch cycles, indicating strong potential for practical applications in food phytate
reduction.[34] According to Coutinho et al. (2019) phytase immobilized on hydroxyapatite (HA)
nanoparticles achieved complete adsorption with over 100% recovered activity. The
immobilized enzyme also exhibited enhanced thermal stability at 80–90 °C, a broader pH
activity range, and increased resistance to acidic conditions and proteolytic enzymes under
simulated fish gastrointestinal conditions.[35] These properties highlight its strong potential for
application in both aquatic and animal feed industry.
According to Khongkomolsakul et al. (2025) chitosan complexation markedly improved the
thermal and gastrointestinal stability of phytase (phyA), increasing residual activity from 3%
in the native enzyme to 40% in the 4:1 CS-phyA complex. Thermal stability was also
significantly enhanced, rising from 20% to 74% at the same complex ratio. The optimized
complexes retained up to 13-fold higher enzyme activity following exposure to heat and
simulated gastric conditions. These results indicate that CS-phyA complexes hold substantial
promise for expanding phytase applications in high-temperature food processing and plant-
based dietary systems.[36] A thermostable phytase from Mucor indicus, optimized using black
gram husk as a substrate, yielded the highest enzyme activity (92.10 U/ml). Further
optimization of media and conditions enhanced activity to 184.03 U/ml. Immobilization
significantly increased catalytic efficiency (17.26 mM/s vs. 5.68 mM/s for free enzyme). When
applied to broiler and layer feed, the immobilized enzyme effectively released phosphorus
(35.45 mg/g and 58.46 mg/g, respectively), highlighting its potential to enhance feed nutrition
through sustainable utilization of agro-waste.[37] In industrial-scale phytase production, the
integration of optimized fermentation strategies is essential to achieve high enzyme yields at
minimal cost. Solid-state and submerged fermentation are the most effective approaches,
depending on the microbial source and downstream processing requirements. Efficient
utilization of agro-industrial residues not only reduces production expenses but also promotes
environmental sustainability. Optimization of media composition, process parameters,
purification methods and enzyme stabilization techniques like immobilization are essential to
enhance viability of phytase in animal nutrition and feed applications.[34]
Next-generation phytases in feed applications: recombinant phytase production
The demand for efficient and sustainable animal nutrition has had a significant impact on global
feed production, driving the development of next-generation phytases. Conventional phytases,
although effective, exhibit several limitations, including a narrow pH activity range, low
stability in the gastrointestinal tract, and poor thermostability at high temperatures, which is an
essential requirement for feed pelleting and extrusion processes. To overcome these challenges,
advanced molecular techniques such as genetic engineering and recombinant expression
systems are being widely employed to enhance phytase yields and improve its functional
properties. Significant progress has been made in the overexpression of phytase genes using
host systems such as Escherichia coli DH5α, Pichia pastoris X-33, Saccharomyces cerevisiae,
and Yarrowia lipolytica. This is a promising area of research, enabling the large-scale
production of engineered enzymes with desired characteristics.[38-40] Attempts have also been
made to express phytase genes directly in transgenic crops to develop self-sufficient feed
sources. Cloning of novel phytase genes from bacteria, fungi, and even metagenomic sources
has expanded the diversity of available phytases, offering enzymes with unique features like
heat resistance, broader substrate activity, and wider pH optima, taking into consideration
industrial-scale needs in animal feed processing, where high temperatures are required and the
acidic gastrointestinal conditions in domesticated animals. Some recombinant phytases now
exhibit dual pH optima, enabling the hydrolysis of phytate across a broad pH range with
different substrates and increased resistance to proteolytic degradation, which helps them
function more efficiently in vivo.
Many ongoing studies in this field aim to enhance phytase thermostability, broaden its pH
activity range, and maximize production yield. For instance, phytase obtained from
Thermoascus aurantiacus under submerged fermentation, using an optimized medium
containing 3.75% (w/v) wheat bran particles and 2% Tween-20 as an additive, yielded a
maximum thermostable phytase activity of 468.22 U/mL after 72 hours of incubation. The
enzyme displayed excellent thermal stability, retaining nearly 80% of its activity at 70°C,
underscoring its potential application as a feed enzyme.[38] Son et al. (2024) found that a novel
phytase, TmPhy, isolated from Turicimonas muris and expressed in Pichia, had dual pH optima
(3.0 and 6.8) with peak activity at 70°C. However, its native thermostability was inadequate
for use in feed. Using genetic engineering and incorporating several mutation strategies,
including random mutagenesis, disulfide bond introduction, and N-terminal modification, an
improved mutant version of the enzyme, named TmPhyMD2, was produced, which exhibited
the highest thermostability, retaining 74.1% of its activity at 80°C.[39] Xing et al. (2023)
enhanced the thermostability of phytase by employing error-prone PCR and site-directed
mutation in E. coli, followed by the expression of selected top mutants in P. pastoris. Five
mutants demonstrated enhanced thermostability, retaining approximately 9.6%, 10.6%, 11.5%,
11.6%, and 12.2% more residual activity than the wild-type enzyme after exposure to 99 °C for
60 minutes. Notably, three mutants D7, E3, and F8 also exhibited significant improvements in
catalytic efficiency (kcat/Km), by 79.8%, 73.2%, and 92.6%, respectively, highlighting their
potential as promising candidates for feed processing and other industrial applications.[40]
Another approach to improve phytase production involved Site-directed mutagenesis using
Quick-Change PCR that introduced T312R and F260R mutations into the active site of Yersinia
intermedia phytase gene. Then the mutated plasmids were then introduced into E. coli DH5α
using the heat shock method. Recombinant enzymes were expressed in the double mutant
T312R/F260R showed a 2.35-fold increase in activity over the wild-type. The lambda Red
recombineering system is a modern genetic engineering technique used to create genetically
modified organisms for the production of recombinant proteins.[41] The study by Arhar et al.
(2025) effectively employed Cupriavidus necator H16 to integrate the Escherichia coli phytase
gene (appA) into the phaC1 locus using linear PCR products, thereby enabling efficient gene
knockouts through electroporation. The resulting strain was marker-free, due to Cre/loxP-
mediated marker recycling, and exhibited a complete loss of PHB granules as a result of
disrupting the phaC1 gene. Functional analysis confirmed the successful expression of phytase,
with the engineered strain releasing approximately 8 μmol of phosphate per unit after 15
minutes of incubation with phytate.[42] These findings highlight the system’s efficiency and
establish Cupriavidus necator as a promising host for sustainable and stable production of
recombinant phytase for industrial applications.
To improve phytase reusability, Saccharomyces cerevisiae was engineered using CRISPR/Cas9
to display a fusion of acid and alkaline phytases on its surface via the α-agglutinin-GPI system.
Two marker-free strains were developed using MFα and Aga2p signal peptides, exhibiting high
activity across a pH range of 1.0–7.0, with dual pH optima and efficient kinetics. The fusion
enzyme showed 3.5–4 times higher activity than the single phytase.[43] A recombinant Pichia
pastoris strain was engineered to secrete alkaline phytase from Bacillus subtilis for potential
aquafeed applications. The phytase gene was cloned into the pPICZαA vector and integrated
into the P. pastoris X33 genome, producing a methanol-utilizing (Mut⁺) strain. Western blot
confirmed phytase secretion, and enzyme assays showed activity at pH 7.5. This highlights the
strain's potential for industrial-scale phytate degradation in aquafeed production.[44]
Strain improvement through genetic engineering is one of the most advanced approaches for
developing recombinant organisms with desired traits. Extensive research is ongoing to refine
techniques such as site-directed mutagenesis, random mutagenesis, and CRISPR-based
systems, all of which have shown promising results. [40,42-44] These advancements pave the way
for more efficient and sustainable phytase applications in the animal feed processing and food
industries and nutrient enhancement of plant-based diets.
Phytase as a sustainable approach for environmental protection
Agriculture and livestock farming are major sources of phosphorus pollution. Undigested
phytate from animal waste, either directly from farms or through manure applied to agricultural
land, can exceed the crop’s capacity to absorb it, leading to soil phosphorus accumulation.
Excess phosphorus runoff contributes to eutrophication, causing algal blooms and oxygen
depletion in aquatic ecosystems. This disrupts natural phosphorus cycling and creates
environmental hazards.[45] Soil microorganisms can slowly degrade phytate and release
phosphorus for plant uptake. Additionally, manure from cattle supplemented with microbial
phytase has been shown to enhance the bioavailability of phosphorus more efficiently than
untreated manure. El Ifa et al. (2024) isolated three strains of phytase-producing rhizobacteria
with the ability to enhance barley growth under phosphate-limited conditions. These strains
should be considered in the development of eco-friendly biofertilizers as alternatives to
conventional phosphorus fertilizers.[46] Therefore, incorporating phytase-producing
microorganisms into biofertilizers could serve as an effective strategy to address the issue of
phosphorus unavailability in soils.
For a sustainable environment, nutrient-rich soil serves as the main ingredient for the growth
of microbes and plants, leading to the development of a healthy ecosystem. Soil is naturally
formed through weathering caused by the disintegration of large rocks and minerals over
centuries. As life forms begin to grow there, the soil becomes more nutrient-rich and adapted
to recycling minerals through different cycles such as carbon cycle, nitrogen cycle, phosphorus
cycle and so on. The phosphorus cycle is one of the most important cycles, playing a vital role
in sustaining phosphorus levels in the environment and ensuring its availability. The
phosphorus cycle lacks a gaseous phase, primarily involving the movement of phosphate from
rocks to soil and water through the process of weathering. Plants absorb phosphate, which then
moves through the food chain and returns to the environment via decomposition and excretion.
Soil microbes play a crucial role in breaking down complex molecules, such as phytate, by
producing phosphate-releasing enzymes, including phytase, thereby converting phosphorus
into a form that plants can absorb. Human activities disrupt the natural phosphorus cycle
through deforestation, excessive use of fertilizers and detergents, and the discharge of
phosphate-rich wastes, causing adverse environmental impacts such as loss of soil fertility,
water acidification, eutrophication, and consequent ecosystem imbalances.[47]
Phytase enzymes can also be used in industrial wastewater treatment processes, where they
facilitate the breakdown of organic phosphorus compounds, thereby reducing phosphorus load
and mitigating environmental pollution. The study by Dalas et al. (2025) highlights the
biotechnological potential of phytase from Bacillus subtilis for wastewater treatment. The
enzyme exhibited broad thermal stability (20–60°C) and pH stability (4–8), with optimal
activity at 30°C (0.83 unit/mL) and pH 6. The application of the enzyme significantly reduced
BOD (64.9% and 56.4%), COD (59.6% and 53%), nitrate, and metal levels in industrial
wastewater.[48]
Several recent studies focus on the immobilization of phytase on biochar as a strategy to
convert organic phosphorus (Org-P) in manure into plant-available inorganic phosphate.
Immobilization of phytase into biochar by covalent grafting accomplished by the carbodiimide
crosslinker method and physical sorption will help to withstand leaching, degradation, or
denaturation. Physisorption was found to be as effective as grafting, with phytase loading
positively correlated with the amount of biochar. Immobilized phytase was stable with minimal
leaching but showed significantly reduced activity compared to free phytase; hence, further
studies are needed to improve its efficiency and to enable industrial-scale usage. Studies using
clay minerals (montmorillonite, kaolinite, and hematite) showed better phytase loading and
activity than biochar.[49,50] Therefore, these methods need to be optimized for better application
of microbial phytase and to reduce organic phosphorus content in the soil.
Conclusion
In recent decades, significant advancements have been made in phytase research, driven by the
need for more efficient and sustainable solutions in the development of animal feed.
International efforts have pushed the boundaries of enzyme engineering to achieve superior
stability and activity, by utilizing agricultural wastes for cost-effective production. Genetic
engineering offers promising strategies for developing more efficient phytase-producing
microorganisms through heterologous gene expression, aiming to enhance thermostability,
broad pH activity, protease resistance, and other desirable traits. Ongoing research is
increasingly focused on the structural and functional characterization of phytase enzymes, with
particular emphasis on identifying novel sources and elucidating their catalytic mechanisms,
active site architecture, and interactions with phytate. Beyond animal nutrition, phytase
applications extend to food, nutrition and processing, development of transgenic plants
incorporating microbial phytase, industrial production of value-added biochemicals such as
myo-inositol, and eco-friendly technologies including the paper industry. As a significant
number of phytases are produced using genetically modified organisms especially transgenic
animals and plants, comprehensive safety evaluations are being conducted to ensure their safe
use in food and animal feed, in accordance with regulatory standards and public health
guidelines. The continued integration of molecular biology, fermentation technology, and
protein engineering is anticipated to drive further advancements in the development of next-
generation phytases with enhanced performance for a wide range of biotechnological
applications.
Financial support and sponsorship
This study was funded by the Council of Scientific and Industrial Research (CSIR), New Delhi,
with Mission Grant No. MMP025201.
Conflicts of interest
There are no conflicts of interest.
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