Review on Desirable Microbial Phytases as a Poultry Feed Additive: Their Sources, Production, Enzymatic Evaluation, Market Size, and Regulation

Poultry's digestive tract lacks hydrolytic phytase enzymes, which results in chelation of dietary minerals, vital amino acids, proteins, and carbohydrates, phytate-phosphate unavailability, and contamination of the environment due to phosphorus. Therefore, it is necessary to use exogenous microbial phytases as feed additive to chicken feed to catalyze the hydrolysis of dietary phytate. Potential sources of microbial isolates that produce desired phytases for chicken feed supplementation have been isolated from agricultural croplands. It is achievable to isolate phytase-producing bacteria isolates using both broth and agar phytase screening media. Potential substrates for submerged fermentation (SmF) for bacterial phytase production and solid-state fermentation (SSF) for fungal phytase production include rice and wheat bran. Following fermentation, saturated ammonium sulphate precipitation is typically used to partially purify microbial culture filtrate. The precipitate is then desalted. Measurements of the pH optimum and stability, temperature optimum and stability, metal ions stability, specificity and affinity to target substrate, proteolysis resistance, storage stability, and in vitro feed dephosphorylation are used to perform an enzymatic evaluation of phytase as an additive for poultry feed. The growth of the feed phytase market is primarily due to the expansion of chicken farms to meet the demand for meat and eggs from humans. The Food and Drug Administration in the USA and the European Food and Safety Authority are primarily in charge of putting rules pertaining to feed phytase use in chicken feed into effect. Conclusively, important components of the production of phytase additives for poultry feed include identifying a reliable source for potential microbe isolation, selecting an economical method of phytase production, thoroughly characterizing the biochemical properties of phytase, and comprehending the size and regulation of the current feed phytase market.


Background
Phytases have attracted considerable attention in the area of animal nutrition, environmental protection, and biotechnology [1,2].In poultry feed, nearly 80% of the total phosphorus content is stored as phytate.Unfortunately, the gastrointestinal tracts (GIT) of poultry exhibit little to no phytase activity [3].Terefore, from the perspective of phosphate bioavailability, phytic acid and its salts, phytates, are considered nutritionally inactive substances.Phytic acid or phytate chelate vital nutrients, carbohydrates, proteins, and essential amino acids [4,5].Furthermore, in regions with intensive livestock production, consuming large amounts of feed high in phytic acid or phytate by monogastric animals leads to phosphorus-related environmental pollution, such as algal blooms [6].Tus, exogenous microbial phytase has been used as monogastric animals' feed additive because the phytase catalyzes hydrolysis of phytic acid improving nutrient bioavailability and uptake and reducing pollution caused by fecal excretion of phosphorus [7,8].
Microbes that produce phytase have been selected from a variety of sources.Phytase-producing flamentous fungi have been reported to be isolated from soil [9], citrus pulp pellets, wheat and rice bran [10], and poultry farms [11].
Tere have been reports of using poultry farms, rhizospheric soils, compost and degraded wood [12], indigenous fermented cheese product [13,14], and the soil surrounding the maize feld (halosphere), the soil surrounding the maize root (rhizosphere), and inside the maize root (endophyte) as sources to isolate phytase-producing bacteria [15,16].Researchers have avoided false positive halo zones on PSM agar plates when screening for phytase-producing bacteria by frst fooding the plate with cobalt chloride solution, then replacing the solution with freshly made coloring reagent (a mixture of aqueous ammonium molybdate and ammonium vanadate), and lastly removing the coloring reagent [13,15,17].Subsequently, the isolate with the highest phytase activity in the broth or the best halozone formation on the agar plate is chosen.16S rRNA gene sequencing is often used to identify the chosen phytase-producing bacterial isolate molecularly [18], whereas Internal Transcribed Spacer (ITS1, 5.8 S, ITS2) region sequencing is typically used for the molecular identifcation of the chosen phytaseproducing fungal isolate.Molecular tools that supplement morphological characterization for fast and accurate fungal identifcation are highly promising [19].
A selected microbial strain that produces phytase has been used to produce phytase through submerged fermentation (SmF) or solid-state fermentation (SSF) of agricultural and agroindustrial waste materials.SSF is the common fermentation type in phytase production from fungal strain [17].On the other hand, SmF has frequently been employed for producing bacterial phytases [20].Microbial culture fltrate is harvested as crude phytase after fermentation, and the obtained crude phytase is partially purifed by precipitating with saturated ammonium sulphate and then desalting the resultant precipitate.Several researchers documented the production of crude phytase [11,[21][22][23][24][25][26] and partially pure phytase [12,13,[27][28][29] with two or more desirable properties for supplementing poultry feed.
In order to establish the novel phytase as a feed additive for nutritional enhancements, extensive biochemical characterization and dephytinization activity of the phytase have been conducted [11].For the purpose of enzymatically evaluating phytase as a poultry feed additive, specifc factors such as pH optimum and stability, temperature optimum and stability, metal ions stability, specifcity and afnity to target substrate, proteolysis resistance, storage stability, and in vitro feed phytate dephosphorylation have been taken into consideration.Harvesting crude phytase from native microbial culture fltrate or partially purifying the crude phytase is a feasible method of using it as an additive in poultry feed in developing countries [30].Accordingly, the sources, isolation, and production of phytase with one or more biochemical characteristics resembling the chicken gut conditions or feed processing conditions are covered in this review.In addition, the review discusses market size and regulations concerning phytase additives in poultry feed.

Sources of Desirable Phytase as Poultry Feed Additive
Monogastric animals are known to have little to no phytase activity in the GIT [6,9], necessitating the addition of exogenous microbial phytases to feed in order to catalyze the hydrolysis of dietary phytate [31].Te major sources of exogeneous phytase are fungal and bacterial strains [20,32].While native plants and animals are also sources of exogenous phytase [33], native algae as a source is hardly available.Tis is a signifcant gap since almost all phytase studies noted in the background that phytate, which is expelled by monogastric animals, is what causes algal blooming [6].If algae are unable to use phytate efectively, which necessitates the synthesis of phytase, the bloom cannot occur.Nonetheless, it has been documented that transgenic microalgae have been developed as a source of phytase for use as a feed additive in monogastric animals [4,34].Phytase-producing native fungi and bacteria have been screened from various source materials.Te sources of the best phytase-producing isolates are presented in Table 1.Woodfordia fruticosa dried fower buds were utilized as a source material to isolate intracellular phytase-producing yeast, Pichia anomalai UDDM-55 [37].Composting soil [9], corn, citrus pulp pellets, wheat, and rice bran [10], soil from the top layer of agriculture croplands, including maize (Zea mays), wheat (Triticum aestivum), black gram (Vigna mungo), and rice (Oryza sativa), and poultry farms [11] were used as source materials to isolate flamentous fungi.Poultry farms, rhizospheric soils, compost and degraded wood [12] and halosphere (soil surrounding the maize feld), rhizosphere (soil surrounding the maize root), and endophyte (inside the root) [15,16] were used as sources to isolate bacteria.Conventionally fermented cheeses, such as Turkey's Lor cheese [14] and India's Kalari [13], have also been identifed as sources of bacteria that produce phytase.It is recommended to isolate probiotic bacteria that produce phytase from dairy products because these bacteria have been classifed as safe (GRAS) by the Food and Drug Administration (FDA) [13].Kali and Kudithi, two more traditionally fermented foods, have been identifed as sources of both yeasts and bacteria that produce phytase [15].Indian rural women use Kali, which is the leftover water from traditionally fermented rice, to cook cereal meals.Kudithi, on the other hand, is fermented liquid cattle feed.

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Isolation and Screening of Phytase-Producing Microbe
Both qualitative and quantitative techniques were used in the isolation and screening of the phytase-producing microorganisms.Te qualitative approach is based on the largest clear zone formation on the PSM agar plate (Figure 1).PSM includes sodium phytate as a solitary source of phosphorus [13], calcium phytate as the only source of phosphorus [12,23,28], or sodium phytate as sources of both carbon and phosphorus [1,10,21,39].If phytate is the only source of carbon and phosphorus, fungal species that can grow on these PSM agar plates must create an extracellular phytase to use the carbon and phosphorus found in phytate [10].Te constituents of commonly used PSM are presented in Table 2.
Unfortunately, false positive reactions (zones of clearing around microbial colonies) can be seen for acid-producing bacteria, which means that PSM lacks specifcity.Because of the dissolution of phytate/phytic acid at low pH levels brought on by acid production, a clear zone forms.To address this issue, a two-step counterstaining procedure was created [40].Agar plates are frst fooded with cobalt chloride solution, which is done on the grounds that it causes the halo zone to vanish by reprecipitating acidsolubilized phytate.Te second step is to decant the cobalt chloride solution.Tis is followed by adding the coloring reagents, aqueous molybdate, and ammonium vanadate solution, based on the idea that a positive reaction will develop a yellow color as a result of the reaction between the reagent and the released phosphate from phytate hydrolysis.Numerous researchers have employed this technique [13,15,17].
Using a quantitative method, isolates are cultivated in PSM broth and their highest level of phytase activity is assessed (Table 1).Certain strains that are identifed as having the ability to produce phytase on an agar plate are unable to do so in broth.Furthermore, some strains that test negative on an agar plate yield high levels of phytase in broth [15].Consequently, the best phytase-producing microbes have been identifed using both the broth and the agar plate [11][12][13]15] (Table 1).

Identification of Phytase-Producing Microbe
Species-level identifcation of the chosen isolates comes next, following the screening and selection of the best phytaseproducing microbes.16S rRNA gene sequencing is typically used for the molecular identifcation of bacteria in general and phytase-producing bacteria in particular [18,38].In one study, 16S rRNA gene sequencing was used to identify the chosen phytase-producing bacterial isolates after microscopic inspection [12].Bhagat et al. [13] used only 16S rRNA gene sequencing in the study to identify the chosen phytaseproducing bacterial isolate (Table 1).According to Johnson et al. [18], sequencing the full-length (∼1500 bp) 16S rRNA gene allows for taxonomic resolution at the species and strain level, which cannot be obtained by targeting 16S variable regions (V1, V2, V3, V4, V5, V6, V7, V8, and V9) with short-read sequencing platforms.Moreover, Durazzi et al. [41] demonstrated that, provided a sufcient number of reads is available, whole genome shotgun sequencing is more powerful than 16S rRNA gene sequencing at identifying less abundant taxa.
Internal Transcribed Spacer Region (ITS1, 5.8 S, ITS2) sequencing is typically used for the molecular identifcation of phytase-producing fungal isolates [19].Alves et al. [23] identifed the selected fungi using ITS region sequences.Besides, 18S rRNA gene sequencing is also used for the identifcation of the highest phytase-producing fungal isolate [11] (Table 1).Te entire ITS region, as well as the ITS1 and ITS2 subregions, is frequently utilized for fungal identifcation; however, the most successful identifcation method was one that relied on the entire ITS region [42].Molecular tools that supplement morphological ones show great promise for fast and accurate species-level fungal identifcation [19].

Phytase Production
Phytase has been produced by submerged, solid, or semisolid-state fermentation.Te production of phytase from fungal strains frequently involves solid-state fermentation (SSF) [17].A better phytase production was reported from SSF than from SmF by fungal strains [43].SSF, as opposed to submerged fermentation (SmF), improves enzyme secretion with ease of recovery and purifcation, uses less expensive substrates, such as agri-waste, and requires less water.It also stimulates fungal growth with fewer nutrient requirements [32].However, SmF can be scaled up very easily for commercial phytase productions, and stirring during the SmF process ensures homogeneity in the phytase properties [17].SmF has been widely used in bacterial strain phytase production [20].In fact, B. subtilis US417 has been reported to produce more phytase on wheat bran SmF (112 U/g of WB) than on SSF (85 U/g of WB) [44].Furthermore, when temperatures are high enough to dry out media and lower water activity below the required level for microbes, SSF is inefective for producing enzymes.Semisolid-state fermentation (semi-SSF) is a preferable choice in this situation [45].
Because it may lower the cost of producing enzymes and result in a less expensive fnal product, the use of agricultural and agroindustrial waste substrates in phytase production has been widely reported [46].Te potential substrates, microbial strains, and types of fermentation along with the amount of produced phytase are shown in Table 3. From afordable substrates SSF such as coconut oil cake by Rhizopus oligosporus [61] and citric pulp bran by A. niger FS3 [51], phytase production of 14.29 U/gds and 0.62 U/mL, respectively, was reported.Te production of 3.91 U/mL of phytase by A. niger F3 from citrus peel SmF was also reported [50].
One frequently utilized agroindustrial residue for the production of phytase is wheat bran (Table 3).It has nutrients that microbes need, including minerals (Ca, K, Mg, and Fe), B vitamins (thiamin, niacin, ribofavin, and folate), insoluble fber, essential fatty acid, starch, protein, and International Journal of Microbiology phytic acid [67].Wheat bran can, therefore, induce and promote the production of phytase [52,58].Maximum phytase production (94 U/mL) was obtained with wheat bran SmF by Klebsiella sp compared to the SmF of rice bran and chickpea by the same bacterial strain [58].From wheat bran SSF by Schizophyllum commune [26] and by A. niger NT7 [11], the highest phytase production of 113.7 U/gds and 208.30 ± 0.22 U/gds, respectively, was reported.A study showed the production of a higher mount of phytase from concentrated (upto 60%) wheat bran extract SmF.Furthermore, the utilization of wheat bran powder resulted in a higher level of phytase than that of wheat bran extract utilization [65].
Because rice bran contains proteins, fats, carbohydrates, and minerals, it has found extensive use in the bioprocess industry to produce phytase (Table 3).R. oligosporus MTCC556 produced 31.3U/gds of phytase from rice bran SSF, according to Suresh et al. 2016 [62].In comparison to wheat bran and oat bran SmF by Aspergillus favus PHY168, Ahmed et al. 2018 [47] reported the highest phytase production from 5% rice bran SmF by the strain.Furthermore, it was reported that Termoascus aurantiacus SL16W produced more phytase from rice bran Sem-SSF than from wheat bran Sem-SSF [45].Based on the aforementioned research, it is unclear whether rice bran or wheat bran promotes the production of every microbial phytase more efectively.

Partial Purification of Phytase
Te production of crude phytase with two or more desirable properties for supplementing poultry feeds has been reported by several researchers [11,[21][22][23][24][25][26].In addition, a number of researchers have reported the production of partially pure phytase [12,13,[27][28][29].In the context of production and characterization of phytase, purifcation has been considered as partial purifcation and purifcation.It has been widely documented that in partial purifcation, saturated ammonium sulphate salting out is followed by desalting procedures (Figure 2).Ammonium sulphate precipitation is the most popular and straightforward technique for partially purifying phytase from microbial sources, according to Bala et al. [28].It is interesting to note that adding fungal desalted phytase precipitated by ammonium sulphate to broiler feed greatly increased P utilization and decreased P excretion into the environment [29].Each inoculum (20 μl � 10 × 10 6 CFU/mL) was inoculated on agar plates using point inoculation, and plates were incubated at 37 °C for bacteria and 30 °C for fungus for 2 days.Ten, all plates with clear zones were washed with distilled water to remove microorganisms.Ten, plates were covered with 2% cobalt chloride solution and incubated for 5 min at room temperature.Ten, the cobalt chloride solution was discarded, and the plates were covered with an equal volume of freshly prepared 6.25% ammonium molybdate and 0.42% ammonium vanadate mixture.After 5 min incubation at room temperature, the solution was discarded and clear zones were still present [17].International Journal of Microbiology

International Journal of Microbiology
When purifying proteins, such as enzymes, the efectiveness of the purifcation processes is typically assessed based on specifc activity, purifcation fold, and percent yield (recovery), which can be calculated using the following equations: Specific activity � the activity of phytase at each steps Total protein content of respective steps  , Purification fold � specific activity of phytase at each steps specific activity of phytase at initial step  , %Yield � phytase activity of each steps phytase activity of initial step   × 100.
Partial purifcation of steps of microbial phytase with suitable properties for poultry feed supplementation and the efcacy of parameters are shown in Table 4. Phytase of A. niger NCIM 563 was partially purifed by ammonium sulphate precipitation (95%) followed by Sephadex G-25 column-based desalting.Specifc activity of 224 FTU/mg, 3.0 purifcation fold, and 95% purifcation yield were obtained from the partial purifcation [68].Partial purifcation of H. nigrescens BJ8 phytase with 20.55 fold and 96.71% yield [28], and crude phytase of A. foetidus MTCC 11682 with 23.4 fold and 12.9% yield [29] were reported.Purifcation fold and yield for partial purifcation of bacterial phytase were reported to be 2.18 and 44.63% for Serratia sp.PSB-15 phytase, and 2.55 and 62.30 for E. cloacae PSB-45 phytase [12].In a diferent investigation, Bhagat et al. [13] reported that the extracellular phytase of P. acidilactici SMVDUDB2 had been partially purifed with a 6.42 fold yield.Te fltrate obtained was centrifuged and administered to ammonium sulphate precipitation followed by desalting [28].(b) Culture broth of Serratia sp.PBS-15 and E. cloacae PBS-45 was centrifuged and administered to ammonium sulphate precipitation followed by desalting [12].(c) Te spent culture media obtained from A. foetidus MTCC 11682 culture were harvested, fltered, and the fltrates were subjected sequentially to ammonium sulphate precipitation, desalting, and gelfltration [29].
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Phytase Enzymatic Evaluation as a Poultry Feed Additives
For the enzymatic evaluation of phytase as a poultry feed additive, factors such as pH optimum and stability, temperature optimum and stability, metal ions stability, specifcity and afnity to target substrate, proteolysis resistance storage stability, and in vitro feed dephosphorylation were taken into consideration.
7.1.Te Efect of pH on the Activity and Stability.Tis review examines the enzymatic evaluation of the stability of microbial phytase and the desirability of an optimal pH in relation to average transit time and pH in various digestive tract segments of broiler chickens, as reported by Ravindran [69].Te efect of pH on the activity and stability of the desirable crude phytase are presented in Table 5.From optimum pH profling, the maximum activities of the phytase of Rhizoctonia sp.(3 FTU/mL) and Fusarium verticillioides (6 FTU/mL) were observed at pH 4.0 and 5.0, respectively, after 30 min incubation of reaction mixture.Te phytase of F. verticillioides maintained ≥50% activity between pH 4.25 to 5.5 [24].Tis fnding suggests that the phytase may withstand the pH of the crop.Immobilised phytase of A. foetidus MTCC 11682 was found stable over pH 2.5 to 7.5.It showed the highest stability at pH 5.5 at which the activity of 47 FTU/mL was observed [22].Tis fnding suggests the phytase can be stable in the pH of upper GIT of poultry despite the preincubation time was less than the average digesta retention time.
Crude phytase was produced by using S. communes LPB 101, and the efect of pH on the phytase activity was tested over 1.0 to 10.0.Te phytase exhibited the highest activity (approximately 165 °FTU/dgs) at pH 5.0 maintaining ≥50% of the maximum activity at pH 5 to 8.5.After 24 h storage in pH 2 to 6 at 4 °C, the phytase showed activity of 65 to approximately 77.75 °FTU/dgs [26].Te fnding of optimum profling implies that the phytase may withstand the pH of the crop.In the same study, with limited information on the temperature as the activity was assayed at 4 °C, pH stability is appropriate for feed supplementation.Te pH stability of phytase of Muscodor sp.UBSX was evaluated after storage in 100 mM citrate bufer (pH 3.0, 4.0, 5.0, and 6.0) at 25 °C for 1 h.At pH 5.0, it exhibited 7.36 FTU/mg (100% relative specifc activity).However, at pH 4.0 and 6.0, the phytase activity was 25% and 35%.Te phytase activity was fully inhibited at pH 3.0 [23].Tese fndings suggest that the phytase is stable enough in the crop of poultry.
When preincubated in pH 2.5 to 10.0 for 1 h, the crude phytase of K. marxianus maintained ≥ 50% of maximum activity.Te highest activity of 3 FTU/mL was obtained at pH 4.0 [25].Phytase of A. niger NT7 was observed to be active at a broad range of pH and was found optimal at pH 2.6 and 4.8.Te relative activity of ≥50% was maintained at pH 2 to 7 [11].Te fndings of Pires et al. and Kumari and Bansal suggest that the phytase under the study might be efcient for poultry feed supplementation as it can withstand the physiological pH of crops, proventriculus, and gizzard.
Te efect of pH on the activity and stability of partially purifed phytase are presented in Table 6.Phytase of the thermophilic fungus, Rhizomucor pusillus, showed an optimum pH of 5.4 and ≈80% activity retained over a wide pH range, 3 to 8 [9].Te phytase of A. niger, NCIM 563, showed two pH optima, i.e., 2.5 (Phy I) with 41.47 FTU/mL maximum activity and 4.0 (Phy II) with 10.71 FTU/mL maximum activity.In the same study, the phytase was found stable at pH 1.5 to 3.5 when preincubated at pH 1.5 to 7.0 for 18 h at 4 C and assayed at pH 2.5.Phytase was stable from pH 1.5 to 7.0 when activity was assayed at pH 4.0 [68].Bala et al. [28] found an optimum pH 5.0 at which phytase activity from H. nigrescens BJ82 was taken as 100%, and the phytase maintained ≥50% relative activity from pH 4 to 6.
Te pH profling for phytase from A. foetidus MTCC 11682 showed pH 3.5 and 5.5 optima.At pH 5.5, the activity of phytase was 1.5-fold higher compared to its activity at pH 3.5, and this activity was considered 100%.Like pH optimum determination, the phytase exhibited two peaks of maximum stability at pH 3.5 and 5.5.At pH 5.5, the enzyme retained 90% of its original activity even when preincubated for 6 h.Half of the phytase activity was lost at pH 2.5, 4.5, and 6.5.A decline in the activity was observed at pH 7.5 [29].Te phytase activity of Serratia sp.PSB-15 and E. cloacae PSB-45 showed the maximum activity of 0.13 FTU/mL and 0.07 FTU/mL at pH 6.0 and 7.0, respectively.Te phytase maintained ≥50% of the original activity from about pH 2.35 to 7.75 (Serratia sp.PSB-15) and about 2.75 to 7.6 (E.cloacae PSB-45) [12].Phytase of P. acidilactici SMVDUDB2 showed 4.5 FTU/mL maximum at pH 5.5 and ≥50% activity was maintained over pH 3 to 8.5 [13].Te phytases under the above studies might act efciently for phytate degradation under pH range in the upper gastrointestinal tract of the broiler [69].

Te Efect of Temperature on the Activity and Stability.
Te desirability temperature optimum and stability of the reviewed microbial phytase was enzymatically evaluated comparing with a body temperature of chicken which is 41 to 42 °C [70].Furthermore, desirability temperature optimum and stability was evaluated in line with commonly used steam conditioning temperature of 65 to 90 °C for 15 s duration [41,42].Steam conditioning and pelleting process of poultry feeds prevent microbial infection [71] and increase body weights [72].
Te efect of temperature on the activity and stability of crude phytase are presented in Table 7. Te crude phytase of Rhizoctonia sp. and F. verticillioides showed the activity of 1.25 FTU/mL and 2.5 FTU/mL, respectively, at a temperature of 50 °C.Te phytase of F. verticillioides maintained ≥50% of the maximum activity at 28 to 60 °C.At 80 °C, almost all phytase activity was lost [24].Te investigation of the efect of temperature on the phytase of S. communes LPB 101 over 30 to 90 °C at 10 °C interval resulted in an optimal temperature of 50 °C [26].Te crude phytase of Muscodor sp.UBSX showed complete thermostability at 40 °C for 2, 5, 10, 15, 20, 30, 45, and 60 min preincubation time.However, the phytase thermostability was reduced by half at 50 and 60 °C International Journal of Microbiology   In line to GIT pH A.foetidus MTCC 11682 3.5 and 5.5 30, 37 100% at pH 5.5 2.5 to 7.5 2.5 to 6.5, at pH 5.5, 100% 6, 37 [29] In line to upper GIT, duodenum, and Jejum pH P. acidilactici SMVDUDB2 International Journal of Microbiology after 10 min and 1.5 min preincubation, respectively [23].Te phytase of A. foetidus MTCC 11682 showed maximum activity at 37 °C.At 70 °C, 74% of the activity was retained after 30 min incubation [22].
In optimum temperature profling, crude phytase of K. marxianus presented approximately 3.25 and 3.5 FTU/ mL maximum activity at 60 and 80 °C, respectively, showing the presence of more than one type of phytase.However, thermostability investigation at 80 °C resulted in 40% residual phytase activity after 5 to 60 min preincubation.Tis paradox might be happened due to the assay performed at pH 7.5 at which the phytase had shown the lowest activity [25].Phytase of A. niger NT7 exhibited activity from 40 to 80 °C.Te optimum temperature was 60 °C at which activity was considered 100%.Interestingly, the phytase was found more stable at 50 °C with a half-life (t 1/2 ) of 240 min.It showed a t 1/2 of 120 min at 60 °C, but preincubation at 70 °C for 60 min resulted in less than half of relative activity [11].Te fndings of Pires et al. [25] and Kumari and Bansal [11] indicated phytases understudy might be stable at feedpelleting temperature and exposure time [12].
Te efect of temperature on the activity and stability of partially purifed phytase are presented in Table 8.Te phytase of R. pusillus was found optimally active at 70 °C [9].Soni and Khire [68] determined the optimum temperature for phytase of A. niger NCIM 563 over the range of 40 to 65 °C.Te optimum temperature was 60 °C for both Phy I (pH 2.5 active) and Phy II (pH 4.0 active).Termal stability was investigated via phytase preincubation for 60 min.At 65 °C, phytase was active at pH 2.5, retained 80% of its original activity after 15 min but at 70 °C activity decreased sharply with only 40% of its original activity remained after 15 min.For Phy II, only 40% of the original activity was retained after 15 min exposure at 65 °C, and 36% of the original activity was detected after 5 min exposure at 70 °C.Interestingly, Phy I retained 65% of its original activity after 10 min at 70 °C, and Phy II retained 60% of its original activity after 10 min at 65 °C.Te optimum temperature for the activity of phytase of H. nigrescens BJ82 was 50 °C at which relative activity of the phytase was considered 100%.Te phytase showed ≥50% relative activity from approximately 43 to 65 °C [28].Phytase of A. foetidus MTCC 11682 exhibited activities in the range of 4 to 80 °C with an optimum activity at 37 °C.Te activity of enzymes did not change much between 37 and 50 °C.Termostability profling in terms of maintaining the residual activity indicated that the enzyme was stable at 37 °C preincubation for 30 min without losing activity, whereas the activity retention was 87% at 50 °C preincubation.Interestingly, about 56% of the residual enzyme activity was retained at 80 °C preincubation after 30 min exposure [29].
Phytase from Serratia sp.PSB-15 was optimally active at 50 °C and stable at 70 and 80 °C after 20 min preincubation.After treating this phytase, 81.1 and 69.8% of the initial activity was retained, respectively.In the same study, phytase from E. cloacae PSB-45 had the maximum phytase activity at 70 °C, stable at 70 and 80 °C after 10 min preincubation, and showed 52.2 and 18.3% residual activity after treatment at 80 °C for 10 and 20 min, respectively [12].Te optimum temperature of phytase of P. acidilactici SMVDUDB2 was 37 °C.Termostability assay of the phytase was determined by preincubating the phytase at 50 °C, 60 °C, 70 °C, and 80 °C for 3 h.Te phytase exhibited good thermostability, and the reduction of the original activity was 25.57% and 32.6% after 3 h at 50 °C and 60 °C, respectively.Interestingly, 100%, 80%, and 60% residual activities were maintained after 30 min preincubation at 50 and 60 °C, 70 °C, and 80 °C, respectively [13].Te studies suggest that phytases understudy may function under poultry physiological temperature and withstand animal feed pelleting temperature range.

Te Efect of Metal Ions on the Activity of Phytase.
Te efect of various metal ions on the crude phytases is shown in Table 9. Salmon et al. [26] reported that the activity of crude phytase of S. communes LPB 101 was stimulated in the presence of 1 mM of K + , Ca 2+ , Mg 2+ , Mn 2+ , Zn 2+ , Cu 2+ , and Na + .At the same concentration, however, Alves et al. reported that the phytase of Muscodor sp.UBSX was inhibited by Na + and Cu 2+ .At both 2 and 5 mM fnal concentrations, Ca 2+ , Cu 2+ , and Mg 2+ increased the activity of crude phytase of K. marxianus [25].In another study, the phytase activity of Aspergillus niger NT7 was augmented by Ca 2+ and Zn 2+ , whereas it was inhibited by Mn 2+ , Mg 2+ , and Cu 2+ at both 1 mM and 5 mM fnal concentration [11].
Te efects of mineral ions on the activity and stability of partially purifed phytase were reported [12,68].In the investigation of Soni and Khire [68], the efect of metal ions at 1 mM fnal concentration on the phytase of A. niger NCIM 563 was addressed.Te phytase retained 79% of its activity at pH 4.0 in the presence of Zn 2+ while at pH 2.5, it retained 61%.Mg 2+ , Mn 2+ , Ca 2+ , and Fe 3+ had a stimulatory efect at pH 4.0 while Fe 2+ and Cu 2+ had a stimulatory efect at pH 2.5.Zn 2+ and Cd 2+ had more inhibitory efects at pH 2.5 than at pH 4.0.Kalsi et al. [12] investigated the efect of metal ions at 5 mM concentration on the phytase of Serratia sp.PSB-15 and E. cloacae PSB-45.After 30 min preincubation, the relative activity profle with ferrous sulphate was about 50% for PSB-15 phytase, while for PSB-45 phytase, it was 35.8%.With copper sulphate, PSB-15 showed 78% relative activity while PSB-45 phytase was least stable.Manganese sulphate caused more inhibition of PSB-45 phytase.Magnesium sulphate inhibited both phytase activities more than 50%.

Specificity and Affinity of Phytase to Target Substrate
Broad substrate specifcity and highest afnity (Michael-Menten constant, K M ) toward a target substrate is a required characteristic of phytase for poultry feed addition [31].Te crude phytase of R. pusillus showed broad substrate specifcity catalyzing the hydrolysis of ribofavin phosphate, AMP, ADP, ATP, paranitrophenol phosphate, NADPH2, phenyl phosphate, and phosphoenol pyruvate, all at concentrations of 5 mM [9].Salmon et al. [26] reported the afnity of crude phytase of S. communes LPB 101 to sodium phytate, 0.4 to 5.2 mM.Te K M of 0.16 mM and maximum 12 International Journal of Microbiology  optimum; max: maximum; IT: incubation time; T °: temperature; PIT: preincubation time; NA: not available; GIT: gastrointestinal tract.≈ is approximate value.Recommendation provided comparing with 41 to 42 °C body temperature of chicken [70] and 65 to 90 °C steam conditioning temperature for 15 s duration [41,42].optimum; max: maximum; IT: incubation time; T °: temperature; PIT: preincubation time; NA: not available; GIT: gastrointestinal tract.≈ is approximate value.I and II stand for phytase I and phytase II, respectively.Recommendation provided comparing 41 to 42 °C body temperature of chicken [70] and 65 to 90 °C steam conditioning temperature for 15 s duration [72,73].14 International Journal of Microbiology velocity (V max ) of 2.087 μmol•mL −1 min −1 were obtained at 1.2 mM Na-phytate concentration (Figure 3).Soni and Khire [68], Kalsi et al. [12], and Bhagat et al. [13] reported the afnity of partially purifed phytase to sodium phytate.Te determined afnity (K M ) for Phy I and II of Aspergillus niger NCIM 563 was 3.18 and 0.514 mM, respectively, while V max was 331.16 and 59.47 μmols/min/mg [68].Using 0.25 to 10.0 mM Sodium phytate, Kalsi et al. obtained 1.25 and 0.48 m values of K M , and 0.157 and 0.140 U/mL values of V max for the phytase of Serratia sp.PBS-15 and E. cloacae PBS-45, respectively.In the determination of K M and V max of the phytase of P. acidilactici SMVDUDB2, different concentrations of sodium phytate (0.1 to 1.8 mM) at pH 5.5 were used resulting in K M and V max values of 0.385 mM and 4.965 μmol/min, respectively [13].

International Journal of Microbiology
8.1.Phytase Resistance to Protease.Proteases are enzymes that can be synthesized in the GIT and that can be classifed into six groups based on their catalytic mechanisms: aspartic, glutamic, metalloproteases, cysteine, serine, and threonine proteases [74].To be used as poultry feed additive, the exogenous phytase must be able to withstand the activity of poultry GIT proteases [75].In one study Bhagat et al. [13], partially purifed phytase of P. acidilactici SMVDUDB2 (10 μg/mL) was incubated in 0.2 M glycine-HCl bufer (pH 2.5) containing pepsin (10 μg/mL) in the ratio of 1 : 1, and 0.2 M Tris-HCl bufer (pH 8.0) containing trypsin (10 μg/mL) in the ratio of 1 : 1 at 37 °C for a period of 3 h.As a control, the phytase was incubated in the same condition as stated above but lacking pepsin or trypsin.Te phytase had strong proteolytic resistance towards pepsin and trypsin.Above 80% of phytase activity was retained after 1 hour incubation period in pepsin and trypsin solution, which reduced to 58.21% and 51.81% after 3 h preincubation in the solutions.

Phytase Storage Stability.
Once produced, storage and distribution of feed phytase is a must before it is mixed with animal feed, therefore, phytase stability during storage must be evaluated [16].Salmon et al. [26] stored crude phytase of S. communes LPB 101 at room temperature (26 ± 2 °C), cooling (4 °C), and freezing temperatures (−18 °C) for 5 months.Te phytase showed great stability at room and cooling temperature storage, losing only less than 10% activity within the 42 days of storage.Te best shelf life was observed at a cooling temperature with 38% of its initial activity maintained after 112 days of storage and still presenting enzymatic activity after 125 days of storage.Ajith et al. [22] stored three phytase samples of A. foetidus MTCC 11682 at −20 °C.Te phytases showed nearly similar stable retention of the enzymatic activity over a period of one year.
Storage stability of partially purifed phytase was reported by Kalsi et al. [12].In the investigation, % residual activity compared to fresh phytase was used for stability evaluation for 30 days of storage at refrigerator (4 °C) or room temperature.Phytase from Serratia sp.PSB-15 was Te efect of metal ions at 1 mM on the activity of phytase of S. communes LPB 101 [26]; b the efect of metal ions at 1 mM on the activity of phytase of Muscodor sp.USBX [23]; c,d the efect of metal ions at 2 and 5 mM, respectively, on the activity of phytase of K. marxianus [25]; RA and NA represent relative activity and are not available, respectively.
International Journal of Microbiology found highly unstable at room temperature (5.6% residual activity).However, the phytase revealed very good stability in the refrigerator (96.5% residual activity).Te authors concluded that the phytase of the isolates could not be stored for longer or if needed, and alternate strategies must be adopted for long-time storage of PSB phytases.

Dephytinization of Monogastric Animal Feed
In vivo studies of the digestive process are long, expensive, and difcult to rationalize, whereas in vitro systems may give more accessible insight into parts of this process [76].As such, feed dephytinization in simulated monogastric animals' digestive system should be conducted for enzymatic evaluation of the produced phytase as poultry feed additive.Kumari and Bansal [11] reported dephytinization activity of crude phytase in which crude phytase (10 FTU) of A. niger NT7 was evaluated for dephytinase activity on wheat bran (10 g) under experimental conditions of 100 mM acetate bufer, pH 4.8 at 50 °C.Te result showed a gradual increase in the release of inorganic phosphorous (2460 ± 102 μg/mL) and proteins (491 ± 20 μg/mL) up to 72 h followed by rapid decline.Te liberation of reducing sugars (2019 ± 61 μg/mL) was found increasing until 60 h and later declined gradually (Figure 4(a)).According to the authors, the reason for the decline attributed to plausibly due to end-product inhibition, limitation of substrates, and denaturation of the phytase.Dephytinization activity of partially purifed phytase was reported by Bala et al. [28] and Kalsi et al. [12].Partially purifed phytase of H. nigrescens BJ8 was evaluated at 10 grams of wheat and gram four to 10 FTU of the phytase.Wheat and gram fours were efciently dephytinized with the concomitant liberation of inorganic phosphate.Tere was progressive increase in the liberation of inorganic phosphate and soluble protein with an increase in reaction time [28].Partially purifed phytase from Serratia sp.PBS-15 and E. cloacae PBS-45 was evaluated for their ability to catalyze hydrolysis of soybean meal phytate.Te use of 250 FTU/kg of each phytase in an incubated feed mixture at 37 °C for 2 h resulted in increased phosphorus compared to the control (Figure 4(b)).Te liberated phosphate was in the range of 45 to 46% [12].

Poultry Feed Phytase Market Size
Every year, there is an increase in demand for meat and eggs.As a result, the world's largest and fastest-growing agrobased production sector is now the poultry industry.Phytase supplements for poultry feed are in higher demand due to the cost of associated feed and the pollution of the environment caused by phosphorus [76][77][78].Te poultry feed phytase market was estimated to be worth US $210 million in 2021 and is projected to grow at a compound annual growth rate (CAGR) of 6.0% in terms of revenue over the forecasting period, 2022-2030, according to a September 2022 Global Marker Insight report [79].Te livestock feed phytase market segment in the report included the markets for poultry, pigs, cattle, aquaculture, and other animals.Of these, the poultry feed phytase market segment is expected to lead the global market (Figure 5).
Te worldwide market will be dominated by the powdered or granular phytase market segment.In addition to holding a combined market share of more than 60%, they are anticipated to grow at the fastest rate in terms of revenue between 2018 and 2026 [80].For them, the revenue trend for the 2022-2030 forecasting period showed the same pattern.Tis market for liquid phytase is predicted to expand at a compound annual growth rate (CAGR) of 6.5%.Because liquid phytase ensures optimal post-feed-pelleting application and keeps heat-sensitive feed phytase from denaturing, it is in high demand [79].

Regional Outlook of the Feed Phytase Market
North America, Europe, Asia Pacifc, Latin America, and the Middle East and Africa (MEA) are the traditional regions into which the feed phytase market is divided [79][80][81][82][83]. Te Asia-Pacifc region is currently experiencing a notable surge in per capita income and rapid economic growth.Te demand for dairy products, meat, and eggs increased as a result  [26] were expressed in the same common unit FTU/gds.16 International Journal of Microbiology of these factors.As a result, the livestock industry in the area has grown, necessitating careful consideration of feed additives such as phytase to enhance animal nutrition [79].However, a high feed phytase cost will limit the market size in developing nations (Figure 6).Te complicated biotechnological process of producing phytase necessitates a large investment in R&D as well as manufacturing, which accounts for the high cost [81].
Te countries that are preferred, according to the Global Market Insight report [79], are North America's USA and Canada; Europe's Germany, UK, Spain, Russia, France, and Italy; Asia Pacifc's China, India, Japan, Australia, Malaysia, Tailand, and Indonesia; Latin America's Brazil, Mexico, and Argentina; and Middle East and Africa's Saudi Arabia, UAE, and South Africa.North America is anticipated to lead the global feed phytase market from 2023 to 2030, driven by the region's well-established livestock industry, which includes swine, poultry, and cattle farms, according to a Business Research Insights report updated on October 2023 [81].Nonetheless, the European animal feed phytase market is expected to gain dominance in the same forecast year and reach a valuation of US $390 million by 2030, according to Global Market Insight [79].Te market participants that were profled during the studies may be the cause of the disparity in feed phytase market analysis trends (Table 10).VTR BioTech was profled by Business Research Insights [81], but not by Global Market Insight [79].Alltech, Novozymes, and Novus International were profled in Global Market Insight [79], but they were not in Business Research Insights [81].International Journal of Microbiology

Conclusions and Recommendations
In isolation, screening and identifcation of native microbes for phytase production for poultry, the selection of phytate enriched source environment, the simultaneous use of both agar and broth PSM, and the use of cultural, morphological, biochemical, and molecular methods for isolate identifcation are very crucial.Fungal phytase is better produced by solid-state fermentation, while bacterial phytase is better produced by submerged fermentation.Te economics of producing phytase is signifcantly impacted by the use of substrates made from agricultural or agroindustrial waste.Te phytase that is produced has been assessed for its enzymatic suitability as an additive for poultry feed based on one or more biochemical properties.Te need to improve the digestibility of poultry feed through phytase supplementation stems from the high demand for an animal protein diet.As a result, the market for poultry feed phytase is expected to grow at a compound annual growth rate (CAGR) of 6.0% between 2022 and 2030.Te cost of the biotechnological process used to produce engineered phytase is causing developing nations to fall behind in the phytase feld.Standards to assess the quality and safety of phytase feed additives for poultry have been ratifed in response to the growth of phytase-producing businesses, despite the difculty in standardizing the process.Terefore, by extracting crude or partially purifed phytase from native microbial culture fltrate, researchers should determine whether using phytase as an additive in poultry feed is feasible in developing nations.Te pH value specifc to the intended breed of poultry, the pepsin secreted from the poultry's GIT, the temperature and exposure duration of the feed pelleting process, the particular feed storage conditions, the stability of the feed's common mineral composition, and the in vitro feed hydrolysis in a reaction mixture that simulates the GIT are all necessary to evaluate this product.It is necessary to take into account hot, acidic, and plant litter-containing environments when isolating and screening appropriate phytase-producing microbes.Researchers and nutritionists should collaborate to create and assess chicken diets that have the right amount of microbial phytase.It is recommended that poultry farmers consult researchers or nutritionists when choosing phytase feed additives for their particular breed of birds.If not, manufacturers ought to share the approval documents for phytase feed additives since they make explicit the usage guidelines, the intended animal, and the minimum level of inclusion.powder/liquid) for all pigs and all avian species (Victory Enzymes GmbH)," EFSA journal.European Food Safety Authority, vol.20, no. 12, Article ID e07701, 2022.

Figure 1 :
Figure 1: Clear zones formed by phytase activity on the phytase screening agar plates.Clear zone of phytase of (a) Aspergillus fcuum (NRRL 3135), (b) Lactobacillus plantarum (B-4496), and (c) Lactobacillus acidophilus (B-4495).Each inoculum (20 μl � 10 × 10 6 CFU/mL) was inoculated on agar plates using point inoculation, and plates were incubated at 37 °C for bacteria and 30 °C for fungus for 2 days.Ten, all plates with clear zones were washed with distilled water to remove microorganisms.Ten, plates were covered with 2% cobalt chloride solution and incubated for 5 min at room temperature.Ten, the cobalt chloride solution was discarded, and the plates were covered with an equal volume of freshly prepared 6.25% ammonium molybdate and 0.42% ammonium vanadate mixture.After 5 min incubation at room temperature, the solution was discarded and clear zones were still present[17].

Step 1 :Figure 2 :
Figure2: Phytase partial purifcation steps: (a) phytase of Humicola nigrescens BJ8 was extracted by mixing 50 mL of distilled water containing 0.1%Tween-80 with solid-state fermented substrate.Te mixture was shaken at 200 rpm for 60 min and fltered through a doublelayer muslin cloth.Te fltrate obtained was centrifuged and administered to ammonium sulphate precipitation followed by desalting[28].(b) Culture broth of Serratia sp.PBS-15 and E. cloacae PBS-45 was centrifuged and administered to ammonium sulphate precipitation followed by desalting[12].(c) Te spent culture media obtained from A. foetidus MTCC 11682 culture were harvested, fltered, and the fltrates were subjected sequentially to ammonium sulphate precipitation, desalting, and gelfltration[29].
line to GIT and feed pelleting T °Opti:

Table 1 :
Screening and identifcation of potential microbial isolates that produce desirable phytase.
a Te value is enzyme index (EI).b FTU/gram of dried substrate (gds) produced through SSF.NA: not available; CZ: clear zone.Te phytase activity units are uniformly converted to FTU/mL for phytase produced through SmF.

Table 3 :
Substrates and fermentation types in production of desirable microbial phytase.

Table 4 :
Summary of partial purifcation steps of desirable microbial phytase.
a one unit of phytase activity was defned as the amount of phytase that releases 1 μmol phosphate per minute under the assay conditions, b determined protein concentration using the Bradford method, and c determined protein concentration using the Lowry method; d protein concentration determination method was not indicated.HIC: hydrophobic interaction chromatography.8InternationalJournal of Microbiology

Table 5 :
pH optimum and stability of crude desirable phytase for poultry feed application.

Table 6 :
pH optimum and stability of desirable partially purifed microbial phytase.

Table 7 :
Temperature optimum and stability of crude phytase for poultry feed application.

Table 8 :
Temperature optimum and stability of desirable partially purifed microbial phytase.

Table 9 :
Te efect of metal ions on the crude desirable phytase for poultry feed application.
Figure 3: Phytase kinetics of crude phytase of S. communes LPB 101 to sodium phytate.Te values of phytase activity production in units of enzyme per gram of dry substrate (U gds −1 )

Table 11 :
Some approved phytases by EFSA for poultry feed additive.