The Ronozyme P (6-Phytase) is usually produced by Aspergillus orzae which carries a gene coding for phytase obtained from Peniophora lycii. The genetic modification safety of the current produced strain has been established in 2004. The products availability is in two: granulate and liquid, featuring 5000 FYT/g and 20000 FYT/g, respectively. Both forms of the additive are said to be equivalent when they are used to deliver equal levels of enzymes. Based on a tolerance study performed in chickens and turkeys meant for fattening laying hens and ducks, piglets, pigs and fattening sows, the FEEDAP Panel concludes that the additive is completely safe for these species at the highest proposed dose of 1000 FYT/kg per feed. The FEEDAP Panel consider these conclusion as one which can be extended to all types of poultry (fattening and laying) and porcine species (growing and breeding) at the same recommended dose. The information discussed in this paper is considered to provide all the sufficient evidence that the said chemical presents no risks for all including the consumer, user or the environment. It also allows for conclusions to be drawn based on the efficacy of this additive when it is supplemented to diets.
Following a European Commission request, the Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) was asked to provide an accurate scientific opinion on the safety and efficacy of Ronozyme P/Bio-Feed Phytase/ZY Phytase (6-phytase) as a component of feed additive for chickens and turkeys which enhances fattening, laying hens, piglets (weaned) and many others (Kumar et al., 2012).
Based on the efficacy studies performed on poultry, the FEEDAP Panel concludes that the efficacy of the Rynozome P additive has been demonstrated as effective in chickens and turkeys for fattening at 250 FYT/kg, 300 FYT/kg in laying hens and 250 FYT/kg in ducks. These conclusions can therefore be extended to all other minor poultry species, that at a minimum dose of 250 FYT/kg growing animals will achieve the target and of 300 FYT/kg all laying/breeding animals will also perform at peak.
Based on the efficacy studies carried out on pigs, the FEEDAP Panel concludes that it is indeed effective as an additive meant for fattening.This is at 500 FYT/kg in piglets and in sows at 750 FYT/kg per feed. These conclusions can therefore be extended to all other minor porcine species at a specified minimum dose of 500 FYT/kg for all growing animals and at 750 FYT/kg for all the breeding animals. The use of this additive paves way for the use of diets which contain a lower level of the inorganic P, which may in turn lower the amounts excreted.
In animal nutrition, phytate has been described as ‘both an anti-nutritional factor and an indigestible nutrient’. However, the presence of phytate in most of the human foods taken may have some positive consequences besides reducing the availability of calcium (Ca), zinc (Zn) and other traceable minerals. The major concern here includes anti-diabetic and anti-carcinogenic effect in humans (Kumar et al., 2012). Phytate is found in potentially usable plant derived ingredients of fish feed including soybean, Jatropha kernel meal found in non-toxic genotype, and detoxified Jatropha kernel meal from the toxic geno-type, rice, wheat, barley, maize, groundnut, sesame, and rapeseed. Soybean meal, rapeseed meal, and sesame meal contain from 50% to 80% of total phosphorus in phytase form. The remaining P is represented by soluble inorganic phosphate and cellular P (phosphorus bound in nucleic acids, phosphorylated proteins, phospholipids, and phospho-sugar). Phytate which is an isolate obtained from plants has its roots based on the group of organic phosphates and is also a component of the mixture of calcium-magnesium salt of the inositol hexaphosphoric acid, called as phytin.
The concentration of phytate and phytase in the feedstuffs varies considerably. Phytate may be an amount between 0.7% and 2% of most cereals as well as oil-seeds (Posters Group 2, 2001). In general plant-derived fish feed ingredients, for example the soybean meal, rapeseed meal, or sesame meal contain 1.0–1.5%, 5.0–7.5% and 2.4% phytate respectively. It has been estimated that about 14.4 million tons of phytate-P is produced annually from worldwide production of seeds and fruits (Kumar et al., 2012). This amount of P is equivalent to 65% of annual sales of P as fertilizers. The activity of indigenous phytase is comparatively higher in cereals and cereal by-products than in legume seeds. Moreover, large variations in phytase activity among feedstuffs have been reported which depend on the genetic and environmental factors.
Table 1: Total phosphorus, phytate phosphorus and endogenous phytase activity in common feed ingredients used in fish diet (Kumar et al., 2012)
|Ingredients||Phytase activity (FTU/kg)||Total P (g/kg)||Phytate P(g/kg)||Proportion of phytate-P in total P (x)|
|Gross defatted com germ and bran||41
|Fine defatted com germ and bran||56
|Rice bran||129 (70–190)||17.51||15.83||90.2|
|Wheat bran||2173 (1700–3090)||10.96||8.36||76.3|
This enzyme enhances phosphate bioavailability in the gut and is widely used as a feed additive, especially for pigs and poultry. Work related rhinitis, conjunctivitis, cough, asthma and dermatitis were reported by workers, who handled phytase in different work-places. Therefore a study was conducted aiming to receive objective data on the frequency of sensitization to phytase and to estimate the allergenic potential of this enzyme (Poster Group 2, 2001). Patients collective and methods: The study included 5 3 phytase-exposed persons, where case history was taken. Our sample was divided in a high and a low exposed group, depending on exposure assessment. We established EAST- and ELISA-tests with phytase coupled on fixed phases to measure IgE- and IgG-antibodies in the sera of exposed and non-exposed persons (negative controls) (Posters Group 2, 2001). Further we performed immunoblotting of the enzyme with pooled sera of five sensitized workers. Results: The high exposed group reported respiratory diseases (87 ‘YO) and conjunctivitis (43 %) more frequently than the low exposed group (60 %, 20%). In the group of 38 subjects with work-related symptoms 14 (37%) had phytase-specific IgE-antibodies and 19 (so”/) p hytase-specsc IgG antibodies, whereas in the group of asymptomatic persons 1 (7 %) had IgE- and 5 (33%) IgG antibodies (Kumar et al., 2012).
The number of IgE positive subjects in the high exposed group was much greater than in the low exposed group. The pooled positive serum showed an IgE-binding to a 65 kDa protein, which possibly represents an allergenic fragment of phytase. Conclusions: The results of this study demonstrate, that powdered phytase has a high allergenic potential (Posters Group 2, 2001). Direct contact should be avoided. Therefore it is suggested, to develop non-powdering capsulated products (e.g. granulates).
Up to date, phytases have been solely used as the main ingredient in animal feed additive in diets largely used for swine and poultry, and to a slight extent in fish diet too. The first commercial product was launched in 1991 (Posters Group 2, 2001). Numerous animal studies have shown the effectiveness of supplemental microbial phytase in improving the utilization of phosphate from phytate. Therefore, including adequate amounts of phytase in the diets for simple-stomached animals reduces the dire need of having this orthophosphate supplementation in feed. As a result, the environment is guarded from pollution with excessive manure phosphorus runoffs because the faecal phosphate excretion of the animals is reduced by up to 50 % (Kumar et al., 2012). Great potential is also registered for the use of this phytases in the processing and manufacturing of food products for human consumption, but still at the moment, no phytase product has been proved relevant for human food application. None has found its way to the market so far. Research in this field focuses on the improvement of the nutritional value of plant-based foods as well as on the technical improvement of food processing. A diet rich in phytate leads to a considerably reduced absorption of dietary minerals and the dephosphorylation of phytate during food processing results in the formation of only partially phosphorylated myo-inositol phosphate esters with a lower capability to impair with the intestinal uptake of dietary minerals. Individual myo-inositol phosphate esters have been shown to have several important physiological functions in man. Therefore, phytases may find application in food processing to produce functional foods, if such biochemically active myo-inositol phosphate esters could be generated by phytases and absorbed in the alimentary tract of humans. Technical improvements by adding phytases during food processing have been reported for bread making, production of plant protein isolates, corn wet milling and the fractionation of cereal bran (Posters Group 2, 2001).
Phytate works in a broad pH range as a highly negatively charged ion. It features a tremendous affinity for food components which have positive charge(s), such as minerals, trace elements and proteins. This interaction does not have only nutritional effects, but also affects the yield as well as quality of most food ingredients for example starch, steep liquor or plant protein isolates. The major concern about the availability of phytate in the human food is its negative effect on mineral uptake. Minerals of concern in this situation are zinc, iron, calcium, magnesium, manganese and copper. The formation of insoluble mineral-phytate complexes at physiological pH values is referred to as the major reason for the poor mineral bioavailability, because these complexes are essentially not available for absorbtion from the human gastrointestinal tract (Narasimha et al., 2013). The human small intestine features a very limited capability to hydrolyze phytate due to the lack of endogenous phytate-degrading enzymes and the limited microbial population in the upper part of the digestive tract. Solubility and stability of the stated myo-inositol phosphate-mineral complexes usually end up decreasing as the number of phosphate residues on the myo-inositol ring decreases (Posters Group 2, 2001).
Therefore, removal of the phosphate residues from phytate will lead to a reduced impairment of the intestinal uptake of essential dietary minerals. In isolated form only myo-inositol pentakisphosphate had the ability to suppress absorption of iron, zinc and calcium in human beings, while myo-inositoltetrakis- and trisphosphates registered no effect on the concentrations under analysis (Posters Group 2, 2001). In the presence of higher phosphorylated myo-inositol phosphates, however, myo-inositol tetrakis- and trisphosphates were shown to contribute equally to the negative effect of phytate on the rates of iron absorption. Because a strong negative correlation was located between zinc absorption and the sum of myo-inositoltris- through hexakisphosphate from cereal and legume meals, such a contribution is probably also the case in the zinc absorption. Phytate is well known to result to complexes with proteins at both acidic and alkaline pH. This interaction is feared to have the capability of affecting changes in the protein structure that can decrease enzymatic activity, protein solubility and proteolytic digestibility.
However, the significance of protein-phytate complexes in nutrition is still under scrutiny (Narasimha et al., 2013). Strong evidence exists that phytate-protein interactions negatively affect protein digestibility in vitro and the extent of this effect depends on the protein source. A negative effect of phytate on the nutritive value of protein, however, was not clearly confirmed in studies with monogastric animals. While some have suggested that phytate does not affect protein digestibility, others have found an improvement in amino acid availability with decreasing levels of phytate. This difference may be at least partly due to the use of different protein sources. Of nutritional significance might also be the inhibition of digestive enzymes such as a-amylase, lipase or proteinases, such as pepsin, trypsin and chymotrypsin, by phytate as shown in in vitro studies.
The inhibitory effect increases with the number of phosphate residues per myo-inositol molecule and the myo-inositol phosphate concentration. This inhibition may be due to the nonspecific nature of phytate-protein interactions, the chelation of calcium ions which are essential for the activity of trypsin and a-amylase, or the interaction with the substrates of these enzymes. The inhibition of proteases may be partly responsible for the reduced protein digestibility. Phytate has also been considered as an inhibitor of a-amylase in vivo as indicated by a negative relationship between phytate intake and blood glucose response (Pirgozliev et al., 2011). Therefore, food rich in phytate has been considered to have great nutritional significance in the prevention and management of diabetes mellitus, one of the most common nutrition-dependent diseases in Western society. The most severe effects attributable to phytate have occurred in populations with unrefined cereals and/or pulses as a major dietary component (Kumar et al., 2012). Especially zinc and iron deficiencies were reported as a consequence of high phytate intakes. To reduce the risk for mineral deficiency in vulnerable groups such as childbearing women, strictly vegetarians, inhabitants of developing countries, especially fast growing children, different strategies have been developed.
The most widely recognized strategies for reducing micronutrient malnutrition are supplementation with pharmaceutical preparations, food fortification, dietary diversification and disease reduction (Posters Group 2, 2001). For various reasons, none has been very successful. An alternative approach would be to increase the total level of micronutrients in the edible parts of staple crops while at the same time increasing the concentration of compounds which promote their uptake and/or decreasing the amount of compounds which inhibit their absorption either by plant breeding or by genetic engineering. Recently, low phytate mutants in maize, barley, rice and soybeans have been isolate and their potential for improving the absorption of iron, zinc and calcium has been shown (Pirgozliev et al., 2011). To improve rice as a source of iron, three proteins were expressed in the central endosperm of the rice seed: a Phaseolus phytoferritin, an endogenous cysteine-rich metallothionein like protein, and an Aspergillus fumigate phytase (Kumar et al., 2012).
If properly targeted, over expression of phytase during seed development can result in reduced phytate levels in the mature seed. Enhanced levels of seed phytase may also contribute to an improvement in mineral absorption by reducing phytate levels in plant-based food during processing and digestion in the human stomach once a meal is consumed (Pirgozliev et al., 2011). In addition, phytate degradation during food processing could be optimized by adding exogenous phytases or by adjusting favorable conditions for the native plant or microbial phytases. Besides enzymatic degradation, no enzymatic hydrolysis of phytate during food processing or physical separation of phytate-rich parts of the plant seed could result in reduced levels of phytate in the final foods. In general, the lower phytate levels must be paid for by a loss of valuable nutrients which are either removed together with the phytate-rich parts of the plant or destroyed by the strong acids or high temperatures needed for no enzymatic phytate ephosphorylation. Enzymatic phytate degradation, however, occurs also under mild conditions and does not affect other food components.
Consumption of phytate, however, does not seem to have only negative aspects on human health. Dietary phytate was reported to prevent kidney stone formation (Kumar et al., 2012), and to protect against atherosclerosis and coronary heart disease as well as against a variety of cancers. The levels of phytate and its dephosphorylation products in urine, plasma and other biological fluids are fluctuating with ingestion or deprivation of phytate in the human diet. Therefore, the reduction in phytate intake in developed compared to developing countries might be a factor responsible for the increase in diseases typical for Western societies such as diabetes mellitus, renal lithiasis, cancer, atherosclerosis and coronary heart diseases.
It was suggested that phytate exerts the beneficial effects in the gastrointestinal tract and other target tissues through its chelating ability, but other mechanisms have also been discussed (Posters Group 2, 2001). Because several myoinositol phosphates, including phytate, are present as intracellular molecules and because the second messenger D-myo-inositol trisphosphate is bringing about a range of cellular functions including cell proliferation via mobilizing intracellular Ca2+, phytate was proposed to exert its anticancer effect by affecting cell signaling mechanisms in mammalian cells (Kumar et al., 2012). An effect of extracellular phytate on the concentration of several intracellular myo-inositol phosphate esters has already been demonstrated in human erythroleukemia cells. Furthermore, it has recently been reported that highly negatively charged myo-inositol polyphosphates can cross the plasma membrane and be internalized by cells (Narasimha et al., 2013). Myoinositol hexakisphosphate was shown to enter HeLa cells followed by an intracellular dephosphorylation to partially phosphorylated myo-inositol phosphates, whereas turnover of myo-inositol pentakisphosphate was quite slow after internalization by SKOV-3 cells. In addition, individual myo-inositol phosphate esters have been proposed to be metabolically active. D-myoinositol trisphosphate, for example, has been studied in respect to prevention of diabetes complications and treatment of chronic inflammations as well as cardiovascular diseases and due to its antiangiogenic and antitumor effects myo-inositol pentakisphosphate was suggested as a promising compound for anticancer therapeutic strategies (Posters Group 2, 2001).
The Saudi Consumer Risk Management Strategy has many directions. Quite a variety of the microbial species are applied in both food and feed production. Some of these have a long history of safe use, while others are not well known or are less understood, hence their use may be considered to represent a risk for all consumers (EFSA News, 2010). Experience from many has proved that there is an urgent need for a tool which will be used for setting priorities within the risk assessment studies of those microorganisms applied in food/feed production referred to EFSA and consequently the subject of a rather formal assessment of safety. To meet this stated need, a certain system was proposed for any pre-market safety assessment study of selected microorganism groups which led to a “Qualified Presumption of Safety (QPS)” (EFSA News, 2010). In essence this claimed that a safety study of a defined taxonomic group (e.g. genus or group of related species) could be determined based on four pillars (establishing identity, body of knowledge, possible pathogenicity and end use). If the taxonomic group being tested did not raise safety concerns, or if safety concerns were existent, but could be defined and excluded (the qualification) the grouping could therefore be granted QPS status. Thereafter, any strain of microorganism featuring the identity of one which could be unambiguously established and assigned to a QPS group would hence be freed from the need for further safety assessment studies other than satisfying any other qualifications specified (EFSA News, 2010). Microorganisms which failed the test and were hence not considered suitable for QPS and they would remain subject to a full safety assessment.
As the number of organisms stated to be suitable for QPS status is sufficiently wide enough to cover a majority of the safety assessments carried out involving microorganisms required of EFSA, the Scientific Committee stated that the introduction of a QPS system for all microorganisms would be the best way to meet the objectives of coming up with a practical tool for setting priorities and also for avoiding the extensive studies of organisms known not to cause any concern (EFSA News, 2010). Although QPS status of most metabolic products of microorganisms cannot be inferred from this QPS status of the production strain, the Committee claimed that the system still had enough value for the analysis of strains used in the production of such products. Further work, however, would be result in instances where the system to encompass those microorganisms is used for biological control purposes. Finally, in reaching its conclusion on the value of QPS as an effective assessment tool, the Scientific Committee acknowledged that there would have to be progressing provisions for reviewing and also modifying the list of organisms given the QPS status (EFSA News, 2010). They recommended that the EFSA through its Science Directorate should be the people responsible for this and hence should review the suitability for any QPS status of all the existing list and ensure additions at least annually (EFSA News, 2010). Reviews may occur more frequently as was required but there should be a formal acknowledgement that even when no changes are proposed, a statement must still be made annually that all QPS status are being maintained for the published list.
In April 2003, responsibility for the safety assessments of food/feed taken care of by the Scientific Committees of the Commission was formally passed on to the European Food Safety Authority (EFSA News, 2010). Shortly after EFSA asked its own Scientific Committee to analyze whether the approach to safety analysis of microorganisms proposed in the QPS document could be safely used to harmonize different approaches to the safety assessment of microorganisms across the various EFSA scientific panels. In doing so, the Committee was expected to take into account the response of all the stakeholders to the QPS approach. Their views had already been sought by the three Commission Scientific Committees in 2002/3 and, subsequently, by EFSA at a Scientific Colloquium carried out at the end of 2004 (EFSA News, 2010).
The Scientific Committee came to a conclusion that QPS as a concept could give a generic assessment system to be used within EFSA that could also be applied to all requests received for the safety assessments of microorganisms which has been deliberately introduced into the food chain (EFSA News, 2010). The benefits of the introduction of QPS system would be a more transparent and therefore more consistent approach across the EFSA panels and hence provide a potential to make better use of all the resources by always focusing on those organisms which posed the greatest risks or uncertainties.
However, the Committee emphasized that the body of knowledge about the organisms for which QPS is sought must be adequate enough to provide relevant assurance that any potential to produce adverse side effects in humans, livestock or the wider environment is well understood and clearly predictable. Judgment as to whether the existing data is sufficient enough as needed, in the view of the Committee, to be considered by an expert group established for this purpose and should be based on a weight-of-evidence approach.
Based on these conclusions, the Scientific Committee recommended that EFSA should come up with another strategy to feature the introduction of an assessment system based on a similar concept or the QPS concept. This should be limited to the new microorganisms introduced into the food chain or used as producer strains for food/feed additives until the value of such a system could be analyzed in practice.
EFSA accepted the stated recommendation of its Scientific Committee and thereafter proposed that the Committee should keep on with its assessment of the QPS system with a clear view to implementation 3. Specifically, the Scientific Committee was asked first to establish which microorganisms would most commonly be referred to EFSA, including those used as a source of microbial products (EFSA News, 2010). Then, based on this survey, to enhance the selection of relevant groups of microorganisms, examine carefully the available data on safety and propose whether QPS status would be an appropriate approach. If this proved possible in a significant number of cases studied, then the Scientific Committee should consider how the implementation of QPS across the various panels could be achieved sensibly.
In conclusion, the Ronozyme P may be considered to feature both benefits and risks. However the risks far outweigh the benefits as the chemical results to food hazards for those who consume products which have already been affected by the chemical. This in return may result to food poisoning as well as other medical conditions such as asthma and meningitis. Therefore to ensure the health of individuals, it is important for the rules to govern the type of food offered for consumption to the people.
Kumar, V. V., Sinha, A. K., Makkar, H. S., De Boeck, G. G., & Becker, K. K. (2012). Phytate and phytase in fish nutrition. Journal Of Animal Physiology & Animal Nutrition, 96(3), 335-364. doi:10.1111/j.1439-0396.2011.01169.x
EFSA News. (2010). European Food & Feed Law Review, 5(6), 369-372.
Posters Group 2 – Environmental and Occupational Health. (2001). Allergy, 56235-250. doi:10.1111/j.1398-9995.2001.tb05144.x
Narasimha, J. J., Nagalakshmi, D. D., Ramana Reddy, Y. Y., & Viroji Rao, S. T. (2013). Synergistic effect of non-starch polysaccharide enzymes, synbiotics and phytase on performance, nutrient utilization and gut health in broilers fed with sub-optimal energy diets. Veterinary World, 6(10), 754-760. doi:10.14202/vetworld.2013.754-760.
Pirgozliev, V. V., Bedford, M. R., Acamovic, T. T., & Allimehr, M. M. (2011). The effects of supplementary bacterial phytase on dietary true metabolisable energy, nutrient digestibility and endogenous losses in precision fed turkeys. British Poultry Science, 52(2), 214-220. doi:10.1080/00071668.2011.560594