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 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 this 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 Additives and Products Panel used in all Animal Feed (FEEDAP) was required to provide an accurate scientific view on the safety along with the level of 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 birds 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 human beings (Kumar et al., 2012). Phytate is usually obtained from potentially usable plant derived ingredients of fish feed including soybean, Jatropha kernel meal found in non-toxic genotype, and detoxified Jatropha kernel meal obtained from the toxic geno-type, rice, 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. 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.
Phytate and phytase concentration in the feedstuffs varie 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 action among most feedstuffs has been reported which are based solely 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 into 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 against excessive manure phosphorus runoff pollution since the animals’ fecal phosphate excretion is reduced by up to 50 % (Kumar et al., 2012). Great potential is also registered for the use of these 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 levels of plant-based foods as well as on the technical improvement of the activity involved for food processing. A phytate rich diet leads to a substantial reduction of the absorption of dietary minerals, and during food processing, the dephosphorylation of phytate results in the formation of partially phosphorylated myo-inositol phosphate esters, which basically have a lower capability to impair the intestinal uptake of dietary minerals. Individual myo-inositol phosphate esters have been shown to have a number of important functions especially physiological in human beings. Therefore, phytases may be applied in food processing to produce functional foods, if such biochemically active myo-inositol phosphate esters were to be made from phytase conjugates and absorbed into the humans’ alimentary tract. Technical improvements by adding phytases during food processing have been reported for bread making, production of isolates from plant protein, corn wet milling and also 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 isolates from plant protein. The major concern features the availability of phytate in the human food is its negative effect on the mineral uptake activity. 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 cause for this poor mineral bioavailability, since these complexes are currently not available for absorption from the human gastrointestinal tract (Narasimha et al., 2013). The human small intestine features a very limited capability to hydrolyze phytate as a result of the lack of endogenous phytate-degrading enzymes and the limited population of microbes in the digestive tracts upper parts. Solubility and stability of the stated myo-inositol phosphate-mineral complexes usually end up decreasing as the phosphate residues on the myo-inositol ring lowers (Posters Group 2, 2001).
Therefore, removal of the phosphate residues from phytate will lead to a reduced impairment of the intestinal uptake of dietary minerals which are essential in the body. In its isolated form, only myo-inositol pentakisphosphate was found to have 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). When there are higher phosphorylated myo-inositol phosphates, however, myo-inositol tetrakis- and trisphosphates were seen to take part 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 structure of proteins that can decrease the activity of enzymes, the solubility of proteins and lastly the proteolytic digestibility.
A factor that is still under scrutiny, however is the significance of protein-phytate complexes in nutrition. (Narasimha et al., 2013). Strong evidence exists that phytate-protein interactions have consequences on protein digestibility in vitro and the level of this negative effect depends on the source of the protein. A negative effect of phytate on the proteins nutritional value, however, was not directly confirmed in the studies carried out on monogastric animals. Though some researchers have found that phytate has no net effect on protein digestibility, other scholarsd have been able to find a correlation between amino acid presence and phytate levels. This difference may be partly because of the use of various protein sources. Another factor of nutritional significance may also be the inhibition of digestive enzymes for example lipases, amylases, or proteinases including trypsin, pepsin, and chymotrypsin by phytate.
The inhibitory effect rises with the number of phosphate residues per myo-inositol molecule and also the concentration of myo-inositol phosphate. This inhibition may be as a result of the nonspecific nature of the interactions of phytate-protein, the chelation of calcium ions which are very important for trypsin and α-amylase activity, or interaction with the substrates of these enzymes. The proteases inhibition may be partly responsible for the reduction in protein digestibility. Phytate has also been considered as an amylase inhibitor as shown by a destructive relationship between phytate intake and blood glucose response (Pirgozliev et al., 2011). Therefore, phytate rich food stuff has been noted to contain great nutritional significance in the prevention as well as the management of diabetes mellitus, one of the most common nutritional diseases in Western society. The most severe consequences whose attribute is to phytate have occurred in populations whose major dietary component is unrefined cereals. (Kumar et al., 2012). Zinc and iron deficiencies were the highest reported consequences of consuming high phytate. To reduce the risk posed includingmineral deficiency in commonly vulnerable groups including childbearing women, strict vegetarians, members of developing countries, especially the fast growing children, different strategies have been implemented.
The widely recognized strategies for reducing micronutrient malnutrition are supplementation with pharmaceutical products, food fortification, diversification of diets and reduction of diseases (Posters Group 2, 2001). For various reasons, none has been successful. A possible alternative approach would be to increase micronutrients levels in the edible parts of crops while at the same time increasing the rates of compounds which promote their uptake. It may also be done by decreasing the compound levels 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 hidden ability for improving iron absorption and also zinc and calcium have been shown (Pirgozliev et al., 2011). To improve rice as iron source, three proteins were spread in the central endosperm of the rice: a Phaseolus phytoferritin, an endogenous cysteine-rich metallothionein like protein, and lastly an Aspergillus fumigate phytase (Kumar et al., 2012).
If properly targeted, over expression of phytase during the development of the seed can cause a reduction in phytate levels in the mature seed. Enhanced seed phytase levels may also contribute to an improvement in absorption of minerals by reducing phytate levels in plant-based foodstuff during processing and thereafter digestion in the human stomach once a contaminated 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 the processing of food or physical separation of phytate-rich parts of the seed could result in a reduction of levels of phytate in the final foods. Generally, the lower phytate levels must be catered for by a loss of valuable nutrients which are either lost along with the phytate-rich parts of the plant, destroyed by the acids or high temperatures needed for enzymatic phytate ephosphorylation. Enzymatic phytate degradation, on the other hand, occurs also under mild conditions and hence does not affect other components of food .
Consumption of phytate not only has negative aspects on human health. Dietary phytate was reported to have preventative effects on kidney stone formation (Kumar et al., 2012), and to protect against atherosclerosis and coronary heart disease and also the variety of cancers. The phytate levels and its dephosphorylation products present in urine, plasma and other biological fluids usually fluctuate with ingestion or a deprivation of phytate present in the human diet. Therefore, the phytate intake reduction in developed countries when compared to developing countries might be a factor fueling the increase in rates of diseases typical for Western societies such as diabetes mellitus, renal lithiasis, cancer, atherosclerosis and coronary heart diseases.
It was suggested that phytate brings on the positive effects in the gastrointestinal tract and other vulnerable tissues through its chelating ability (Posters Group 2, 2001). Because several myoinositol phosphates, including phytate, are available as intracellular molecules, and because the second messenger D-myo-inositol trisphosphate is giving room for a range of cellular functions including cell proliferation via mobilizing intracellular Ca2+, phytate exerts its anticancer effect through annexing of signaling mechanisms in mammalian cells (Kumar et al., 2012). Previously, it had been demonstrated how the extracellular phytate could affect the concentration of various intracellular myo-inositol phosphate esters. Furthermore, highly negatively charged myo-inositol polyphosphateshave been recently reported to have the capability of crossing the plasma membrane and eventually be internalized by cells (Narasimha et al., 2013). Myoinositol hexakisphosphate was noted to enter HeLa cells, after which an intracellular dephosphorylation occurred, followed by 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 claimed to be metabolically active. D-myoinositol trisphosphate, for example, has been studied while looking at the possible prevention of diabetes complications as well as treatment of chronic inflammations and cardiovascular diseases, and as an effect of its antiangiogenic and antitumor effects myo-inositol pentakisphosphate was seen as a potential 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. A number of these have been used for long and are considered safe, while the rest 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 researchers has proved that a tool which will be used for setting priorities within the risk assessment studies of those microorganisms applied in food production known as EFSA are urgently needed, and consequently, the main 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, for example genus or group of related species, could be determined based on four sections; establishing its identity, the body of knowledge, all possible pathogenicity and end use. If, however, 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 established unambiguously and thereafter 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 featured in the production of such feed products. Further work, however, would result in instances where the system to encompass those microorganisms is used for biological control purposes only. Finally, once the conclusion was reached 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 at the times when no changes are proposed or implemented, a statement must still be made annually that all QPS statuses are being maintained for the published list.
In April 2003, safety assessments responsibility of food taken care of by the Scientific Committees of the Commission was formally handed over 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 studies techniques of microorganisms across the various EFSA scientific panels. By taking this path, the Committee was expected to take into account the response of all the QPS stakeholders who introduced the approach. In the past, their views had already been sought by the three Commission Scientific Committees in 2002/3 and, thereafter 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 the possible products 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 various organisms for which QPS is looked at 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 expertees established for this main purpose and hence should be based on an approach focusing on weight-of-evidence.
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 exact QPS concept. This should be focused on the new microorganisms recently introduced into the food chain or used as strains for production of 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 the possible implementation number three. Specifically, the Scientific Committee was requested to first of all establish which microorganisms would most commonly be referred to EFSA, including those needed as a source of microbial products (EFSA News, 2010). Then, based on this survey, to enhance the selection of relevant groups of microorganisms, carefully examine the available data on safety and hence propose whether QPS status is indeed 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.
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EFSA News. (2010). European Food & Feed Law Review, 5(6), 369-372.
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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