Utilization of Aspergillus niger Phytase Preparation for Hydrolysis of Phytate in Foods

The majority of phosphorous in plant seed, especially cereal grains and legumes, is myoinositol hexaphosphate (phytate or IP6) (Rosa et al., 1999). Structure of IP6 is shown in Figure 1. Figure 2 shows distribution of IP6 in some plant foods. Unrefined cereals and soybean products contain high levels of IP6. Rice bran contains IP6 more than 6,000 mg/100 g of dry matter. IP6 has a strong capability to chelate multivalent metal ions, particularly zinc, calcium, and iron ions, which results in the formation of highly insoluble salts (Nolan et al., 1987; Hotz et al., 2001). The IP6 can be hydrolyzed by enzyme (phytase) and converted to lower myo-inositol phosphates; from inositol pentaphosphate (IP5) to inositol monophosphate (IP1) and myo-inositol. It has been demonstrated that the metal complexes with IP1−IP4 are more soluble than those with IP5 and IP6 (Sandberg et al., 1989). Animal experiments have demonstrated that the ingestion of the meal that contains more than 1% IP6 decreases intestinal absorption of metal ions and induces the metal ion deficiency (Hirabayashi et al., 1998; Grases et al., 2001). In some communities, unrefined cereals are still main ingredients for the diet. In addition, the unrefined grains of rice, wheat, rye, etc., are richer in various minerals, dietary fiber, vitamins, and other bioactive components than refined ones. Therefore, these unrefined cereals are used as food ingredients for bread,


Introduction
The majority of phosphorous in plant seed, especially cereal grains and legumes, is myoinositol hexaphosphate (phytate or IP 6 ) (Rosa et al., 1999). Structure of IP 6 is shown in Figure  1. Figure 2 shows distribution of IP 6 in some plant foods. Unrefined cereals and soybean products contain high levels of IP 6 . Rice bran contains IP 6 more than 6,000 mg/100 g of dry matter. IP 6 has a strong capability to chelate multivalent metal ions, particularly zinc, calcium, and iron ions, which results in the formation of highly insoluble salts (Nolan et al., 1987;Hotz et al., 2001). The IP 6 can be hydrolyzed by enzyme (phytase) and converted to lower myo-inositol phosphates; from inositol pentaphosphate (IP 5 ) to inositol monophosphate (IP 1 ) and myo-inositol. It has been demonstrated that the metal complexes with IP 1 −IP 4 are more soluble than those with IP 5 and IP 6 (Sandberg et al., 1989). Animal experiments have demonstrated that the ingestion of the meal that contains more than 1% IP 6 decreases intestinal absorption of metal ions and induces the metal ion deficiency (Hirabayashi et al., 1998;Grases et al., 2001). In some communities, unrefined cereals are still main ingredients for the diet. In addition, the unrefined grains of rice, wheat, rye, etc., are richer in various minerals, dietary fiber, vitamins, and other bioactive components than refined ones. Therefore, these unrefined cereals are used as food ingredients for bread, Fig. 1. Structure of phytic acid (IP 6 ) www.intechopen.com breakfast cereals and so on due to their high health-promoting activities (Fukui et al., 1997;Haros et al., 2001;Lopez et al., 2001;Porres et al., 2001). In addition to the unrefined cereals, legumes, especially soybean have long history as food ingredient. Recently, soy flour, soy protein isolate, its protease digest, etc. have been formulated and used as protein source for infant formulae, sports drinks, enteral nutrients and also diets for animal, poultry, and fish. However, as mentioned above, these products are also rich in IP 6 . Then, nutritional disturbances in the absorption of iron and zinc may occur by ingestion of IP 6 -rich foods (Shaw et al., 1995;Sandberg et al., 1996;Minihane et al., 2002). refined oats bread* raw brown 30% soybean SPI soybean natto salted natto wheat flour rice flour brown-rice flour* curd bread * Data from Rosa et al. 1999. Natto; steamed soybean fermented with Baccillus subtillis. Salted natto; steamed soybean fermented with Aspergillus oryzae and further fermented with wild yeast and lactic acid bacteria in the presence of salt (more than 5%).

Fig. 2. Phytate contents of some cereals and soybean products
It has been demonstrated that the IP 6 in foods can be degraded by phytase, which would improve mineral absorption in humans (Sandberg et al., 1996;2002). On the basis of these facts, some phytase preparations have been formulated and used to reduce IP 6 and IP 5 levels of some plant-based foods (Haros et al., 2001;Saito et al., 2001;Porres et al., 2001: Matsuo et al., 2005 and also animal and fish diets (Pallauf & Rimbach, 1997;Sajjadi & Carter, 2003).
In this chapter, the recent application of fungal phytase preparation as food additive and its problem are reviewed. In addition, recent studies for control of the contamination of other enzymes in the phytase preparation are focused.
Indeed, some bacteria, yeasts, and fungi have been demonstrated to produce phytases. As mentioned above, the enzymatic hydrolysis of the IP 6 in unrefined cereals and legumes improves mineral absorption in humans (Sandberg et al., 1996;2002). These facts can lead an idea that microbial phytase can be used as food additive to reduce IP 6 level in the plantbased foods. So far to now, phytase preparations have been prepared from Asperigillus niger, recombinant Aspergillus oryzae, Pichia pastoris etc. in an industrial scale and used as food additive (Greiner & Konietzny, 2006). However, these microbial also produce protease, amylase and etc. As demonstrated in the following sections, a food additive-grade Asperigillus niger phytase preparation has significant protease and amylase activities (Matsuo et al., 2010). In some cases, occurrences of protease and amylase in the food additive-grade phytase preparation exerts adverse effects on texture and appearance of the final products.
In the following sections, application of a food additive-grade Aspergillus niger phytase preparation on some plant-based foods are introduced. Effect of the phytase treatment not only on IP 6 content but also on the other properties will be discussed.

Application for improvement of nutritional value of animal, poultry, and fish diets
Plant seeds and grains have been used for animal, poultry, and fish diets. As shown in Figure 2, the unfermented soy and cereal products contain high levels of IP 6 . The occurrence of IP 6 in diet may interfere mineral absorption (Sandberg, 2002). In some cases, it may induce serious nutritional problem. Indeed, feeding IP 6 -containing soy protein isolate retarded growth of fish larvae. To solve these problems, Aspergillus niger and Pichia pastoris phytase preparation was used to decrease IP 6 in the plant-based diet. This treatment significantly improve feeding efficacy. Now, formulated Aspergillus niger and Pichia pastoris phytases for improvement of feeding efficacy for fish, chicken, animals are commercially available (Pallauf & Rimbach, 1997;Sajjadi & Carter, 2003).

Application of phytase to isolate conglycinin and glycinin from soy protein isolate
Phytase treatment of soy protein isolate can be carried out in aqueous solution. During the Asperigillus niger phytase treatment, protein precipitation occurred (Saito et al., 2001). As well known, major soluble proteins in soybean are glycinin and β-conglycinin, which are classified as globulin. Both proteins are precipitated in acidic condition at approximately pH 4.5. However, predicted isoelectric point of glycinin based on the protein sequence is neutral pH. The glycinin forms complex with IP 6 and then it has apparent acidic isoelectric point, which makes glycinin precipitate in acidic condition. The phytase treatment degrades the glycinin-bound IP 6 and shifts isoelectric point to neutral pH. Then selective precipitation of glycinin occurs at neutral pH after the phytase treatment of soy protein isolate. This technique is successfully applied to sepration of glycinin and β-conglycinin for food ingredient. It has been demonstrated that β-conglycinin can reduce blood neutral lipid level of animal and human by ingestion (Fukui et al., 2004;Moriyama et al., 2004;Kohno et al., 2006). Now, food-grade β-conglycinin fraction is prepared on the basis of phytase treatment www.intechopen.com and following selective precipitation technique. The β-conglycinin fraction is approved to present health claim on lipid metabolism in "Food for the Specific Health Use" in Japan.

Application for unrefined cereal flour-containing bread making.
Whole grains of wheat, rice, rye, etc., are richer than refined grains in various minerals, dietary fiber, and vitamins. Besides minerals, vitamins, and dietary fiber, the unrefined grains are rich in bioactive components that have health-promoting activities; for example, γ-aminobutyric acid (GABA), γ-oryzanol, polyphenols, and feruric acid. These components show antihypertensive (GABA), antioxidant, and anti-hypercholesterolemic (γ-oryzanol and feruric acid) activities (Sugano et al., 1997;Xu et al., 2001;Hayakawa et al., 2002). However, the consumption of the unrefined whole grains as the steamed and fried forms, are not very prevalent due to the poor swelling and textural properties of the unrefined whole grains. Then, the unrefined whole grains are usually processed to flour and used as bread ingredient, which has been gaining popularity worldwide (Fukui et al., 1997;Haros et al., 2001;Lopez et al., 2001;Porres et al., 2001). As shown in Figure 3, IP 6 in the dough mix containing 30% brown rice flour decreases during processing by the action of yeast phytase. However, the relatively higher levels of IP 6 remains in the bread containing brown rice flour. As shown in Figure 3, addition of food additive-grade Aspergillus niger phytase preparation significantly decreased IP 6 level of brown rice-added bread. Aspergillus niger phytase has been also used to decrease IP 6 level in the whole wheat fours (Haros et al., 2001). At early attempt, the flour was pretreated with phytase in water suspension system. It has been, however, demonstrated that direct addition of the phytase preparation into dough mix can reduce IP 6 content, which is the easier approach to decrease IP 6 level in the unrefined grain flour-containing bread (Haros et al., 2001;Matsuo et al., 2005;. However, addition of high dose of the phytase preparation (3000 U) induced collapse of whole bread crust and deteriorates texture and taste of the final product as shown in Figure 4 E (Matsuo et al., 2005). Two grams of the crude phytase preparation was dissolved in 50 mL of 0.02 M sodium acetate buffer, pH 6.0 and loaded on a column (15 cm × 10 mm i.d.) packed with TSK gel Super Q-Toyopearl 650S (Tosoh Co., Tokyo, Japan) that had been pre-equilibrated with the same buffer. The absorbed proteins were eluted using 200 mL of a linear gradient of 0-0.5 M NaCl in the same buffer at 1.5 mL/min. Fractions were collected every 1 min. From Matsuo et al., 2010. Figure 5 shows that the food additive-grade phytase preparation contains significant activities of protease and amylase, which can be separated from the phytase activity by anion-exchange column chromatography (Matsuo et al., 2010). Addition of the purified phytase fraction free from protease and amylase activities into the dough mix decreased IP 6 level without affecting bread volume ( Figure 6). These facts imply that amylase and/or protease are responsible for the collapse of the bread crust by addition of high dose of the commercial phytase preparation. However, it has been difficult to isolate phytase, protease, and amylase activities in enough amounts for the additive purpose by conventional chromatography technique due to high cost of preparative LC system. Then it is difficult to control protease and amylase activities in the phytase preparation, which has limited the use of the fungal phytase preparation as food additive. To solve this problem, a large-scale isolation method for food additive-grade enzyme is necessary. This method should be inexpensive, biocompatible, easy to scale-up. The following section introduces recent advance in purification of phytase by new technique. Wheat bread 30% brown-rice bread

Crude phytase preparation
Purified phytase preparation

Control of protease and amylase activities in phytase preparation by autofocusing
As mentioned in the previous section, it has been demanded to develop a method for largescale fractionation of enzymes in crude extract and fermentation broth in order to control contamination of other enzymes in phytase preparation. For fractionation of peptides in the crude digests of food proteins, a preparative ampholyte-free isoelectric focusing has been developed (Hashimoto et al., 2005), which has a potential for purification of enzymes. This technique depends on amphoteric nature of the sample compounds. The crude enzymatic digest of food protein, crude extract, fermentation broth, etc. contain numerous compounds with different isoelectric points. Then these compounds can act as ampholine for isoelectric focusing. This phenomenon is referred to autofocusing (Yata et al., 1996;Akahosi et al., 2000;Hashimoto et al., 2005). This technique does not require harmful reagents and solvents just requires water and agarose gel in sample compartments and diluted phosphoric acid and sodium hydroxide in the electrode compartments. All of them can be used for food processing. Assembly of the apparatus is illustrated in Figure 7. A plastic plate with window is covered with nylon screen and the screen is wetted with hot agarose solution. After standing for few minutes, thin agarose gel layer is formed on the screen. The plates with agarose gel layer are inserted into the slots of the tank as shown in Figure 7. Then the tank can be separated into 12 compartments. Both ends of the compartments are used as electrode compartments. Others are used as sample compartments (10 compartments Fig. 7. Schematic drawing for assembly of Autofocusing apparatus. The tank is divided into 12 compartments separated with thin agarose gel layer. Volume of compartment can be changed from 100 mL to 5 L. From Hashimoto et al., 2005 Water-solution of sample is applied into the sample compartments. 0.1 N phosphoric acid and sodium hydroxide are loaded into cathode and anode, respectively. By loading direct electric current into the electrode compartments at constant voltage mode at 500 -1000 V, the compounds in sample start to migrate to their own isoelectric points. This technique has been successfully used for peptide fractionation (Hashimoto et al., 2005;Higaki-Sato et al., 2006;Park et al., 2008;Murota et al., 2010;Elbarbary et al., 2010).
The food additive-grade phtase preparation also contain many compounds. Then autofocusing was used to purify phytase in the commercial preparation. As shown in Figure  8, pH gradient approximately from 2 to 11 was formed by autofocusing of food additivegrade Aspergillus niger phytase preparation. The protease activity was migrated to acidic fractions (Fr. 1-6). The amylase and phytase activities were widely distributed. Fr. 7 has the Fraction number Fig. 8. Fractionation of phytase, amylase, and protease in a food additive-grade Aspergillus niger phytase preparation. pH gradient (upper) was formed by autofocusing for 24 hours. Fraction No. 7 and 9 were free from protease activity and used as high amylase and low amylase fractions, respectively highest phytase and amylase activities. In the Fr. 9, the amylase activity dropped to 15% of the maximum activity, whereas relatively high level of the phytase activity (more than half of the activity in Fr. 7) was recovered in the Fr. 9. Then, Fr. 7 and 9 were collected and used as high amylase and low amylase fractions, respectively. These fractions were free from protease activity. The crude phyase preparation, which contains significant protease activity and two protease free autofocusing fractions were added to the 30% brown rice flour-added bread dough mix. Phytate content decreased by addition of all fractions. As shown in Figure 9, addition of the low amylase fraction did not significantly affect the loaf volume and did not induce collapse of crust up to 3000 U of phytase activity. Addition of the high amylase fraction showed positive effect on loaf volume up to 514 U of amylase activity. However, addition of amylase, more than 1029 U, decrease of loaf volume and partial collapse of crust (arrow) were observed. Addition of the crude preparation at 791 U of protease activity induced partial collapse of crust even amylase activity was less than 500 U. Addition of protease more than 1582 U resulted in extensive collapse of whole crust and deteriorated texture properties (mash-like texture).  On the basis of these results, it can be concluded that IP 6 content in the unrefined grain flour-containing bread can be reduced by direct addition of the fungal phytase. However, presence of high protease activity in the phytase preparation can deteriorate swelling and textural properties and collapse the crust. On the other hand, amylase can improve the swelling property at relatively low activity (<500 U), while it deteriorates the swelling property at high activity (> 1000 U). These phenomena can be explained as follows. Protease in the preparation degrades gluten and deteriorates gluten network. Consequently, the dough can not retain the CO 2 gas generated by fermentation, which decreases loaf volume and finally collapses whole crust. The amylase in the preparation may induce oligosaccharides from starch and consequently enhance fermentation by yeast at the low amylase activity. On the other hand, the high amylase activity can deteriorate starch gel network after cooking process. Indeed, fungal amylase has been used in the baking industry to accelerate fermentation. However, addition of excessive amylase and protease has been demonstrated to make the dough slack (Fred et al., 1975). These facts clearly indicate that the control of protease and amylase activities is crucial for the application of the fungal phytase preparation to bread making to reduce IP 6 and obtain good quality of the product.

Conclusion and future prospects
It has been demonstrated that fungal phytase can be used to reduce IP 6 level of the soybean based products and also unrefined grain flour-containing products, especially bread. However, fungal phytase may contain protease and amylase, which may affect quality of the final products. In some cases, the product may be unacceptable due to excessive degradation of protein, which induces unfavorable effects on texture and appearance. Then it is crucial to control the contaminated enzyme activity in the phytase preparation. In the present chapter, ion exchange column chromatography and autofocusing can at least partially control contamination of protease and amylase activities. In regardless of the high resolution, column chromatography requires relatively expensive apparatus and high running cost. In addition, it may require salts, buffer, solvents etc, which should be removed from the final product. It also increases preparation cost. On the other hand, autofocusing does not require organic solvents and salts for fractionation and has inherited potential for scale-up. Recently, continuous type autofocusing apparatus has been developed . Then this technique would be useful to control protease and amylase activities in the phytase preparation. By using this technique, a food additive phytase optimized for bread making and other applications could be produced.
IP 6 also suppresses divalent ions-induced Fenton's reaction; producing hydroxyl radical from hydrogen peroxide. Then IP 6 has been used to suppress oxidation of food (Graf and Eaton, 1990). However, little is known for the suppressive effect of IP 1 -IP 5 against Fenton's reaction. If degradation products of IP 6 by phytase also suppress Fenton's reaction without reducing mineral bio-availability, phytase can be used for production of anti-oxidant food ingredient with excellent nutritional value. In addition, functional food based on suppression of Fenton's reaction without disturbing nutritional could be also produced by the limited digestion of IP 6 by phytase. For these purposes, further studies on control of hydrolysis of IP 2-6 by phytase are necessary. A food additive is defined as a substance not normally consumed as a food in itself and not normally used as a characteristic ingredient of food whether or not it has nutritive value. Food additives are natural or manufactured substances, which are added to food to restore colors lost during processing. They provide sweetness, prevent deterioration during storage and guard against food poisoning (preservatives). This book provides a review of traditional and non-traditional food preservation approaches and ingredients used as food additives. It also provides detailed knowledge for the evaluation of the agro-industrial wastes based on their great potential for the production of industrially relevant food additives. Furthermore the assessment of potential reproductive and developmental toxicity perspectives of some newly synthesized food additives on market has been covered. Finally, the identification of the areas relevant for future research has been pointed out indicating that there is more and more information needed to explore the possibility of the implementation of some other materials to be used as food additives.