The positive impacts of microbial phytase on its nutritional applications

Accepted Manuscript The positive impacts of microbial phytase on its nutritional applications Hai-Yan Song, Aly Farag El Sheikha, Dian-Ming Hu

PII:

S0924-2244(18)30575-2

DOI:

https://doi.org/10.1016/j.tifs.2018.12.001

Reference:

TIFS 2375

To appear in:

Trends in Food Science & Technology

Received Date: 15 August 2018 Revised Date:

25 September 2018

Accepted Date: 1 December 2018

Please cite this article as: Song, H.-Y., El Sheikha, A.F., Hu, D.-M., The positive impacts of microbial phytase on its nutritional applications, Trends in Food Science & Technology, https://doi.org/10.1016/ j.tifs.2018.12.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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ABSTRACT

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Phytic acid is the main reservoir of phosphorous in plants and accounts for more than 80% of the

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total phosphorous in cereals. It is well known to possess anti-nutritional behavior because it has

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bound positively charged proteins and multivalent cations or minerals in foods. Therefore, phytic

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acid and its salts have been considered as a threat in the human diet. However, we should not

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ignore their potential health benefits, e.g., anticancer and antioxidant agents. The anti-nutritional

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effects of phytic and its derivatives are known to be reduced through an enzyme namely phytase,

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which originated to many sources, the microbial origin is one of them. This review narrates the

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positive nutritional impacts of using the microbial phytases via its food applications.

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Furthermore, highlighting the role of modern molecular tools and genetic engineering to express

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phytase genes of microbial origin for phytase production in lieu of directly supplementing

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microbial phytase. This approach deserves more attention, as it is able to open up new research

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prospects in the future.

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Keywords: Phytic acid, phytase-producing microorganisms, nutritional impacts, health effects,

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food applications, transgenic plants

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COMMENTARY

2 The positive impacts of microbial phytase on its nutritional applications

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Hai-Yan Song1, Aly Farag El Sheikha1,2,3* and Dian-Ming Hu1*

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Tel./ Fax: +86 (791) 838-13459 Tel. : +86 (151) 801-09061

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E-mail: [email protected]

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E-mail: [email protected]

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Utilization of Fungal Resources, 1101 Zhimin Road, Nanchang 330045, China

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L8S 4K1, Canada

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Technology, 32511 Shibin El Kom, Minufiya Government, Egypt

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McMaster University, Department of Biology, 1280 Main St. West, Hamilton, Ontario,

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Minufiya University, Faculty of Agriculture, Department of Food Science and

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Jiangxi Agricultural University, Jiangxi Key Laboratory for Conservation and

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1. Phytic acid: Antinutrient and beneficial roles

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1.1. What is phytic acid?

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The discovery of phytic acid (myo-inositol-hexakis-dihydrogenphosphate, IP6) (Figure

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1) and its salts, phytates dates from 1855 to 1856 when Hartig first reported small round

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molecules in different plant seeds (Hartig, 1855; Hartig, 1856). It is a chelating agent for

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cations and a form of cations as well as for phosphorous storage in many plant seeds

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(Cosgrove, 1966).

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1.2. Food sources of phytic acid and its salts

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Phytic acid and its salts represent the majority of the phosphorus as the energy source and

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antioxidant for the germinating seed in cereals, legumes, nuts and oil seeds which

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account 60-90% of the total phosphorous (Loewus, 2002). Phytate is therefore widely

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distributed as a common constituent in plant-derived foods (Table 1; Greiner &

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Konietzny, 2006; Schlemmer, Frolich, Prieto, & Grases, 2009; Afinah, Yazid, Anis

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Shobirin, & Shuhaimi, 2010). It should be noted the daily intake of phytate can be as high

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as 4500 mg (Reddy, 2002). On average, daily intake of phytate was estimated to be

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2000–2600 mg for vegetarian diets as well as diets of inhabitants of rural areas in

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developing countries and 150–1400 mg for mixed diets (Reddy, 2002). As for the

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developed countries represented in the United Kingdom, the study conducted by

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Amirabdollahian and Ash (2010) estimated the median daily intakes of phytate for

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children, adolescents, adults and the elderly population were 496, 615, 809 and 629 mg/

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day, respectively.

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1.3. Antinutritional effects of phytic acid and its derivatives

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Phytic acid has a potential for binding positively charged proteins, amino acids, and/or

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multivalent cations or minerals in foods. The resulting complexes are insoluble, difficult

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for humans to hydrolyze during digestion, and thus, typically are nutritionally less

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available for absorption (Afinah, Yazid, Anis Shobirin, & Shuhaimi, 2010). Phytate as a

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polyanionic molecule of IP6 makes a strong chelating agent with nutritionally important

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minerals such as calcium, potassium, sodium, magnesium, copper, iron, zinc, cobalt and

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manganese (Ruckebusch, Knap, Umar Faruk, & Upton Augustsson, 2013). Equation 1

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shows the interaction of phytate with the minerals. The formation of insoluble mineral-

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phytate complexes is regarded as the major reason for the poor mineral bioavailability

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because these complexes are essentially non-absorbable from the human gastrointestinal

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tract (Greiner & Konietzny, 2006). This could be one of the attributing reasons to

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malnutrition in Asian countries where up to 75% of the total calorie intake is from cereals

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(Sands, Morris, Dratz, & Pilgeram, 2009), whereas, most of the cereals are low in

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proteins and minerals but high in starch and phytate (Savita, Yallappa, Nivetha, &

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Suvarna, 2017).

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1.3.1. Is it possible to overcome the antinutritional effects of phytic acid and its

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derivatives?

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Through two strategies, it is possible to overcome the antinutrient effects of phytic acid

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and its derivatives in foods: 1) indirect-enzymatic processes such as soaking, cooking,

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germinating, fermentation and addition of vitamin C; 2) direct enzymatic processing by

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adding the phytase enzyme for phytate hydrolysis (it will be discussed later).

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70 Soaking. It is often used as a pre-treatment to facilitate processing of legume grains and

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cereal seeds. Soaking period is varied for a short period (15 to 20 minutes) to a long

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period (12 to 16 hours or overnight in household technique), which based on the type of

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grain and targeted process after. Because phytate is water soluble, a significant phytate

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reduction can be realized by discarding the soak water (Greiner & Konietzny, 2006;

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Karkle & Beleia, 2010).

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Cooking. As known, phytic acid and its salts are heat-stable compounds, hence the heat

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can destroy small amounts of these compounds. Therefore, considerable phytate

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destruction during cooking only takes place in two situations (Greiner & Konietzny,

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1998; Karkle & Beleia, 2010):

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- discarding the cooking water, or

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- intrinsic plant phytases hydrolysis action during the early phase of cooking.

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Germinating. It is a process widely used in legumes and cereals, which aims to increase

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their palatability and nutritional value. Also. the germination process responsible for

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increasing the activity of intrinsic plant phytases hydrolysis action in which the positive

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conversion of certain antinutrients, e.g., phytates to improve mineral bioavailability

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(Greiner, 2002).

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Fermentation. Fermentation process involves a wide range of microorganisms that

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responsible for many bioreactions. One of these bioreactions, the enzymatic reactions,

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which enrich the food product via many aspects, i.e., nutritional, sensorial, safety and

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promotion of health (El Sheikha & Hu, 2018). Phytate hydrolysis occurs throughout the

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different stages of fermentation as a result of the enzymatic activity of phytases

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associated with this process (Greiner & Konietzny, 2006).

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Addition of vitamin C. Ascorbic acid appears strong enough to overcome phytic acid and

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its derivatives. The addition of 50 mg of vitamin C can counteract the antinutritional

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effects of the phytic acid and its salts content in a meal (Sharma & Mathur, 1995;

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Banerjee, Adak, Adak, Ghosh, & Chatterjee, 2015). Precisely, the study conducted by

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Siegenberg and others (1991) suggested that greater than or equal to 80 mg ascorbic acid

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would be required to overcome the antinutritional effects of the phytic acid of any meal

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containing greater than 25 mg phytic acid.

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1.4. Potential benefits of phytic acid and its derivatives

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Although there are many studies that point to the negative impacts of phytic acid and its

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salts as mentioned-above, there are, on the other hand, other studies that highlight their

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positive effects such as as prevent kidney stone formation (Grases et al., 2000), protect

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against coronary heart disease (Jariwalla, Sabin, Lawson, & Herman, 1990), anticancer

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agent (Vucenik & Shamsuddin, 2003) as well as antioxidant agent (Burgess & Gao 2002;

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Mittal, Gupta, Singh, Yadav, & Aggarwal, 2013). Moreover, phytate has the protective

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effect on diabetes mellitus, renal lithiasis and arteriosclerosis in developing countries,

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which their meals constitute a considerable amount of phytate (Jenab & Thompson, 2002;

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Afinah, Yazid, Anis Shobirin, & Shuhaimi, 2010; Kumar, Sinha, Makkar, & Becker,

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2010). However, in western countries, these diseases are common because of their foods

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characterized by low phytate content. The proofs also mentioned that phytate inhibitory

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effects on mineral absorption are not seen in diets containing animal protein. These

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pieces of evidence lead that phytate is beneficial as a dietary antioxidant in an animal

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protein diet (Cornforth, 2002). Figure 2 summarizes all these beneficial effects of

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phytate on the human health.

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2.1. Phytase’s functions and sources

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In simple words, phytase (myo-inositol-hexakis-phosphate-phosphohydrolase) is known

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as an enzyme that has the ability to release the phosphate and other minerals from phytic

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acid and its salts (phytates). Endogenous phytases formed during the maturation process

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of plant seeds and grains that are commonly found in plant-based foods. About two-thirds

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of the phosphorus present in plant-based foods (legumes, cereals and other grains) is

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bound in the form of phytic phosphate (Group, 2017).

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Phytase is one of the many primary enzymes necessary for the digestive process and a

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key enzyme for human health. The functional role of phytase is its ability in preventing

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the antinutritional effects of phytic acid and its salts (phytates) by forming complexes

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with minerals and proteins, this meaning that the phytase enzyme increases the

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bioavailability of these important nutrients which reflected positively on the body

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physiology and general health (Shivanna & Venkateswaran, 2014; Group, 2017).

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Knowing that the enzymatic degradation of phytic acid and its derivatives will not

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produce toxic by-products (see Equation 1; Rebello, Jose, Sindhu, & Aneesh, 2017).

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Both endogenous and exogenous phytases are present in plants, animal tissues and

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microbial sources (bacteria, yeasts and molds) (Gaind & Singh, 2015; Ribeiro Correa, de

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Queiroz, & de Araajo, 2015). The catalytic mechanisms, optimal parameters, substrate

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specifications and stability of the enzyme are different depending on: a) the enzyme

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originated source; b) the enzyme food application. The typical phytase for all food

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applications does not exist. Generally, plant phytases exhibit maximum activity at lower

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temperatures compared to their microbial counterparts. The higher pH and thermal

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stability, as well as higher specific activity of microbial compared to plant phytases,

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make the former more favorable for an application in food processing (Greiner &

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Konietzny, 2006). Table 2 illustrates the comparison of phytases characteristics that

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produced from plant and microbial sources.

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supplementation are industrially and commercially demanded. Table 3 shows some

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commercial phytases that originated from microbial sources.

importance

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utmost

Hence, the importance is raising of answering the following question ….

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the

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2.2. Why is the microbial phytase the best source?

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In order for the answer to be completed to this question, it should be addressed from

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many aspects as follows.

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2.2.1. Efficiency point of view

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Phytases from microbial origin may achieve complete phytate degradation, while

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endogenous phytases just reduce phytates by 73–80% (Sandberg, Rossander Hulthén, &

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Türk, 1996). Of the microorganisms, phytase fungal sources are preferred to bacterial

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sources due to high chitinase activity and thermostability. Bacterial phytases differ from

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fungal counterparts in their activity from acidic to alkaline pH, protease resistance in the

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gastrointestinal tract, high substrate specificity, and calcium ions (Ca2+) dependence (Jain

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& Singh, 2016).

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2.2.2. Economic and commercial points of view

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Consumer vision. From the point of view of the consumer that any addition, i.e., enzyme

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(e.g., phytase) during the manufacture of food will reflect negatively on the consumer by

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increasing the price of this food product. Of course this conclusion is true but the

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consumer should take into considerationn determining whether an enzyme will be a

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commercial success is the economics of its use—will the cost of using the enzyme be at

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least equal to the value of the changes rendered through its use?

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To fully answer this question, the following questions must be answered:

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1- What is the need to add the enzyme?

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Today, nearly all commercially prepared foods contain at least one ingredient that has

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been made with enzymes.Some of the typical applications include enzyme use in the

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production of sweeteners, chocolate syrups, bakery products, alcoholic beverages,

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precooked cereals, infant foods, fish meal, cheese and dairy products, egg products, fruit

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juice, soft drinks, vegetable oil and puree, candy, spice and flavor extracts, and liquid

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coffee, as well as for dough conditioning, chill proofing of beer, flavor development, and

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meat tenderizing (Enzyme Technical Association, 2001).

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The increase in global demand for processed food, especially in the growing economies,

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has led the food manufacturing industry to move to new developments and to optimize

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their processes, leading to a continuous demand in enzyme application. Food and feed

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applications account for 55%–60% of the global enzymes market, and the market is still

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growing at an estimated 6%–8% annual growth (Guerrand, 2018).

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2. What is the amount of enzyme? Hence, what is the expected increase in the final

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product price?

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Because enzymes are catalysts, the amount added to accomplish a reaction is relatively

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small. For example, an enzyme preparation in most food uses is equal to 0.1% (or less)

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of the product (Enzyme Technical Association, 2001), which can be expected through a

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slight increase in price especially with the use of modern biotechnology tool "enzymes-

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producing microorganisms" (Singh & Satyanarayana, 2015). Under these circumstances,

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which proves that whatever the additional cost to the consumer as a result of using an

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exogenous enzymes is cheap compared to to the value of the desirable changes rendered

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through its use.

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The endogenous phytase is obviously not enough to perform its function efficiently, i.e.,

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mitigate the antinutritional properties of phytic acid and its salts. Additionally, such

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technological processes (germination, soaking, etc.) could improve the endogenous

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phytase activity is questionable from an economic point of view. Hence, efforts are

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required to produce cost-effective phytase that can be achieved via microbial production

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of phytases (Żyta, 1992; Bhavsar & Khire, 2014; Singh & Satyanarayana, 2015).

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Although phytases from several species of bacteria, yeast and fungi have been

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characterized (Pandey, Szakacs, Soccol, Rodriguez-Leon, & Soccol, 2001; Konietzny &

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Greiner, 2002; Vohra & Satyanarayana, 2003), however, fungal sources are more

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promising for the production of phytases on a commercial scale (Konietzny & Greiner,

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2004; Vats & Banerjee, 2004; Singh, Kunze, & Satyanarayana, 2011). For instance, the

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production of phytase-producing fungi has been performed using three different

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cultivation methods, namely, solid-state (Ebune, Al-Asheh, & Duvnjak, 1995), semi-solid

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(Han, Gallagher, & Wilfred, 1987), and submerged fermentation (Ullah & Gibson, 1987;

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Nair & Duvnjak, 1990). Whereas, the solid-state fermentation (SSF) system has

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generated a great deal of interest in recent years because of its economic and practical

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advantages including high product concentration, improved product recovery, simple

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cultivation equipment, and lower plant operational cost (Becerra & González Siso, 1996;

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Pandey, Szakacs, Soccol, Rodriguez-Leon, & Soccol, 2001; Singh & Satyanarayana,

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2015).

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2.2.3. Applicability point of view

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The phytases with desirable properties have been generated through the microbial

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production since native phytases do not possess all ideal properties required for the

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industrial applications. Several studies have shown that microbial phytases are most

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promising in food and feed applications. Moreover, thanks to recent developments in the

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biotechnological researches, the microbial phytases capable of promoting plant growth

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and combating environmental phosphorus pollution (Pandey, Szakacs, Soccol,

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Rodriguez-Leon, & Soccol, 2001; Vohra & Satyanarayana, 2003; Cao et al., 2007; Singh,

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Kunze, & Satyanarayana, 2011; Bhavsar & Khire, 2014; Singh & Satyanarayana, 2015).

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Hence, many companies already have launched the production of the microbial phytase

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products on the commercial scale (Mittal, Gupta, Singh, Yadav, & Aggarwal, 2013;

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Rebello, Jose, Sindhu, & Aneesh, 2017; see Table 3).

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Below, we focus on the significant impacts of microbial phytases on food applications.

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3. Potential food applications of microbial phytases

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The microbial enzymes are of great importance in the food industry due to their substrate

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and product specificity, minimal by-product formation and high yield. They are important

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ingredients in several food products and production processes (Sandberg, Rossander

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Hulthén, & Türk, 1996; Greiner & Konietzny, 2006; Ravindran & Jaiswal, 2016;

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Rebello, Jose, Sindhu, & Aneesh, 2017; Hua, El Sheikha, & Xu, 2018).

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From the industrial vision, the global enzyme industry is growing at a fast pace. It is

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estimated that by 2018 will be worth a staggering $7.1 billion (BCC Reasearch, 2014).

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Enzymes are predominantly used for the food production of several products that we use

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in our day-to-day lives (e.g., cereal-based and fermented foods) (Jegannathan & Nielsen,

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2013).

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3.1. How has the microbial phytase as a biotechnological tool been recognized in the

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food industry?

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Since the first commercial phytase product Natuphos® launch in 1991, the market volume

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has reached €150 million and is likely to expand with new applications. Organic

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phosphorus hydrolysis by microbial phytases has been extensively considered in diverse

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biotech applications, including environmental protection and agricultural, animal and

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human nutrition (Menezes-Blackbur, Jorquera, Gianfreda, Greiner, & Mora, 2014).

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Because of the powerful value of phytases in improving the efficiency of phosphorus use,

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biotechnology has led the rapid development of the field to its current state. With the

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development of heterologous gene expression, large amounts of enzymes can be

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produced at relatively low cost. The importance of phytases as potential biotechnological

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tools has been recognized in various fields, including the food industry (Bhavsar &

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Khire, 2014). Here two important questions will arise:

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1. How have biotech contributions reflected on the nutritional role of microbial phytase?

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The biotechnology reflected positively on the nutritional role of microbial phytase via

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increasing phosphorus utilization, metal bioavailability and technical improvement of

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food processing.

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2- What challenges can this technology face? It will be a challenge to minimize the

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negative effect of phytate on iron and zinc nutrition without losing its potential health

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benefits.

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3.2. The industrial significance of microbial phytase

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The expansion of industrial microbial phytase applications should be pursued vigorously

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to improve the productivity and reduce agricultural practices costs (e.g., fertilizer

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application rates) and hence the cost of food production. The use of microbial phytases is

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thus envisioned as an effective means to improve plant growth and its yield that reflect

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positively on the food industry (Balaban et al., 2017).

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Overall, the industrial significance of microbial phytase is quite evident in the food

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industry, biofuel production, and industrial waste detoxification (Liu, Jong, & Tzeng,

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1999; Fredrikson, Biot, Alminger, Carlsson, & Sandberg, 2001; Shetty et al., 2008;

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Mittal, Gupta, Singh, Yadav, & Aggarwal, 2013; Luangthongkam, Fang, Noomhorm, &

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Lamsal, 2015).

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As mentioned by Mittal, Gupta, Singh, Yadav and Aggarwal (2013) that the applications

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of microbial phytases will be a breakthrough in the field of food industries and can be

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nicknamed as boom of food industry. Collectively, Figure 3 illustrates the food

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applications of microbial phytases.

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In this context, the positive impacts of nutritional applications of microbial enzymes can

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be addressed in two ways:

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1- Improving the nutritional value of plant-based foods.

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2- Technical improvement of food processing.

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3.3.1. Improving the nutritional value of plant-based foods

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In regard of human nutrition, the consumption of plant rich in phytates leads to a

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considerable reduced absorption of dietary minerals and proteins. One possible solution

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for this nutritional problem seems to be the hydrolysis of food phytates by exogenous

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phytases (Żyta, 1992), which the microbial phytases are the best sources (especially the

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fungal ones) (Żyta, 1992; Konietzny & Greiner, 2004; Vats & Banerjee, 2004; Singh,

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Kunze, & Satyanarayana, 2011; Singh & Satyanarayana, 2015; Jain & Singh, 2016).

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Research has proved that phytase directly from microorganisms can improve the

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nutritional value of plant-based foods (Żyta, 1992; Vohra & Satyanarayana, 2003; Singh,

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Kunze, & Satyanarayana, 2011; Dahiya, 2016; Savita, Yallappa, Nivetha, & Suvarna,

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2017). Such as the study conducted by Van Den Berg, Kumar, Wilson, Heath and Smith

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(1980) proved significant enhancement of mineral mobilization; approximately 20-28%,

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26-37% and 24-42% of Zn2+, Fe2+ and Ca2+, respectively after the addition of phytase

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enzyme to chickpea flour. There was the significant reduction (75-88%) in phytic acid

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content.

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Novel strategies (transgenic plants). Crop plants as bioreactors have been used as a cost-

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effective and innovative option for phytase production in lieu of directly supplementing

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microbial phytase (Ushasree, Shyam, Vidya, & Pandey, 2017). Plants engineered to

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express phytase genes of microbial origin are expected to have lower phytate content and

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thus represent a more nutritious food (Balaban et al., 2017).

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Prokaryotic and eukaryotic microbial phytase genes were transformed successfully into

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different crop plants. Microbial phytases from Aspergillus niger, A. ficuum, A. fumigates

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and from other fungi and yeast are widely used because of their stability in a broad range

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of pH and temperature (Hamada et al., 2005; Yao, Huang, & Tan, 2012). Bacterial

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phytase genes, such as 168phyA from Bacillus subtilis and appA from E. coli, have also

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been used successfully to generate transgenic plants (Hong et al., 2004; Hong et al.,

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2008). In some laboratory experiments these genes have been successfully expressed in

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transgenic soybeans, corn, wheat, sweet potato and canola, often indeed improving

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utilization of phytate as the source of phosphorus (Brinch-Pedersen, Hatzack, Sorensen,

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& Holm, 2003; Chiera, Finer, & Grabau, 2004; Chen et al., 2008; Shen, Wang, & Pan,

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2008; Li, Yang, Li, Qiao, & Wang, 2009). Furthermore, Li, Yang, Li, Qiao and Wang

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(2009) noticed that the transgenic soy roots expressing A. ficuum phytase (аfрhyA) were

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shown to have 6 and 3.5 fold higher catalytic activity and inorganic phosphate content

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than wild-type control plants. Accordingly, an important question has come to mind …

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What are the constraints of using phytase engineered crop plants?

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Although phytase expressing transgenic plants are beneficial for plant and animal

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nutrition and ecological development (Raboy, 2009; Rao, Rao, Reddy, & Reddy, 2009;

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Reddy et al., 2013); there are issues like loss of seed viability, yield, susceptibility for

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environmental stress (Yip et al. 2003; Bowen, Souza, Guttieri, Raboy, & Fu, 2007;

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Perera, Hung, Moore, Stevenson-Paulik, & Boss, 2008; Raboy, 2009; Dhole & Reddy,

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2016), non-acceptance of Genetically Modified Organisms (GMOs) (Zilberman, Kaplan,

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Kim, Hochman, & Graff, 2013). Although GM crops are beneficial, the protest or

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skeptical attitude from environmental nongovernmental organizations (NGO), societal

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frame workers and policy makers, affect the consumer attitude towards GM crops

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(Zilberman, Kaplan, Kim, Hochman, & Graff, 2013). For example, most of the nations

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from Europe are strictly opposing GM food crops, thus, implementing this technology is

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not that much easy (Reddy, Kim, & Kaul, 2017).

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But is it possible to overcome this problem and why? In terms of the possibility, globally

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more than 70% of the GM crops are used as animal feed to feed 100 billion animals, but

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there is no detrimental effect against health and performance (Fernandez-Cornejo,

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Wechsler, Livingston, & Mitchell, 2014). Therefore, production of transgenic crops

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expressing phytase may not be an issue. In terms of the urgent need, numerous studies as

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mentioned-above have proven negative effects of phytate in human, plant, animal, and

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environment. Hence, it is mandatory to pursue further research to improve the phytate

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related problems through genetic engineering.

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3.3.2. Technical improvement of food processing

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The addition of exogenous phytase, i.e., microbial phytase during food processing was

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reported to affect the economy of the production process as well as yield and quality of

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the final products (Greiner & Konietzny, 2006). Therefore, microbial phytases may find

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application in food processing to produce functional foods (Greiner & Farouk, 2007). For

354

example, tempeh as a popular oriental fermented food made from soybeans inoculated by

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Rhizopus oligosporus in the koji process. The digestibility, vitamin content, and flavor of

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soybean improved with the mold fermentation (Fardiaz & Markakis, 1981). Moreover,

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Fujita et al. (2001) have proved that the alcohol fermentation for sake brewing was

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promoted in terms of its yield of alcohol production via using the high phytase producing

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strain of A. oryzae.

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As follows, we will discuss in details the technical improvements of microbial phytases

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addition during food processing….

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Bread processing. Around 3,500 BC the ancient Egyptians made the bread. From that

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time the heritage of bread, its economic, political and religious importance has persisted

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worldwide (El Sheikha, 2015). From the nutritional vision, bread is considered an

367

important source of both iron and the phytate (Afinah, Yazid, Anis Shobirin, &

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Shuhaimi, 2010). This fact already has proved from more than 2500 years ago by ancient

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Egyptians through the following hieroglyphics which meant in English "Let me Live

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upon Bread and Barley of White my Ale Made of Grain Red” (El Sheikha, 2015).

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In whole wheat bread, phytic acid is present at levels of 0.29 to 1.05% (w/w). During

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breadmaking, the addition of microbial phytases, i.e., mold ones the whole phytates

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content of dough could be roughly removed. The desirable conditions of adding

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microbial phytase would have to be Ca2+ independent, pH optimum of 4.5 to 5.0 and have

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a high reaction rate at 30 °C (Knorr, Watkins, & Carlson, 1981). Also, Sandberg,

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Rossander Hulthén and Türk (1996) reported that the improvement of iron absorption

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after the addition of phytases obtained from Aspergillus niger to wheat bran flour.

379

Furthermore, the addition of exogenous microbial phytase as one of breadmaking

380

additives has improved both baking and physical bread parameters (e.g., proofing time,

381

specific volume, crumb firmness) (Haros, Rosell, & Benedito, 2001). In 2008, Palacios,

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Haros, Sanz, and Rosell noted that incorporating of the phytase-producing bifidobacteria

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in the wheat dough as the microbial source of phytate-degrading enzymes and as a

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fermentation starter as the same time could replace the lactic acid bacteria. The

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Bifidobacterium strains from infants could be good starters for being used in

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breadmaking. Similarly, Sanz-Penella, Laparra, Sanz, and Haros (2012) have investigated

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the use of a Bifidobacterium strain, as a starter in the baking process, to increase the

388

available iron through phytate degradation. Therefore, the study conducted by Rodriguez-

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Ramiro et al. (2017) aimed to compare the effect of three commercial baking processes

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[sourdough, conventional yeast and Chorleywood Bread Making Process (CBP)] on the

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phytate content of wholemeal bread. This study demonstrated that the sourdough process

392

leads to the full degradation of phytate in wholemeal bread and increased the

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bioavailability of iron in the cell model when the bread was digested in combination with

394

other iron sources, compared with CBP and conventional fermented breads. The wider

395

use of sourdough processes could therefore have significant benefits for iron nutrition.

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Isolation of plant proteins. Because of the good nutritional and functional properties of

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isolated plant proteins, their applications have been found increasingly interesting in food

399

production. But the technologically, the high content of phytates present in plant seeds

400

and grains prevents access to high quality isolated proteins in acceptable quantities

401

(Greiner & Konietzny, 2006). Therefore, embedding an exogenous phytase (e.g.,

402

microbial source) into the isolation process was reported to result in significantly higher

403

protein yields and an almost complete removal of phytates from the final isolated plant

404

protein (Wang, Hettiarachchy, Qi, Burks, & Siebenmorgen, 1999; Fredrikson, Biot,

405

Alminger, Carlsson, & Sandberg, 2001).

406

In this respect, the soybean stands out, since it contains higher protein content (∼35–

407

40%) than most other legumes (de Souza Ferreira, Silva, Demonte, & Neves, 2010). The

408

isolated soybean protein has shown activity on cholesterol metabolism in studies in vitro

409

and in vivo (Torres, Torre-Villalvazo, & Tovar, 2006; Scarafoni, Magni, & Duranti,

410

2007). This utmost importance of isolated protein has led the American Food and Drug

411

Administration (FDA) to authorize 25 g of soy protein a day (FDA, 1999). As known, β-

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conglycinin and glycinin are the two major soybean proteins, representing approximately

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40% and 30% of the total protein, respectively (Krishnan & Nelson, 2011). Hence, the

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importance of isolating them was the main target of the study conducted by Saito, Kohno,

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Tsumura, Kugimiya and Kito (2001), where they proposed a separation method using

416

phytase for efficient isolation of both protein fractions.

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Grains wet milling. One of the strategic grains is maize. Maize, also known as corn, is a

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grain plant cultivated for food, which has a global production in 2016, 18 million tons

420

(FAOSTAT, 2016). But as most of the grains, maize comprises phytate, which ends up in

421

the corn steep liquor and constitutes an undesirable component. For this reason, Caransa,

422

Simell, Lehmussaari, Vaara and Vaara (1988) have used the microbial phytase to

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accelerate the steeping process as well as improve the properties of corn steep liquor.

424

This positive results already proved also by Antrim, Mitchinson and Solheim in 1997.

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Cereal bran fractionation. Bran is the outer layer of the grain. Nutritionally, bran

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fractions produced by milling are rich in fiber, minerals, vitamin B6, thiamine, folate,

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vitamin E and some phytochemicals, in particular antioxidants such as phenolic

429

compounds (Shewry, 2009). Bran is used in the production of brown and wholemeal

430

flours, hence retaining some of the valuable nutritional components that are depleted

431

when these fractions are further removed in the refinement of white flour. The enzymatic

432

treatment (by phytase addition) using to economically separate the main fractions of the

433

bran in order to produce high-value protein, soluble non-starch carbohydrates, oil

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fractions, and insoluble fiber (Kvist, Carlsson, Lawther, & DeCastro, 2005). All these

435

fractions have much broader market applications and greater value than the original bran.

436 Phytase-producing microorganisms could be probiotics? The term probiotic was

438

technically defined by an Expert Committee of FAO as ‘‘live microorganisms which

439

upon ingestion in certain numbers exert health benefits beyond inherent general

440

nutrition’’ (FAO/WHO, 2002; Vasiljevic & Shah, 2008). The probiotics have been used

441

for food fermentation since the ancient time; can serve a dual function by acting as food

442

fermenting agent and potential health benefits provider (Ray, El Sheikha, & Kumar,

443

2014).

444

From this vision, the microbial phytase extracted from the gastrointestinal tract of sea

445

cucumbers, Holothuria scabra can be used for hydrolyzing phytates as well as probiotics

446

(Hirimuthugoda, Chi, & Wu, 2007). Following the same research direction, Miao, Xu,

447

Fei, Qiao and Cao (2013) have applied the genetic engineering technology to produce the

448

enzyme phytase from a gene (phyC) native to Bacillus subtilis GYPB04 for the

449

development of functional, healthy fermented dairy products that provide both active

450

phytase and viable probiotics to the consumer.

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4. Future trends

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To feed the ever-growing world population, modern agriculture will continue to rely on

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the improvements in biotechnology. One of the recent technologies is applying the

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genetic engineering to increase the nutritional value, i.e., nutrients bioavailability from

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plant food-based foods (raw or processed) via reduction of phytic acid content and its

457

derivatives.

458

A focused platform for microbial production, downstream processing and application-

459

oriented research will help in developing an integrated technological solution to phytase

460

production. This will present new insights in the biological and engineering facets of

461

phytase producing microbes and reveal a new era in phytase biotechnology (Bhavsar &

462

Khire, 2014). Two promising strategies that should be considered as potentially viable

463

options (Ushasree, Shyam, Vidya, & Pandey, 2017):

464

- Genetically engineered plants with low phytic acid content by using antisense

465

approaches to disrupt genes involved in phytic acid biosynthesis.

466

- Transgenic plants expressing microbial phytase.

467

Regarding the nutritional impacts of the microbial phytases applications, the ideal

468

phytase for all food applications does not exist. Thus, screening nature for phytases with

469

more favorable properties for food applications and engineering phytases in order to

470

optimize their catalytic and stability features are suitable approaches to make a proper

471

phytase available for a specific application in food processing. However, only a limited

472

number of microbial phytases have been reported and studied, and our knowledge of the

473

mechanisms and factors regulating microbial phytase activity is limited. Further research

474

for developing new technologies and identifying the most efficient phytases-producing

475

microorganisms must continue and should be directed towards food application-oriented

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research. Also, more studies required in terms of the microbial strains engineered for

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food enzyme production from a safety point of view. Only nine microorganisms are

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Generally Recognized As Safe (GRAS) based on FDA regulations, primarily A. oryzae,

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A. niger, B. subtilis and B. licheniformis (Hua, El Sheikha, & Xu, 2018).

480 5. Conclusions and remarks

482

The potentials of industrial uses of microbial enzymes have increased greatly in the 21st

483

century and continuously increasing to meet the demand of a rapidly growing population

484

and cope exhaustion of natural resources. Currently, phytase has emerged as the world’s

485

most widely used food enzyme.

486

The impacts of phytic acid in foods have become the major concerns due to its negative

487

effect on mineral bioavailability and protein digestibility in human nutrition. Thus, the

488

inclusion of exogenous phytase, i.e., microbial phytase in food medium has been seen as

489

a promising solution to overcome this problem. But why the microbial phytase is the best

490

source of exogenous phytase? Because the microbial phytases exhibit desirable activity

491

profile over a broad pH range, excellent thermal stability, and broad substrate

492

specificity, are more promising nutritionally and economically.

493

It had been showed here that the phytase enzyme has enormous potential in food

494

industrial sector as reported for bread processing, isolation of plant proteins, grains wet

495

milling and cereal bran fractionation. Recently, some studies suggest phytase producing

496

microorganisms as novel probiotics.

497

Although, a significant progress has been achieved in microbial phytase research during

498

the last few years, scientists have to do more efforts in isolating novel and best phytate-

499

hydrolyzing enzymes microorganisms and optimizing their catalytic features, thermal

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tolerance and specific activity via genetic engineering to generate an idyllic phytase for

501

food application.

502 Authors Contributions Statement

504

H-Y S and A F E are considered as the first author. H-Y S collected and analyzed the

505

literature. A F E designed and wrote the manuscript. D-M H revised the manuscript. All

506

authors read and approved the final manuscript.

Conflicts of interest

509

The authors declare no competing financial interests.

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510 Acknowledgments

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This research was supported by the National Natural Science Foundation of China

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(31460009, 31500021) and Key Research and Development Program of Jiangxi Province

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(20161BBF60078). Dr. Dian-Ming Hu thanks China Scholarship Council (CSC) for

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financial support.

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Figure 2 Potential health benefits of phytic acid and its derivatives.

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Figure 1 Phytic acid (myo-inositol-hexakis-dihydrogenphosphate, IP6).

Figure 3 Food applications of microbial phytases.

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Equation 1 How to prevent the antinutrional effects of phytate in human body using

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Table 1 Phytic acid/phytate content in common plant-based foods Plant-based food Phytic acid/phytate content (%) as DW basis Cereal-based food Wheat 0.39-7.30 Corn 0.72-6.39 Rice 0.06-8.70 Sorghum 0.57-3.35 Legume-based food Beans 0.61-2.38 Peas 0.22-1.22 Chickpeas 0.28-1.60 Lentils 0.27-1.51 Nut-based food Almonds 0.35-9.42 Walnuts 0.20-6.69 Brazil nuts 0.29-6.34 Cashew nuts 0.19-4.98 Oil seed-based food Soybean 1.00-10.7 Rapeseed 2.50-7.50 Sesame seed 1.44-5.36 Sunflower 2.10-4.30 Others Tomato seed 1.66 Eggplant seed 1.42 Kiwi fruits 1.34 Cucumber seed 1.07 Sources: Greiner and Konietzny (2006), Schlemmer et al. (2009), Afinah et al. (2010)

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Table 2 The comparison of phytases characteristics that produced from plant and microbial sources Produced phytase characteristics Source pH optimum Temperature optimum Michaells-Menten Reference (° C) constant (mM) Plant-based source Wheat 5.2 55 0.3 Pears (1953) Soybean 4.8 60 2.4 Sutardi and Buckle (1986) Microbial-based source Bacteria Aerobacter aerogenes 4.0-5.0 25 0.135 Greaves et al. (1967) Bacillus sp. DS11 7.0 70 0.55 Kim et al. (1998) Escherichia coli 4.5 55 0.13 Greiner et al. (1993) Klebsiella aerogenes 4.5-5.2 60 0.11 Tambe et al. (1994) Yeasts Pichia spartinae 4.5-5.5 75-85 0.33 Nakamura et al. (2000) Pichia rhodanensis 4.0-4.5 70-75 0.25 Nakamura et al. (2000) Arxula adeninivorans 4.5 75-85 0.25 Sano et al. (1999) Pichia anomala 4.0 60 0.20 Vohra and Satyanarayana (2002) Molds Aspergillus niger van Teighem 2.5 55 0.606 Vats and Banerjee (2004) Aspergillus ficuum 1.3 67 0.295 Zhang et al. (2010) Aspergillus niger 5.5 55 0.20 Berka et al. (1998) Sporotrichum thermophile 5.0 60 0.156 Singh and Satyanarayana (2009)

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Danisco, Brabrand/ Denmark

Allzyme

Alltech/ USA

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Better phytase efficacy, heat stability, and cost efficacy Activity equivalent to minimum 500 FTU g−1, excellent stability, good heat resistance, high biological efficiency, flowability, and mixability Patented technology with unrivalled thermotolerance

E. coli species bacterium and is expressed in a Saccharomyces pombe A. niger

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Significant features Active over wide range of pH from 6.5 to 5.5

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Table 3 Common commercial phytases from microbial sources Commercial name Company/ Country Source NatuphosTM BASF/ Germany Aspergillus niger NRRL 3135 phyA gene cloned in multiple copies in a PluGBug® system RonozymeTMP Novo Nordisk/ Peniophora lycii produced in an Denmark A. niger system Optiphos JBS/ USA Escherichia coli-based phytase enzyme

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Non-GMO used in solid-state fermentation, used in pigs, poultry, and aquaculture Source: Adopted from Rebello et al. (2017). Reproduced with permission of Springer Nature

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phytase the best source? < Microbial phytase as a boom of the food industry. < Phytaseproducing microorganisms as could be probiotics. < Genetic engineering reflects positively on

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the nutritional applications of microbial phytases.

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