Phytases of Probiotic Bacteria: Characteristics and Beneficial Aspects

Abstract

Probiotics play a vital role in clinical applications for the treatment of diarrhea, obesity and urinary tract infections. Phytate, an anti-nutrient, chelates essential minerals that are vital for human health. In the past few decades, research reports emphasize extensively on phytate degradation in animals. There is a growing need for finding alternate strategies of phytate utilization in human, as they are unable to produce phytase. At this juncture, probiotics can be utilized for phytase production to combat mineral deficiency in humans. The main focus of this review is on improving phosphate bioavailability by employing two approaches: supplementation of (1) fermented food products that contain probiotics and (2) recombinant phytase producing bacteria. In addition, several factors influencing phytase activity such as bacterial viability, optimal pH, substrate concentration and specificity were also discussed.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-017-0647-3) contains supplementary material, which is available to authorized users.

Introduction

The joint initiative of Food and Agricultural Organization (FAO) as well as the World Health Organization (WHO), defined probiotics as mono or mixed cultures of “live microorganisms which when administered in adequate amounts beneficially affect the host” [1]. The history of probiotics dates back to 1907 when Russian scientist Ellie Metchnikoff postulated the idea of using lactic acid bacteria for modulating intestinal flora. Since then intensive research was undertaken in the field of probiotics for improving human health and the timeline of these applications are presented in Fig. 1.

Fig. 1.

Timeline of probiotics

Phytate also known as myo-inositol hexaphosphate is the principal storage form of phosphorus in plant-based foods.

The chemical structure of phytate consists of six phosphate groups (five of them present in equatorial position with the last one placed in axial position) attached to its inositol ring. The overall negative charge contributed by phosphates (Fig. 2) help in chelating divalent and trivalent metal ions. Therefore, phytate acts as an anti-nutrient and limit mineral bioavailability in human. Despite causing a major nutritional deficiency, phytate can be regarded for its beneficial effects especially in treating colon cancer, AIDS, Alzheimer’s disease, Arthritis and Parkinson’s diseases [2].

Fig. 2.

Chemical structure of phytate (retrieved from PubChem)

Phytases of plants and microorganisms degrade phytate into inositol and free orthophosphates. These enzymes differ in their structure. Plant-based phytases predominantly exist in alpha/beta form whereas, bacterial phytases belong to the beta class of proteins [3].

The crystal structure of bacterial phytase consists of antiparallel β-sheets (Fig. 3). The active site of enzyme consists of “catalytic” (phosphate 1) and “affinity” (phosphate 2) binding sites. The two phosphates of the substrate (phytate) bind to these sites causing structural conformations via both ionic and hydrogen bonding. Then the catalytic site is activated by three Ca²⁺ ions, which are vital for substrate binding. Besides, three more Ca²⁺ bind to the affinity site that improves the substrate specificity and overall stability of the enzyme. Binding of phosphate ions also activates the presence of a seventh Ca²⁺ ion in the structure. Catalysis is initiated by an attack of hydroxide ion of water molecule on the first phosphate group, triggering its cleavage. Thereafter, every alternate phosphate is cleaved leading to the final product. Phytases cleave equatorial phosphate groups readily as compared to axial ones [4]. Probiotic bacteria can be efficiently utilized for phytate utilization as compared to their plant-based counterparts owing to the former’s increased substrate specificity and catalytic efficiency [5].

Fig. 3.

Structural configuration of phytase (PDB id: 1H6L)

Humans have a monogastric digestive system which lacks sufficient phytase producing bacteria in their intestine for phytate utilization. This leads to a serious nutritional deficiency in the host. The undigested phytate from food passes into the soil and lead to environmental phosphate pollution [2].

Hence, this review is focused on probiotic bacteria which are capable of degrading phytate in humans.

Probiotic Selection Criteria

Probiotics have been used for centuries due to their broad spectrum of biological activities in human. For bacteria to be used as probiotics, they must be non-pathogenic, non-invasive, non-carcinogenic, adhere to intestinal epithelium, resistant to gastric acidity, stable in food matrix during intestinal digestion, aid in immunomodulation, and colonize for a stable time period. Moreover, they must have verified strain properties, be effective against the specified target and exhibit bile salt hydrolase activity [6].

These probiotics can be administered through enteral route or by enema. Enteral route is widely preferred for consumption of fermented food products. Whereas, an enema is preferred for fecal microbiota transplantation (FMT) [7]. The review is focused only on fermented food products for administering probiotics via an enteral route as it is cost-effective and convenient when compared to FMT [8, 9]. The choice of fermented foods that contain viable bacteria, which are capable of degrading phytate are listed in Table 1.

Table 1.

Fermented food products as the source of probiotics

Microorganisms	Fermented foods	References
Bacillus subtilis (natto) N-77, B. subtilis CF92, Bacillus coagulans IDCC 1201	Commercial natto (traditional Japanese food), fermented soybean, pharmaceuticals	[10–12]
Lactobacillus strains acidophilus 16A, brevis 14G, fermentum 6E, plantarum DC400	Italian sourdough	[13]
L. acidophilus BS, L. casei 1 K, L. casei DSM20011, L. fermentum DSM 20052, L. plantarum 110	Commercial fermented milk, cheese, fermented beets, fermented plant food	[14]
L. acidophilus 1C5, 4C1 and 1C3 and 5C2, L. caseiImmunitass 4D2, 4D1 and Lactobacillus rhamnosus 4C3, 2C2, T, 3C15 and 3C23, L. caseiShirota 6D1, 6D2, Bifidobacterium longum T, Lactobacillus delbreuckii 5D1, Bifidobacterium bifidum MF, L. plantarum 6C1, 1D1, 3C12, 1D2, 6C3, 1C1, 6C4, 6C5	Dairy and pharmaceutical products	[15]
L. acidophilus, L. fermentum and L. plantarum	Fermented sorghum–Irish potato gruel	[16]
L. casei, L. fermentum, L. plantarum and Pediococcus pentosaceus	(Eragostis tef) Atmit	[17]
L. casei NRRL B-1445, L. delbrueckii NRRL B-445, L. fermentum NRRL B-4524, L. fermentum NRRL B-4524, Leuconostoc mesenteriodes NRRL B-512F, P. pentosaceus NRRL B-14009 and L. plantarum NRRL B-4496	Natural vegetable fermentation	[18]
L. casei MF50, 54, L. fermentum MF25, P. pentosaceus MF32, 33 and 35 and L. plantarum MF79	Ethiopian injera (African soft pancake)	[19]
Enterococcus sp. hirae, faecium and durans	Indian fermented soybean foods	[20]
E. faecium RJ16	Goat cheese	[21]
E. faecium A86, L. plantarum H5, L. plantarum L3	Pizza dough, sourdough, sausages	[22]
L. brevis, L. plantarum	Southern Italian sourdough	[23]
L. brevis, P. pentosaceus, E. durans and L. plantarum	Fermented Himalayan vegetables	[24]
L. brevis	Hatay boiled cheese	[25]
Lactobacillus reuteri and L. plantarum	Iranian Sangak bread	[26]
Lactobacillus pentosus, L. fermentum, L. brevis and L. plantarum Lb 29, L10	Carper berry	[27]
L. plantarum strains 17bp30, 17bp31, 17bp48, Mb25, Mb26, 17bp29, Mb46, Mb50, Mb61, Mb67 and Lactobacillus paracasei strains C3-70, C3-89	Spanish farmhouse cheese	[28]
L. brevis G11, G25, L. fermentum N33, N25 and L. plantarum A6	Fermented corn	[29]
L. plantarum, L. mesenteriodes	Moroccan sourdough bread	[30]
L. plantarum	Greek dry fermented sausages	[31]
L. acidophilus, L. plantarum and L. mesenteroides	Iranian Sangak bread	[32]
L. plantarum	Fermented food (Shalgam)	[33]
L. plantarum, L. mesenteroides	Italian Cornetto di Matera sourdough	[34]
P. pentosaceus KTU05-8 and KTU05-9	Wholemeal wheat bread	[35]
L. acidophilus EF7, L. plantarum 299v, L. rhamnosus GG B103, L. reuteri M 14-C, CF2 7-F, DSM 20016, SD 2112, MM7 and MM2-3	Sweet potato	[36]
L. plantarum DPC2739, L. plantarum W723	Dairy products, Sorghum-Ogi	[37, 38]

Probiotic bacteria in fermented food products have an impact on human health. Probiotics isolated from a wide variety of functional foods ranging from sourdough to fermented bread, dairy products are capable of effective phytate degradation thereby aiding in improved mineral uptake in humans.

In general, probiotics competitively bind to the host intestinal epithelium and stimulate an immune response by activating specific signaling cascades and cell-based reactions. Each bacterial genera has a unique action mechanism for the corresponding biological functions.

Probiotics belonging to Bacilli group are gram-positive, facultative aerobes which produce non-pathogenic spores. The spores play an important role in increased shelf-life of the probiotic product as they are thermostable, recalcitrant to a wide range of pH fluctuations and viable under extreme intestinal conditions. The presence of an outer thick peptidoglycan layer protects bacterial spores from extreme heat, organic acids, lysosomal degradation and helps them in several beneficial activities on human health including treatment of rheumatoid arthritis and blood clotting.

Escherichia coli are gram-negative probiotic microbes which predominately participate in anti-inflammatory activity through modulation of the host immune system. They are also helpful in treatment of colitis and constipation.

Microbes belonging to genera Lactobacilli, Enterococci, Bifidobacteria, Pediococcus and Leuconostoc represents a group of non-pathogenic, facultative anaerobes which are commonly present in the human gut. In addition to their gastric viability, they efficiently colonize and have prolonged epithelial adherence. They also produce bacteriocins which competitively inhibit other pathogenic bacteria and maintain a healthy balance of beneficial microbes. These bacteria find applications ranging from preparation of fermented food products to clinical therapeutics such as inflammatory bowel disease (IBD), diarrhea, and cholesterol regulation.

Functional Foods for Phytase Production

Fermented foods like cheese, sausages and caper berry contain viable lactic acid producing bacteria that colonizes in the human intestine. In addition, it enables the sustenance of enzyme source and stability by continuous multiplication. Probiotic bacteria from fermented foods recorded high phytate degradation (Table S-1, see supplementary material). Fermented soybean containing Bacillus subtilis produced phytase activities ranging up to 1,354,906.6 U/mL.

Lower phytase, phosphatase activities were recorded for Lactobacilli sp. grown in sweet potato base medium (SPM) with sodium phytate, p-nitrophenyl phosphate as substrate. Lactobacillus casei 1 K produced highest phosphatase activity of 162,119.2 U/mL with 0.48% phytate hydrolysis.

The level of phytase activity in vivo depends on factors like; the concentration of phytate, bacterial viability, optimum pH, the accessibility of phytate, presence of inorganic phosphate and other organic acids. Human intestine normally maintains a constant temperature of 37 °C with pH ranging from 5 to 7. Probiotic bacteria isolated from humans showed efficient phytate degradation (Table S-2, see supplementary material).

Bifidobacterium sp. BIF longum 12R, catenulatum 31S and breve 211 were isolated from human infants and adults after consumption of whole wheat bread. They produced high specific phytase activities of 6.92, 6.59 and 6.57 U/mg at pH 7.2. L. reuteri CECT 9025 produced the highest phosphatase activity in modified MRS medium containing sodium phytate.

In addition to above-mentioned parameters, molecular mass of phytase, substrate specificity and the presence of minerals also influence phytate degradation in the human intestine.

Microbial phytases may be monomeric or dimeric depending on the conformational state of the enzyme. The characteristics of phytases and phosphatases from probiotic bacteria are tabulated (Table 2), where most of them contain monomeric chains. The exception being L. brevis, which has dimeric chains each with different molecular masses 73 and 34 kDa, respectively.

Table 2.

Characteristics of probiotic phytases and phosphatases

Enzyme	Microorganisms	Substrate specificity	Molecular mass (kDa)	References
Phytase	B. subtilis (natto) N-77	Sodium phytate	38	[10]
	B. subtilis CF92	Sodium phytate (high), α-naphthyl phosphate and Adenosine triphophosphate (ATP) (low)	46	[11]
	B. coagulans IDCC 1201	Sodium phytate	–	[12]
	L. brevis	–	73, 34	[25]
	L. plantarum	–	46	[33]
Phosphatase	L. plantarum NRRL B-4496	Acetyl phosphate, sodium phytate, p-nitrophenyl phosphate and α-d-glucose-1 phosphate	–	[18]
		–	52	[29]
	L. plantarum DPC2739	–	27	[37]

Phosphorylated compounds are generated during digestion of food, mineral metabolism and their presence in the intestinal cavity may interfere with phytate degradation. Most of the bacterial phytases are highly specific towards sodium phytate.

Functional foods contain a good amount of trace elements and minerals important for human nutrition. These metal ions in different concentrations might affect probiotic phytate degradation. The effect of different minerals on phytase activity are tabulated (Table 3).

Table 3.

Effect of minerals on phytate degradation

Microorganisms	Activators	Inhibitors	References
B. subtilis (natto) N-77	Ca2+	Zn2+, Cd2+, Ba2+, Cu2+, Fe3+ and Al3+	[10]
B. subtilis CF92	–	Mn2+, Zn2+, Fe2+, Cu2+, Mg2+ and Co2+	[11]
B. coagulans IDCC 1201	Co2+	–	[12]
L. plantarum	–	Ca2+, Hg2+, Mg2+, Mn2+, Zn2+, Ni2+, Cu2+, Co2+ and Fe2+	[33]
L. plantarum DPC2739		Fluoride, hexametaphosphate at 0.5 mM and orthophosphate, tripolyphosphate and pyrophosphate at 5 mM (phosphatase)	[37]

Ca²⁺ plays an important role in stimulating, stabilizing bacterial phytases at optimum concentrations. However, excess Ca²⁺ can lead to competitive inhibition of enzymatic activity by binding to the enzymatic active site. The presence of inorganic phosphate also inhibits phytate degradation.

Role of Recombinant Phytases and Their Expression

Oral consumption of fermented food products and FMT are effective strategies for probiotic delivery. However, these strategies are associated with certain disadvantages including lack of site specific action and short-term efficacy. This may be overcome by recombinant phytases (Table 4).

Table 4.

Recombinant phytases

Source of phytase gene	Vector plasmid	Host microorganism	References
B. subtilis NCDC-070, NCIM-2712	Ins T/A	E. coli JM-109	[39]
B. longum subsp. infantis and B. pseudocatenulatum	pNGPHYpseudso, pNGPHYlongum	L. casei	[40]
B. licheniformis PB-13	pET32a(+)	E. coli BL21	[41]
E. coli	pET22b	Pichia pastoris	[42]
B. amyloliquefaciens DSM 1061		B. amyloliquefaciens DSM 1061	[43]

Probiotics belonging to Bacilli genera are efficiently utilized for expression of cloned phytases. Phytase from B. licheniformis, when expressed in E. coli efficiently degraded phytate producing a phytase activity of 0.96 U/mL with a recombinant protein having a molecular mass of 66 kDa.

Lactic acid bacteria (LAB) can be effectively used for recombinant phytase expression [44]. They are safe, cost-effective and also yield enzyme with high purity and stability. On the contrary, high species diversity, obscurity in the route of administration, and stringent monitoring due to lack of clinical trial data pose critical challenges in employing these recombinant probiotic phytases [45].

Well-known probiotics belonging to genus Bacilli and Lactobacilli are capable of adherence and colonization in the human intestine. Most often they are inferred by their high cell counts. They produce bacteriocins, which competitively inhibit pathogens thereby aiding overall improvement of gut microbiome. Moreover, consumption of fermented foods containing viable bacteria provides a scope for phytate degradation.

Conclusion and Future Scope

Phytases from probiotics provide a solution for phosphate utilization in humans. This can be achieved by consuming fermented food products, an ideal vehicle for delivering probiotics. It was noted that L. brevis and B. subtilis in fermented food products are capable of producing higher amount of phytase. Most of the probiotic phytases are very specific towards phytate and are stimulated by Ca²⁺ ions for effective activity. Alternatively, recombinant phytases can also be used for intestinal phytate utilization. Despite the wide usage of probiotics, it is yet to be approved by US Food and Drug Administration (FDA). Therefore, more research needs to be done on improvising probiotic strains for phytase production as well as optimizing probiotic dosage for phytate utilization.

Electronic Supplementary Material

Below is the link to the electronic supplementary material.

Compliance with Ethical Standards

Conflict of interest

None.