Use of Bacterial 3-Phytase for Feed or Food Products

Abstract

The present invention relates to the use of bacterial 3-phytases for the production of feed or food products. Further, the present invention relates to the use of bacterial 3-phytases for feeding animals and for the production of a food or feed additive.

Claims

1. A method of preparing a feed or food product comprising adding at least one bacterial 3-phytase to an animal feed stuff to produce said feed or food product.

2. The method according to claim 1, wherein the phytase is characterized by less than 25% (wt./wt. free phosphate) accumulation of the intermediate product inositol-tetraphosphate after 35% inorganic phosphate release within an assay of 2.7 mmol/L phytate at 37 C. and pH 5.5 using an enzyme dosage of 0.2 U/mL.

3. The method according to claim 1, wherein the phytase is characterized by a phosphate release of at least 15% from 2.7 mmol/L phytate at pH 5.5 after 1 hour using an enzyme concentration of 0.21 g/mL.

4. The method according to claim 1, wherein the phytase is a phytase from the genus Dickeya or Serratia.

5. The method according to claim 4, wherein the phytase is a phytase from Dickeya sp., Serratia sp., Dickeya zeae, Dickeya chrysanthemi, Dickeya dadantii, Dickeya solani or Dickeya paradisiaca.

6. The method according to claim 1, wherein the phytase shows at least 70% sequence identity to SEQ ID NO 1.

7. The method according to claim 1, wherein the phytase shows a temperature optimum of from 30 to 65 C. and/or a pH optimum of from pH 2.5 to 5.5.

8. The method according to claim 1, wherein the feed or food product further comprises at least one compound selected from the group consisting of vitamins, minerals, organic acids, probiotic components, oils, fats, pigments, growth factors, antimicrobial agents, cellulose, starch.

9. The method according to claim 1, wherein the feed or food product is in dry or liquid form.

10. A method of feeding an animal, comprising offering to the animal a feed or food product produced by the method of claim 1.

11. A method of preparing a feed or food additive comprising adding at least one bacterial 3-phytase to a composition to produce said food or feed additive.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention is in the following further described by examples and figures. It is emphasized that the examples and figures have only exemplary character and do not limit the scope of the present invention.

[0030] FIG. 1 shows a comparison between bacterial 3-phytases according to the present invention and bacterial 6-phytases currently used within the state of the art for feeding animals with respect to accumulation of the intermediate product inositol-tetraphosphate at pH 5.5 and 37 C.

[0031] FIG. 2 shows a comparison of bacterial 3-phytases according to the present invention and fungal 3-phytases which are also known within the state of the art to be used for feeding animals and have been used in the past relating to a phosphate release pertaining to the same amount of phytase used within the assay at pH 5.5 and 37 C.

[0032] FIG. 3 shows a comparison of bacterial 3-phytases according to the present invention and bacterial 6-phytases currently used within the state of the art for feeding animals with respect to phosphate release from the intermediate product inositol-tetraphosphate at pH 5.5 and 37 C.

[0033] FIG. 4 shows a comparison between bacterial 3-phytases according to the present invention and bacterial 6-phytases currently used within the state of the art for feeding animals with respect to accumulation of the intermediate product inositol-tetraphosphate at pH 5.0 and 40 C.

[0034] FIG. 5 shows a comparison of bacterial 3-phytases according to the present invention and fungal 3-phytases which are also known within the state of the art to be used for feeding animals and have been used in the past relating to a phosphate release pertaining to the same amount of phytase used within the assay at pH 5.0 and 40 C.

[0035] FIG. 6 shows a comparison of bacterial 3-phytases according to the present invention and bacterial 6-phytases currently used within the state of the art for feeding animals with respect to phosphate release from the intermediate product inositol-tetraphosphate at pH 5.0 and 40 C.

DETAILED DESCRIPTION

Methods

[0036] The following methods have been applied within the examples:

State of the Art Phytases

[0037] The following phytases have been tested within the examples:

TABLE-US-00001 Short name Species SEQ ID NO E. coli Escherichia coli 17 Cit. braaki Citrobacter braakii 19 Butt. sp Buttiauxella sp. 21 A. niger Aspergillus niger 23

Determination of Enzyme Concentrations

[0038] SEQ ID NO 1 concentration was determined via UV absorbance using its molar extinction coefficient. All other phytase solutions and extracts were quantified via an in house SDS gel quantification method using a SEQ ID NO 1 calibration curve. Phytase samples were applied to a SDS gel which was subsequently stained with Sypro Ruby (Thermo Fisher: S12000). The gel image was recorded on a standard Bio-Rad gel documentation instrument. Image analysis was performed using ImageLab software (Bio-Rad). Protein concentration was determined by signal integration of the phytase specific SDS gel bands using a SEQ ID NO 1 calibration curve on the same SDS gel.

Determination of Phytase Activity

[0039] 5 L enzyme solution (in 100 mmol/L sodium acetate buffer, pH 5.5, 0.05% (w/v) Triton X-100) were incubated with 95 L 2.88 mmol/L sodium phytate (Sigma 68388, lot BCBM4006V) in 100 mmol/L sodium acetate buffer, pH 5.5 at 37 C. for 15 min. The reaction was stopped by adding 100 L 10% (w/v) trichloroacetic acid. Subsequently, 100 L of the stopped enzymatic reaction was mixed with 100 L molybdate reagent (aqueous solution of 1.2% (w/v) ammonium molybdate; 4.4% (v/v) sulfuric acid, and 27 mg/mL (w/v) ferrous sulfate) and the solution was incubated for 15 min at room temperature. Absorbance at 700 nm was determined and the amount of released inorganic phosphate was calculated using inorganic phosphate standard solutions (0-1.5 mmol/L). One unit of phytase activity was defined as the amount of enzyme that releases 1 mol phosphate per min at 37 C.

Determination of Phytate Content in the Commercial Phytate Preparation

[0040] The standard substance phytic acid sodium salt hydrate (Sigma Art. 68388, lot BCBM4006V) contains phytate, sodium and water. The phytate content is not available by a certificate of analysis and was determined by acid hydrolysis and subsequent determination of free phosphorus by ICP-OES. 200 mg phytate sodium salt hydrate (Sigma Art. 68388, lot BCBM4006V) was weighted in a teflon tube. Subsequently, 1 mL water, 1 mL sulfuric acid 98%, and 4 mL hydrogen peroxide solution 30% were added. The tube was closed with the lid and bursting cap and incubated at 300 C. for 1 h in an autoclave. The hydrolysate was transferred quantitatively to a 50 mL volumetric flask and fill up to 50 mL with water. This solution was analysed for the content of phosphorus by ICP-OES according DIN EN ISO 11885 E22. The content of phytate was calculated by the weight, the content of phosphorus and molar masses. The content of free phytate in phytic acid sodium salt hydrate (Sigma Art. 68388, lot BCBM4006V) was 54.7%.

Determination of Different Inositol-Phosphate Species by HPAEC-UV

[0041] The determination of the different inositol-phosphate species was performed by high performance anion exchange chromatography with post column derivatisation using an UV detector at 290 nm (HPAEC-UV). The chromatographic system was a Dionex ICS-3000 with dual pump (IC-3000 DP), dual column oven (ICS-3000 DC), cooled autosampler (ICS-3000 AS) and UV detector (ThermoFisher MWD-3000). Instead of a post column delivery system a knitted reaction coil (4 mm system, 375 L) was used for mixing the flows of both pump systems to perform the derivatisation reaction before detection. System 1 was used to separate the different inositol-phosphate species by using a Dionex CarboPac PA100 Guard column 450 mm and a Dionex CarboPac PA100 analytical column 4250 mm. For gradient elution, HPLC grade water (pump system 1/canal A) and 0.5 M hydrochloric acid (pump system 1/canal B) were used at a flow rate of 1.0 mL/min. The gradient conditions were as follows: 0 min. 5% B; 0-8 min. 5-10% B; 8-25 min. 10-35% B; 25-35 min. 35-100% B; 35-42 min. 100% B; 42-43 min 100-5% B; 43-55 min 5% B. Pump system 2 provides the post column reaction solution (0.33 M perchloric acid with 0.1% Fe(NO.sub.3).sub.3) to a tee connector in combination with the knitting coil at a flow rate of 0.4 mL/min. The injection volume was about 50 L at a column temperature of 30 C. and an autosampler temperature of 10 C. The run time was 55 min. The signals were detected at 290 nm and manually intregrated. The standard stock solution was prepared as follows: 100 mg phytic acid sodium salt hydrate (Sigma Art. 68388, lot BCBM4006V) was diluted in 100 mL Phosphate Standard Solution 1000 mg/L (Merck Art. 119898). The dilutions to several calibration levels were performed with water (HPLC grade) to end up with the following concentrations: 0.1 mg/mL Phytic acid sodium salt hydrate/Phosphate, 0.2 mg/mL Phytic acid sodium salt hydrate/Phosphate, 0.4 mg/mL Phytic acid sodium salt hydrate/Phosphate, 0.6 mg/mL Phytic acid sodium salt hydrate/Phosphate, and 0.8 mg/mL Phytic acid sodium salt hydrate/Phosphate.

List of Retention Times of the Inositol-Phosphate Species:

[0042]

TABLE-US-00002 Substance Retention time [min] Phsophate 3.45 1,4-Inositol-diphosphate 11.81 4,5-Inositol-diphosphate 12.33 1,2,3-Inositol-triphosphate 17.86 1,4,5-Inositol-triphosphate 18.27 1,5,6-Inositol-triphosphate 18.69 1,2,4,5-Inositol-tetraphosphate 24.38 1,3,4,5-Inositol-tetraphosphate 25.39 2,4,5,6-Inositol-tetraphosphate 27.63 1,4,5,6-Inositol-tetraphosphate 28.82 1,2,3,4,5-Inositol-pentaphosphate 32.12 1,2,4,5,6-Inositol-pentaphosphate 34.40 1,2,3,4,5,6-Inositol-hexaphosphate (Phytate) 39.07

EXAMPLES

Example 1 Determination of Phytate Degradation Pattern

[0043] Enzymatic reactions were performed by incubating a 2.7 mmol/L phytate solution (Sigma 68388, lot BCBM4006V) in 100 mmol/L sodium acetate buffer, pH 5.5 at 37 C. with 0.2 U/mL of phytase. Time resolved enzymatic phytate degradation data was recorded by stopping the enzymatic reaction at different time points via immediate incubation at 99 C. for 10 min in a thermo shaker. The samples were analyzed according to method Determination of different inositol-phosphate species by HPAEC-UV. Peak areas of inositol-phosphate isomers (inositol-phosphates with same amount of phosphate residues) were summed up. Subsequently, peak areas were corrected by normalization of the signals to the amount of phosphate residues of the different inositol-phosphate species (i.e. phytate contains six phosphate residues resulting in a six times higher detector response factor than the one of inorganic phosphate). For comparison of the degradation pattern, data of kinetic time points were taken at which equal amounts of phosphate had been released.

[0044] The results are shown in FIG. 1.

Example 2 Determination of Phosphate Release Data at Same Enzyme Amounts of Phytase

[0045] 5 L of enzyme solution (in 100 mmol/L sodium acetate buffer, pH 5.5, 0.05% (w/v) Triton X-100) are incubated with 95 L 2.88 mmol/L sodium phytate (Sigma 68388, lot BCBM4006V) in 100 mmol/L sodium acetate buffer, pH 5.5 at 37 C. for 1 h. The final enzyme concentration in the reaction is 0.21 g/mL. The reaction is stopped by adding 100 L 10% (w/v) trichloroacetic acid. Subsequently, the stopped enzymatic reaction is diluted 1/10 and 100 L of the dilution is mixed with 100 L molybdate reagent (aqueous solution of 1.2% (w/v) ammonium molybdate; 4.4% (v/v) sulfuric acid, and 27 mg/mL (w/v) ferrous sulfate). The solution is incubated for 15 min at room temperature. Absorbance at 700 nm is determined and the amount of released inorganic phosphate is calculated using inorganic phosphate standard solutions (0-1.5 mmol/L).

[0046] The results are shown in FIG. 2.

Example 3 Determination of Phosphate Release from Inositol-Tetraphosphate

[0047] An Inositol-tetraphosphate preparation was produced by incubation of 2.7 mmol/L phytate in 100 mmol/L sodium acetate buffer, pH 5.5 at 37 C. for 1 h with 0.1 U/mL of SEQ ID NO 17.

[0048] After the reaction, the enzyme was inactived by incubation at 95 C. for 30 min. The resulting 2.7 mmol/L inositol-tetraphosphate solution was used as a substrate in an enzymatic reaction according to Example 2 with a final phytase concentration of 0.2 U/mL.

[0049] The results are shown in FIG. 3. The abbreviations used in the figure are: IP6: Inositol-hexaphosphate; IP5: Inositol-pentaphosphate; IP4: Inositol-tetraphosphate; IP3: Inositol-triphosphate; IP2: Inositol-diphosphate; IP1: Inositol-monophosphate; Pi: phosphate.

Example 4 Determination of Phytate Degradation Pattern

[0050] Example 4 has been conducted as example 1, however, pH was set to 5 and incubation temperature was 40 C.

[0051] Results are shown in FIG. 4.

Example 5 Determination of Phytate Degradation Pattern

[0052] Example 2 has been conducted as example 1, however, pH was set to 5 and incubation temperature was 40 C.

[0053] Results are shown in FIG. 5.

Example 6 Determination of Phytate Degradation Pattern

[0054] Example 3 has been conducted as example 1, however, pH was set to 5 and incubation temperature was 40 C.

[0055] Results are shown in FIG. 6.

[0056] A sequence listing is provided as an ASCII text file named 2016DE604_adjusted_SequenceListing.txt created on 7 Oct. 2019 and having a size of 61.4 kilobytes. The ASCII text file is hereby incorporated by reference in the application.