Intestinal phytase activity of transgenic corn studied

Intestinal phytase activity of transgenic corn studied

*Dr. William A. Dudley-Cash is a poultry and fish nutritionist and has his own consulting firm in Modesto, Cal. To expedite answers to questions concerning this column, please direct inquiries to Feedstuffs, Bottom Line of Nutrition, 7900 International Dr., Suite 650, Bloomington, Minn. 55425, or email [email protected]

THE majority of phosphorus in plant feed ingredients is present as phytate-bound phosphorus, the primary storage form of phosphorus.

Poultry and swine lack adequate levels of endogenous phytase, the enzyme that is necessary to release this phytate phosphorus. Traditionally, the nutritional requirement for phosphorus has been satisfied by the addition of inorganic sources of phosphorus and/or animal byproducts. However, inorganic phosphorus sources have become very expensive, and the use of animal byproducts in feeds is sometimes not desirable or not permitted.

These circumstances have promoted the development of a major industry based on developing exogenous sources of phytase. The addition of an exogenous source of phytase has become a nearly universal practice in poultry and swine feeds.

There are a large number of commercially available products that may be used to supply phytase to the feed. All require the incorporation of an additional ingredient that must be measured and mixed with reasonable care. An alternative would be the incorporation of a transgenic source of phytase into a major feed ingredient that is already a part of the feed formulation, like corn.

Advances in biotechnology have enabled the production of genetically modified plants expressing microbial-derived phytase in tobacco seeds, soybeans, canola and wheat, with papers dating as far back as 1993 at least.

 

Transgenic corn

The Chinese Academy of Agricultural Sciences has developed a phytase transgenic corn (PTC), an Aspergillus niger-derived phytase expressed in the endosperm of corn. This corn was developed as an innovative way to deliver microbial phytase to poultry feeds.

Gao et al. (2012) reported research comparing PTC and isogenic conventional corn for nutritional content and comparing PTC with an exogenous source of microbial phytase in terms of phytase activity. This research was reviewed last month (Feedstuffs, Nov. 4).

Results of the analysis of the two sources of corn are shown in Table 1. The analytical values were nearly identical, with the exception of moisture and phytase activity. PTC contained 8,047 phytase units of activity (FTU) per kilogram. PTC was also numerically higher in fat content.

The authors concluded that transgenic phytase expressed in corn appears to be as efficient as comparable microbial phytase at releasing phytate phosphorus in corn/soybean meal-based diets. These results confirmed that PTC has the potential to serve as a practical source of phytase activity in poultry feeds.

A follow-on paper was just published from the same laboratory, Gao et al. (2013), reporting research designed to evaluate the residual phytase activity of PTC as it transits the intestinal tract of laying hens and comparing this residual activity with two commercial sources of phytase activity. At the same time, the research measured the ability of phytase in PTC to release phosphorus from phytate and made comparisons with two commercial sources of phytase activity.

 

Experimental design

In the study, 504 Hy-Line brown laying hens 60 weeks of age, with a mean egg production of 87.83%, were randomly allocated to one of seven dietary treatments, with eight replicates for each treatment. Each of the eight replicates consisted of a suspended stainless steel cage containing three hens.

Following an adaptation period of three weeks, the birds were fed the treatment diets for 21 days. The laying hens were maintained on a 16-hour light schedule and had ad libitum access to the diets and water.

The experimental diets are shown in Table 2. The conventional corn and PTC were the same materials used in Gao et al. (2012). The two commercial phytase sources were PA — Natuphos (BASF AG), a recombinant enzyme from A. niger — and PB — Phyzyme (DuPont Danisco Animal Nutrition), a recombinant enzyme from Escherichia coli.

The seven experimental treatments consisted of a negative control (NC) using conventional corn and no added phytase; two levels of phytase (500 and 5,000 FTU/kg) provided by PTC (PTC500 and PTC5000), and two levels of phytase (500 and 5,000 FTU/kg) provided by each of the commercial phytase sources (PA500, PB500, PA5000 and PB5000). The levels of the ingredients that changed are indicated. All other ingredients were present at the same level in all seven diets. The level of corn oil added was adjusted to accommodate the somewhat higher level of oil contained in PTC.

The nutrient content of the seven diets was very similar, except for the phytase activity. Titanium dioxide was added as an indigestible marker.

On day 21 of the experiment, all of the birds were killed by intracardial injection of sodium pentobarbitone. Digesta samples were collected from the crop, proventriculus and gizzard, jejunum and ileum. The samples were pooled by replicate.

The jejunum was defined as the section of intestine extending from the distal end of the duodenal loop to Meckel's diverticulum. The ileum was defined as the portion of the small intestine extending from Meckel's diverticulum to a point approximately 40 mm proximal to the ileo-cecal junction. The digesta samples were frozen immediately after collection, lyophilized and ground to pass through a 0.5 mm sieve and stored at -20 degrees C before chemical analysis.

Phytase activity in the diets and digesta samples were determined according to the method of Engelen et al. (2001). One FTU is defined as the quantity of enzyme that releases one micromole of inorganic phosphorus per minute from 1.5 mM sodium phytate at pH 5.5 and 37 degrees C. The phytate phosphorus content of the samples was determined according to the method described by Rounds and Nelson (1994) using high-performance liquid chromatography.

 

Experimental results

Table 3 shows the results for phytase activity analyzed in the feed and in the digesta at each level of the intestine. Also shown is the calculation of percent of phytase in the feed that was recovered at each level of the intestine (percent residual).

The conventional corn used in the NC treatment contained a small amount of phytase activity. This phytase activity was relatively resistant to destruction in the intestinal tract, with 61% of the activity recovered at the level of the proventriculus and gizzard, 53% recovered in the jejunum and 32% recovered in the ileum.

When compared with either the PA or PB commercial sources of phytase, the phytase contained in PTC was roughly twice as resistant to destruction at the level of the crop (72-75% versus 31-36%), proventriculus and gizzard (28-38% versus 14-18%) and jejunum (15% versus 8-10%). At the level of the ileum, the percent residual phytase activity was very low for all of the experimental sources of supplemental phytase.

Of interest, at the level of the ileum, the percent residual phytase activity was much higher at the lower 500 FTU/kg level of supplemental phytase (8-6%) than at the higher level of supplemental phytase (2-3%).

These results show that there is definitely a significant loss of measured phytase activity as the feed progresses along the length of the intestinal tract. The phytase activity contained in PTC is much more resistant to degradation than either of the commercial sources of phytase. The researchers speculated that the phytase activity, as expressed in the endosperm of PTC, may be more protected in undigested feed particles.

Table 4 shows the phytate phosphorus recovered in the diets and digesta. The story here is quite different. In Table 3, it was clear that the phytase activity in PTC was much more resistant to degradation in the intestinal tract compared with either of the commercial phytase sources.

In Table 4, we see that it doesn't seem to matter. Even though the level of residual phytase is much higher for the PTC treatments, the percent of both phytate phosphorus and residual phytate phosphorus found at each level of the intestinal tract shows that there appears to be no extra benefit from the higher level of residual phytase found in the PTC treatments. The phytase activity in PTC may be more protected from destruction, but it may also be less active in releasing phytate phosphorus. Also, the assay for phytase activity is a standardized set of conditions and may not accurately represent what is happening in the intestinal tract.

Table 4 shows that the level of phytase added to the diet has a significant effect on the amount of phytate phosphorus released. However, the difference in phytate phosphorus released by 5,000 FTU is not as different from the phosphorus released by 500 FTU as might have been expected. This is just one experiment and a comparison of only three sources of phytase activity, but the results suggest that you don't get as much bang for your buck when you increase the level of phytase by a factor of 10.

 

Comment

The research reported in these two papers clearly shows that PTC (as well as other transgenic feed ingredients) have the potential to be an alternative source of phytase. However, transgenic corn will come with its own unique set of issues.

The use of transgenic feed ingredients is at least a 2.5-crop year project, requiring the production of the seed and the crop and then use of the ingredient. Do you contract for it or buy it as you use it? Transgenic ingredients are naturally produced and may vary depending on any number of agrarian practices. Transgenic ingredients must be produced, shipped, stored and used on an identity-preserved basis.

I personally find it interesting that the concept of PTC is being studied in China, a society where major changes — as long as they are safe, effective and make economic sense — can be made "easier." In a society that traces its heritage back several-thousand years, a few years devoted to changing the way things are done is not a big deal.

 

The Bottom Line

PTC definitely has potential as an alternative source of phytase activity in poultry feeds. It is not clear how and at what level transgenic corn will compete with other commercial sources of phytase.

 

References

Gao, C.Q., C. Ji, H. Zhao, J.Y. Zhang and Q.G. Ma. 2013. Phytase transgenic corn in nutrition of laying hens: Residual phytase activity and phytate phosphorus content in the gastrointestinal tract. Poult. Sci. 92:2923-2929.

Gao, C.Q., Q.G. Ma, C. Ji, X.G. Luo, H.F. Tang and Y.M. Wei. 2012. Evaluation of the compositional and nutritional values of phytase transgenic corn to conventional corn in roosters. Poult. Sci. 91:1142-1148.

 

1. Composition of conventional corn and PTC, % dry matter (DM)

Item

Conv. corn

PTC

Moisture

13.64a

12.39b

Gross energy, MJ/kg

18.62

18.68

Crude protein

9.17

9.23

Total fat

4.37

4.85

Ash

1.54

1.46

Calcium

0.01

0.01

Phosphorus

0.33

0.33

Phytase activity, FTU/kg

37b

8,047a

Amino acid content

 

 

Lysine

0.26

0.26

Methionine

0.35

0.38

Cysteine

0.28

0.32

Threonine

0.34

0.33

Arginine

0.52

0.50

Valine

0.45

0.40

Leucine

1.06

1.08

Isoleucine

0.33

0.30

a,bMeans within a row with no common superscript are significantly different (P < 0.05).

 

2. Ingredient composition* and nutrient content of experimental diets, % as fed

 

-Treatments-

Ingredient

NC

PTC500

PA500

PB500

PTC5000

PA5000

PB5000

Conv. corn

62.40

56.16

62.40

62.40

 

62.40

62.40

PTC

6.24

62.40

Corn oil

1.20

1.15

1.20

1.20

0.70

1.20

1.20

Zeolite powder

0.20

0.25

0.20

0.20

0.70

0.20

0.20

Nutrient

 

 

 

 

 

 

 

AME (MJ/kg)

11.18

 

 

 

 

 

 

Crude protein

16.6

 

 

 

 

 

 

Lysine

0.78

0.76

0.79

0.79

0.77

0.78

0.77

Methionine

0.38

0.37

0.36

0.38

0.37

0.37

0.39

Methionine + cysteine

0.66

 

 

 

 

 

 

Calcium

3.5

3.52

3.51

3.51

3.51

3.51

3.51

Total phosphorus

0.34

0.33

0.34

0.33

0.34

0.34

0.32

Non-phytate phosphorus

0.10

0.10

0.10

0.11

0.12

0.11

0.10

Phytase activity, added (FTU/kg)

0

500

500

500

5,000

5,000

5,000

Phytase activity, total (FTU/kg)

34

533

512

514

5,094

5,117

5,068

*Each diet also contained 25% soybean meal, 9.10% limestone, 0.30% salt, 0.20% DL-methionine, 0.10% choline chloride, 1.00% vitamin and trace mineral mix and 0.50% titanium dioxide.

AME = apparent metabolizable energy.

 

3. Phytase activity in diets and digesta

 

-Phytase activity, FTU/kg, DM intake basis-

 

Feed,

-Crop-

-Proventriculus and gizzard-

-Jejunum-

-Ileum-

Treatment

FTU/kg

FTU/kg

% residual

FTU/kg

% residual

FTU/kg

% residual

FTU/kg

% residual

NC

38

43e

113

23d

61

20d

53

12d

32

PTC500

599

429c

72

170c

28

91c

15

48c

8

PA500

575

208d

36

82cd

14

47d

8

36c

6

PB500

577

203d

35

97cd

17

51d

9

38c

6

PTC5000

5,729

4,315a

75

2,155a

38

876a

15

156a

3

PA5000

5,744

1,803b

31

1,036b

18

593b

10

135b

2

PB5000

5,704

1,756b

31

975b

17

574b

10

125b

2

a,b,c,d,eMeans within a column with no common superscripts are significantly different (P < 0.05).

 

4. Phytate phosphorus content in diets and digesta

 

-Phytate phosphorus, % DM intake basis-

 

Feed,

-Crop-

-Proventriculus and gizzard-

-Jejunum-

-Ileum-

Treatment

%

%

% res.

%

% res.

%

% res.

%

% res.

NC

0.272

0.222a

82

0.198a

73

0.164a

60

0.154a

57

PTC500

0.260

0.205b

79

0.134b

52

0.071b

27

0.053b

20

PA500

0.266

0.190c

71

0.136b

51

0.072b

27

0.057b

21

PB500

0.250

0.186c

74

0.133b

53

0.069bc

28

0.054b

22

PTC5000

0.246

0.169d

69

0.091c

37

0.058d

24

0.034c

14

PA5000

0.260

0.170d

65

0.099c

38

0.060cd

23

0.036c

14

PB5000

0.252

0.163d

65

0.102c

40

0.057d

23

0.033c

13

a,b,c,dMeans within a column with no common superscripts are significantly different (P < 0.05).

 

Volume:85 Issue:49

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