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Application of “Protozyme” (Aspergillus orizae) to Poultry Feeding.*
APPLICATION OF "PROTOZYMB" (Aspergillus orizae) TO POULTRY FEEDING.* F. H. CLICKNER and E. H. FOLLWEL·L
There have been several changes in the industrial methods of preparing the enzyme since the original process outlined by Takamine, (Eflfront & Prescott) (4) the essential changes being those which would increase the amyloelastie, peptonizing or saccharifying power, in order to satisfy the conditions required for its various applications. The history of the production and application of diastatie enzymes from fungus sources is not without interest and is worthy of brief comment in a paper of this nature. Nageli C. (1879) is credited with being the first to demonstrate the production of diastase by micro-organisms, his experiments being later followed by the works of Kuscheldt who produced the enzyme in workable quantities and applied the name ' ' Eurotin ' ' to the product. The various workers, as they made improvements in culture methods giving greater and more specific application to the enzymes of Aspergillus orizae, attached the names of "Taka-diastase," "Neo-diastase," and "Protozyme." The two former products find their application in the medicinal field, being highly purified and concentrated so as to give rapid action, while the last is used extensively in the textile industry for the de-sizing of cloth (Waksman, S. A.) (18) ; clarification of fruit juices, due to its ability to work in an acid solution (Waksman, S. A.) (19) ; and is now being tested for its effect on digestion and assimilation in feeding of poultry. The preliminary results will be reported later in this paper. *Read at the annual meeting of the A. A. of I. and I. In P. H.. Man hattan, Kansas, August, 1925.
241
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 17, 2015
"Protozyme" is a trade name given to a group of vegetable enzymes of a fungus origin ; namely, those produced by cultures of Aspergillus orizae.
POULTRY
242
SCIENCE
ENZYMES OF PROTOZYME (Aspergillus Orizae) Demonstrated by
Cytase Amylase Maltese
Newcombe, F. C. Davis, W. A. & Daish, A. J. Dubourg, E. Pringsheim, H. & Zemplen, G. Sanquinettl, M. J. Waksman, S. A.
Invertase Emulsin Rennet Protease Oxidase Reductase Catalase Inulase Tannase
Reference
Saito, K. Okada, S. W a k s m a n , S. A. W a k s m a n , S. A. Follwell E,. H. & Clickner, F . H. Kellner, 0 . Mori, Y. Nagaoka, M. Kita, G.
8 2 3 11 13 20 12 9 20 20 5 6
Physical Properties of Protozyme 1. A p p e a r a n c e is similar to toasted bread crumbs, due to the fact t h a t it is dried a n d milled. 2. Color shading from light to dark brown, depending on intensity a n d r a p i d i t y of drying. 3. Odor is a characteristic yeasty odor (when p r e s e n t ) . 4. Taste similar to wheat bran. Chemical Properties of Protozyme 1. Reaction range. Enzyme
pH. Value
Amylase
2.6-8.0
Maltase 47 deg. C. Maltase 35 deg. C. Protease
7.2 3.0 5.1
Determined by Sherman, H. C. & Associates (14) Compton, A. (1) Compton, A. Okada, S. (9)
2. Amyloclastic a n d saccharifying power. 138 L i n t n e r Units (Sample used in t e s t ) . Ranges from 145-200 L i n t n e r Units as p r e p a r e d commercially. Diastase from Protozyme converts starch into maltose a n d chiefly into dextrose. 3. Milk coagulating factor .066 c. c. Protozyme ( 2 % Sol.) equivalent to one Lab. u n i t as measured by Effront ( 4 ) , Wohlgemuth (2) methods. 4. Gelatin liquifying power. E x t r a c t from 1 gram of P r o tozyme will liquify 15 grams of gelatin in two hours. Stronger proteolytic properties t h a n malt Lwow S. ( 7 ) .
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 17, 2015
Enzyme
APPLICATION
Of
"PROTOvYMË"
TO FEED
243
AVERAGE W E E K L Y W E I G H T S OP LEGHOEÎÎ P U L L E T S F E D PROTOZYME
(The Data a r e pounds) Week 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
A m o u n t of Protozyme in Mash by W e i g h t 5% 3% 2% 1% .13 .23 .34 .49 .67 .79 .96 1.18 1.43 1.75 2.00 2.12 2.23 2.31 2.40 2.50 2.58 2.67 2.93 3.24
.12 .20 .30 .42 .61 .80 .92 1.11 1.29 1.65 1.89 1.94 1.97 2.01 2.10 2.23 2.34 2.48 2.64 3.03
.12 .20 .30 .39 .61 .80 .89 1.04 1.25 1.55 1.78 1.89 1.97 2.04 2.12 2.20 2.27 2.37 2.53 2.89
.12 .19 .30 .38 .58 .72 .83 1.02 1.23 1.30 1.72 1.85 1.92 1.97 2.05 2.11 2.18 2.35 2.50 2.80
-none .12 .18 .28 .36 .56 .69 .76 .93 1.11 1.35 1.57 1.62 1.66 1.70 1.73 1.91 2.04 2.20 2.48 2.65
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 17, 2015
5. Temperature. Stable while dry. Enzymic action decreased or destroyed above 60 degrees C. Protozyme as a Means of Promoting Growth A test has been made with Protozyme, and the preliminary data of the first trial are incorporated in this paper. One thousand White Leghorn chicks, hatched on March 1st, were divided into five groups. Each group was fed a commercial chick scratch mixture until the sixth week, when it was changed to one consisting of equal parts by weight, of cracked corn and whole wheat. All pens were started on a commercial starting mash, which was changed after the sixth week to the folloAving growing mash, consisting of bran 300 lbs., standard wheat middlings 100 lbs., finely ground oats 100 lbs., corn meal 100 lbs., and meat scraps 100 lbs. Protozyme was included in the ration as follows : 5 percent of the weight of the mash in pen 1, 3 % in pen 2, 2% in pen 3, 1% in pen 4 and none in pen 5. At the end of the sixth week, the cockerels were removed, and thereafter growth figures secured on the pullets, 100 in each group.
244
POULTRY
SCIENCE
PRELIMINAKY AVERAGE FEED CONSUMPTION OF LEGHORN PROTOZYME FOR 20 WEEKS.
Birds Receiving:
Scratch Grain Mash Consumption Consumption Lbs. Lbs. 7.65 7.67 7.68 7.70 7.77
8.31 8.31 8.27 8.21 7.73
P r i o r to the seventh week, mortality in the respective pens was as follows: 5, 4, 10, 11, and 16. The birds were placed on range at the end of the seventh week, after which none died in a n y pen. E g g production was first noted in the 1% and check pen, d u r i n g the 17th week. At the end of the 20th week, total production in the respective pens was the following number of eggs: 16, 4, 2, 11, 24. Since this is of a preliminary n a t u r e only, it is given without comment. Protozyme as a Means of Promoting Egg Production. On December 1st, four groups of birds in t h e i r first laying year, 25 in each group, were placed on a test to ascertain what value protozyme might have as a means of i n d u c i n g increased egg production. All groups were fed the regular New J e r s e y laying rations and housed in the long laying house on the Station plant, where they M'ere subjected as closely as possible to conditions prevailing on commercial plants. Protozyme was ineluded in the laying mash in the different groups as follows : 5% by weight of the mash, 3 % , 1% and none at a l l ; this latter group serving as a check to the other three. The figures are of a preliminary n a t u r e , a n d it is too early to make a definite recommendation concerning the possibilities of accessory feeding of this n a t u r e as a means of obtaining an e x t r a profit in flock management.
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 17, 2015
5% Protozyme in Mash Z% 2% 1% " " " No
PULLETS FED
APPLICATION
OF 'ΨΚΟΤΟυΎΜΕ"
TO
FEED
SUMMARY OF SCRATCH GRAIN CONSUMPTION OF INDIVIDUAL
245 BIRDS
F E D PROTOZYME.
A m o u n t ot Protozyme in Mash by Weight
Total
5% 3.75 4.59 3.57 3.30 3.01 3.10 3.00
3% 3.79 4.02 3.48 3.30 3.00 3.10 3.00
1% 3.74 4.19 3.30 3.31 3.00 3.10 3.00
Check 3.98 4.33 3.41 3.32 3.00 3.10 3.00
24.31
23.69
23.64
24.14
SUMMARY OF M A S H CONSUMPTION OF INDIVIDUAL BIRDS CONSUMING
PIROTOZYME I N
MASH
A m o u n t of P r o t o z y m e in Mash by Weight Month
5%
3%
1%
December January February March April May June
2.21 2.77 2.83 2.85 3.00 2.97 3.00
1.80 2.81 2.76 2.87 3.26 3.30 3.52
1.91 2.78 2.66 2.90 2.98 2.93 2.95
1.55 2.41 2.10 2.33 2.85 2.67 2.70
19.63
20.32
18.81
16.61
Total
Check
SUMMARY OF EGG PRODUCTION OF INDIVIDUAL BIRDS CONSUMING PROTOZYME IN M A S H .
Amount of P r o t o z y m e in Mash by Weight Month
5%
3%
1%
Check
December January February March April May June
5.20 6.17 11.87 17.50 17.56 16.60 18.13
4.24 8.68 11.50 17.00 16.81 16.92 21.12
3.44 7.04 11.88 17.16 17.16 16.40 15.30
3.08 6.69 8.56 14.76 16.50 16.70 17.93
Total
93.03
6.27
88.38
84.22
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 17, 2015
Month December January February March April May June
246
POULTRY iiCIENCE
Mortality to date has been practically negligible. Two birds have been lost from the δψο and 1% pens Avith prolapsis of the oviduct. BIBLIOGRAPHY
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 17, 2015
1. Compton, A. Studies in t h e m e c h a n i s m of enzyme action. Role of the, reaction of the m e d i u m in fixing the o p t i m u m tem p e r a t u r e Proc. Roy. Soo. 3 B, 1915, 88, 250, 408; 1921, 92, 1-6. 2. Davis, W. A. & Daish, A. J. Methods of e s t i m a t i n g carbohydrates. The estimation of starch in plant m a t e r i a l . The use of Taka-diastase. J. Agric. Sc. 1914, 6, 152, -108; also 1916, 10, 3-48, 56, 76. 3. Dubourg, B. De le F e r m e n t a t i o n des saccharides. Compt. rend. Acad, desc. Par, 1899, 128, 440-442. 4. Effront, J. Biochemical Catalysts in life and i n d u s t r y . Proteolytic' enzymes. Transi. S. C. Prescott J. Wiley & Sons, New York. 1917. 5. Kellner, O. Mori, Y, & Nagaoka, N. Beitraze zur K e n n t n i s der invertuenden F e r m e n t e . Ztzcher, physiol. Chem. 1890. 6. K i t a G. Uber die E n z y m e des asperzillus oryzae. Wchnschr. B r a n 1912, 29, 460, 463, 1913, 30, 563; J. I n d u s t & E n g i n . Chem. 1913, 5, 220-222. 7. Lwow, S. Zaymase & R e d u k t a s e in ihren gegenseltigen Beziehungen, Ber. Dent. bet. Gesell, 1913, 31, 141-147; Bull. Acad, desc, St. Petersburg, 1913, 501-532. Ztschr. Garungsphysiol, 1913, 3, 289-320. 8. Newcombe, F . C. Cellulose enzymes Aun Bot 1899, 3, 49, 80. 9. Okada, S. On the optimal conditions for the proteoclastic action of taka-diastase. Biochem J. 1916, 10, 130-136. 10. Saito, K. Der Emplusz der N a h r u n g auf die. Diastase bildung durch schammeppilze. 11. Pringshein, H. and Zemplen, G. Studies uber die Polysacharîdes paltenden F e r m e n t e in pelzpreazafte, Ztscha Physiol Chem, 1907, 62, 367, 389. 12. Saito, K. Uber de Eiweisszersetzung durch Schimm&lphilze. Labenzym und Katalase bei aspergilluss oryzae. Bot. Magas. Tokyo, 1902, 17. Biochem C'entrallbe, 1904, 2, 1370. 13. Sanquinette, M. J. Cantupution a I'etude de Γ amylomycee Rouxii de la levure shinolse et des moississures ferments de Γ amidon. A n n . de r Inst. Pasteur, Par. 1897, 11, 204-276. 14. Sherman, H. C. & W a l k e r F . Influence of certain a m i n o acids upon the enzymio hydrolyses of s t a r c h . J . Am. Chem, S o c , 1921, 43, 2454-2461. 15. T a k a m i n e J. Enzymes of Aspergillus orzae and t h e application of its amyloclastic enzyme to t h e fermentation i n d u s t r y . J. I n d u s t & Engin, Chem., 1914, 6, 825-828. Chem. News, 1914, 109, 215. 16. T a k a m i n e , H. Diastatic ssubstances from fungus growths. J. Soc. Chem. Ind. 1898, 17, 118-120; also Country Brewers Gaz. 1894. 17. Vines, S. H. The proteases of Dants, Ann. Bot., 1904, 18, 289-317; 1909, 23, 1-18; 1910, 24. 213-222.
APPLICATION
OF "PROTOvYME"
TO FEED
247
18. Waksman, S. A. The use o£ enzymes in the textile industry. The textile recorder. Dec. 15, 1919. 19. Waksman, S. A. The use of enzymes in the clarification of fruit juices. The Gamier, April 25, 1922. 20. Waksman, S. A. Paper No. 86, of the Jour. Ser. N. J. Agr. Expt. Sta. Dept. Soil Chem. & Bact 21. Wohlgemuth, J. Zur kenntnis der Takadiastase. Biochem. Ztaschr. 1912, 39, 324-338. Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 17, 2015
There have been several changes in the industrial methods of preparing the enzyme since the original process outlined by Takamine, (Eflfront & Prescott) (4) the essential changes being those which would increase the amyloelastie, peptonizing or saccharifying power, in order to satisfy the conditions required for its various applications. The history of the production and application of diastatie enzymes from fungus sources is not without interest and is worthy of brief comment in a paper of this nature. Nageli C. (1879) is credited with being the first to demonstrate the production of diastase by micro-organisms, his experiments being later followed by the works of Kuscheldt who produced the enzyme in workable quantities and applied the name ' ' Eurotin ' ' to the product. The various workers, as they made improvements in culture methods giving greater and more specific application to the enzymes of Aspergillus orizae, attached the names of "Taka-diastase," "Neo-diastase," and "Protozyme." The two former products find their application in the medicinal field, being highly purified and concentrated so as to give rapid action, while the last is used extensively in the textile industry for the de-sizing of cloth (Waksman, S. A.) (18) ; clarification of fruit juices, due to its ability to work in an acid solution (Waksman, S. A.) (19) ; and is now being tested for its effect on digestion and assimilation in feeding of poultry. The preliminary results will be reported later in this paper. *Read at the annual meeting of the A. A. of I. and I. In P. H.. Man hattan, Kansas, August, 1925.
241
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 17, 2015
"Protozyme" is a trade name given to a group of vegetable enzymes of a fungus origin ; namely, those produced by cultures of Aspergillus orizae.
POULTRY
242
SCIENCE
ENZYMES OF PROTOZYME (Aspergillus Orizae) Demonstrated by
Cytase Amylase Maltese
Newcombe, F. C. Davis, W. A. & Daish, A. J. Dubourg, E. Pringsheim, H. & Zemplen, G. Sanquinettl, M. J. Waksman, S. A.
Invertase Emulsin Rennet Protease Oxidase Reductase Catalase Inulase Tannase
Reference
Saito, K. Okada, S. W a k s m a n , S. A. W a k s m a n , S. A. Follwell E,. H. & Clickner, F . H. Kellner, 0 . Mori, Y. Nagaoka, M. Kita, G.
8 2 3 11 13 20 12 9 20 20 5 6
Physical Properties of Protozyme 1. A p p e a r a n c e is similar to toasted bread crumbs, due to the fact t h a t it is dried a n d milled. 2. Color shading from light to dark brown, depending on intensity a n d r a p i d i t y of drying. 3. Odor is a characteristic yeasty odor (when p r e s e n t ) . 4. Taste similar to wheat bran. Chemical Properties of Protozyme 1. Reaction range. Enzyme
pH. Value
Amylase
2.6-8.0
Maltase 47 deg. C. Maltase 35 deg. C. Protease
7.2 3.0 5.1
Determined by Sherman, H. C. & Associates (14) Compton, A. (1) Compton, A. Okada, S. (9)
2. Amyloclastic a n d saccharifying power. 138 L i n t n e r Units (Sample used in t e s t ) . Ranges from 145-200 L i n t n e r Units as p r e p a r e d commercially. Diastase from Protozyme converts starch into maltose a n d chiefly into dextrose. 3. Milk coagulating factor .066 c. c. Protozyme ( 2 % Sol.) equivalent to one Lab. u n i t as measured by Effront ( 4 ) , Wohlgemuth (2) methods. 4. Gelatin liquifying power. E x t r a c t from 1 gram of P r o tozyme will liquify 15 grams of gelatin in two hours. Stronger proteolytic properties t h a n malt Lwow S. ( 7 ) .
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 17, 2015
Enzyme
APPLICATION
Of
"PROTOvYMË"
TO FEED
243
AVERAGE W E E K L Y W E I G H T S OP LEGHOEÎÎ P U L L E T S F E D PROTOZYME
(The Data a r e pounds) Week 1. 2. 3. 4. 5. 6. 7. 8.
9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
A m o u n t of Protozyme in Mash by W e i g h t 5% 3% 2% 1% .13 .23 .34 .49 .67 .79 .96 1.18 1.43 1.75 2.00 2.12 2.23 2.31 2.40 2.50 2.58 2.67 2.93 3.24
.12 .20 .30 .42 .61 .80 .92 1.11 1.29 1.65 1.89 1.94 1.97 2.01 2.10 2.23 2.34 2.48 2.64 3.03
.12 .20 .30 .39 .61 .80 .89 1.04 1.25 1.55 1.78 1.89 1.97 2.04 2.12 2.20 2.27 2.37 2.53 2.89
.12 .19 .30 .38 .58 .72 .83 1.02 1.23 1.30 1.72 1.85 1.92 1.97 2.05 2.11 2.18 2.35 2.50 2.80
-none .12 .18 .28 .36 .56 .69 .76 .93 1.11 1.35 1.57 1.62 1.66 1.70 1.73 1.91 2.04 2.20 2.48 2.65
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 17, 2015
5. Temperature. Stable while dry. Enzymic action decreased or destroyed above 60 degrees C. Protozyme as a Means of Promoting Growth A test has been made with Protozyme, and the preliminary data of the first trial are incorporated in this paper. One thousand White Leghorn chicks, hatched on March 1st, were divided into five groups. Each group was fed a commercial chick scratch mixture until the sixth week, when it was changed to one consisting of equal parts by weight, of cracked corn and whole wheat. All pens were started on a commercial starting mash, which was changed after the sixth week to the folloAving growing mash, consisting of bran 300 lbs., standard wheat middlings 100 lbs., finely ground oats 100 lbs., corn meal 100 lbs., and meat scraps 100 lbs. Protozyme was included in the ration as follows : 5 percent of the weight of the mash in pen 1, 3 % in pen 2, 2% in pen 3, 1% in pen 4 and none in pen 5. At the end of the sixth week, the cockerels were removed, and thereafter growth figures secured on the pullets, 100 in each group.
244
POULTRY
SCIENCE
PRELIMINAKY AVERAGE FEED CONSUMPTION OF LEGHORN PROTOZYME FOR 20 WEEKS.
Birds Receiving:
Scratch Grain Mash Consumption Consumption Lbs. Lbs. 7.65 7.67 7.68 7.70 7.77
8.31 8.31 8.27 8.21 7.73
P r i o r to the seventh week, mortality in the respective pens was as follows: 5, 4, 10, 11, and 16. The birds were placed on range at the end of the seventh week, after which none died in a n y pen. E g g production was first noted in the 1% and check pen, d u r i n g the 17th week. At the end of the 20th week, total production in the respective pens was the following number of eggs: 16, 4, 2, 11, 24. Since this is of a preliminary n a t u r e only, it is given without comment. Protozyme as a Means of Promoting Egg Production. On December 1st, four groups of birds in t h e i r first laying year, 25 in each group, were placed on a test to ascertain what value protozyme might have as a means of i n d u c i n g increased egg production. All groups were fed the regular New J e r s e y laying rations and housed in the long laying house on the Station plant, where they M'ere subjected as closely as possible to conditions prevailing on commercial plants. Protozyme was ineluded in the laying mash in the different groups as follows : 5% by weight of the mash, 3 % , 1% and none at a l l ; this latter group serving as a check to the other three. The figures are of a preliminary n a t u r e , a n d it is too early to make a definite recommendation concerning the possibilities of accessory feeding of this n a t u r e as a means of obtaining an e x t r a profit in flock management.
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 17, 2015
5% Protozyme in Mash Z% 2% 1% " " " No
PULLETS FED
APPLICATION
OF 'ΨΚΟΤΟυΎΜΕ"
TO
FEED
SUMMARY OF SCRATCH GRAIN CONSUMPTION OF INDIVIDUAL
245 BIRDS
F E D PROTOZYME.
A m o u n t ot Protozyme in Mash by Weight
Total
5% 3.75 4.59 3.57 3.30 3.01 3.10 3.00
3% 3.79 4.02 3.48 3.30 3.00 3.10 3.00
1% 3.74 4.19 3.30 3.31 3.00 3.10 3.00
Check 3.98 4.33 3.41 3.32 3.00 3.10 3.00
24.31
23.69
23.64
24.14
SUMMARY OF M A S H CONSUMPTION OF INDIVIDUAL BIRDS CONSUMING
PIROTOZYME I N
MASH
A m o u n t of P r o t o z y m e in Mash by Weight Month
5%
3%
1%
December January February March April May June
2.21 2.77 2.83 2.85 3.00 2.97 3.00
1.80 2.81 2.76 2.87 3.26 3.30 3.52
1.91 2.78 2.66 2.90 2.98 2.93 2.95
1.55 2.41 2.10 2.33 2.85 2.67 2.70
19.63
20.32
18.81
16.61
Total
Check
SUMMARY OF EGG PRODUCTION OF INDIVIDUAL BIRDS CONSUMING PROTOZYME IN M A S H .
Amount of P r o t o z y m e in Mash by Weight Month
5%
3%
1%
Check
December January February March April May June
5.20 6.17 11.87 17.50 17.56 16.60 18.13
4.24 8.68 11.50 17.00 16.81 16.92 21.12
3.44 7.04 11.88 17.16 17.16 16.40 15.30
3.08 6.69 8.56 14.76 16.50 16.70 17.93
Total
93.03
6.27
88.38
84.22
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 17, 2015
Month December January February March April May June
246
POULTRY iiCIENCE
Mortality to date has been practically negligible. Two birds have been lost from the δψο and 1% pens Avith prolapsis of the oviduct. BIBLIOGRAPHY
Downloaded from http://ps.oxfordjournals.org/ at Kainan University on April 17, 2015
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